Disinfection of Water for Aquaculture
ABSTRACT International Commemorative Symposium, 70th Anniversary of The Japanese Society of Fisheries Science. 1-5 October 2001. Yokohama, Japan. Disinfection of water for aquaculture is critical for preventing the introduction and spread of infectious disease. A pathogen-free water source is essential for success in aquaculture. Typical treatment systems make use of high efficiency sand filters to clarify the water before treatment with ultraviolet (UV) light or ozonization. Fish pathogens are divided into two groups based on their sensitivity to UV and total residual oxidants (TROs) produced by ozonization of seawater. Hypochlorite produced by electrolysis of seawater (salt water) showed bactericidal and viricidal effects. This method can easily treat large volumes of water, and is suitable for disinfecting wastewater before discharging.
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ABSTRACT: Salmonid fish and flounders are economically important fish cultured in northern Japan. Viral diseases remain one of the limitations to the successful propagation in aquaculture. The incidence of disease, especially viral disease, has been increasing. Methods currently used to control viral disease in hatcheries are: (a) hygiene and sanitation, (b) disinfection of water supplies and wastewater, (c) selection of pathogen- free broodstock, (d) health monitoring of hatched fry, (e) control of normal intestinal flora, (f) vaccination, and (g) temperature control.
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ABSTRACT: Special Issue: International Symposium on Koi Herpesvirus Disease : Strategy for Koi Herpesvirus Disease Control. 13 March 2004. Yokohama, Japan. Survival of three salmonid viruses and two marine fish viruses in fish rearing water or coastal sea water were observed at 0, 5, 10 and 15 ℃ for 7 or 14 days. Interaction between viruses and microorganisms present in the rearing water was observed. Infectious pancreatic necrosis virus (IPNV) and fish nodavirus (BF-NNV) were stable in waters used at every temperature tested for 14 days, but it was observed that, for infectious hematopoietic necrosis virus (IHNV), Oncorhynchus masou virus (OMV), and hirame rhabdovirus (HIRRV), as the temperature increased, the loss of infectivity also increased. When IHNV and OMV were suspended in filtrated and autoclaved rearing water, infectivity was reduced in comparison with the untreated water. Subsequently, adsorption of IHNV to mud or small particles was studied. IHNV adsorbed to several clays (kaolin, bentonite, Japanese acid clay) and diatomaceous earth in sterilized water with a wide range of pH (5-11) at concentrations of 1, 10, and 100 mg/mL. Except for bentonite, infectivity of clay-adsorbed IHNV persisted for at least 9 weeks. The clay-adsorbed IHNV also persisted in infectivity to rainbow trout Oncorhynchus mykiss, causing cumulative mortality rates of more than 73 %. Then, inactivation effects of UV irradiation, ozonization, and electrolyzation of water were studied against six fish rhabdoviruses, three fish herpesviruses, one fish birnaviruses, one fish iridovirus, and one fish nodavirus. Six rhabdoviruses, three herpesviruses, and lymphocystis disease virus were found to be sensitive to UV irradiation, ozonization, and electrolyzation. Susceptibility of IPNV, chum salmon virus (CSV), and BFNNV to UV was found to be low. IPNV and CSV were low sensitive to ozonization and electrolyzation. Virucidal effects of six kinds of disinfectants were examined against OMV, IPNV, IHNV, and HIRRV at 15 and 20℃ for 30 sec and 20 min. At 15℃ for 20 min, minimum concentrations showing 100 % plaque reduction of viruses tested by iodophore, sodium hypochlorite solution, benzalconium chloride solution, saponated cresole solution, formaldehyde solution, and potassium permanganate solution were 40, 50, 100, 100, 3500, and 16 ppm, respectively.
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TitleDisinfection of Water for Aquaculture
Author(s)Kasai, Hisae; Yoshimizu, Mamoru; Ezura, Yoshio
Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
Proceedings of International Commemorative Symposium 70111 Anniversary of The Japanese Society of Fisheries Science,
F i . ~ h . Sci. 2002; 68 Supplement I: 821'824. October 1'5, 2001. Yokohama. Japan
DISINFECTION OF WATER FOR AQUACULTURE
HISAE KASAl,1 MAMORU YOSHIMIZU1 AND YOSHIO EZURA1
Laboratory of Biochemistry and Biotechnology, Division of Marine Bioscience, Graduate School of Fisheries
Science, Hokkaido University, Minato 3-1-1, Hakodate, Hokkaido 041-8611, Japan.
SUMMARY: Disinfection of water for aquaculture is critical for preventing the introduction and spread of
infectious disease. A pathogen-free water source is essential for success in aquaculture. Typical
treatment systems make use of high efficiency sand filters to clarify the water before treatment with
ultraviolet (UV) light or ozonization. Fish pathogens are divided into two groups based on their
sensitivity to UV and total residual oxidants (TROs) produced by ozonization of seawater. Hypochlorite
produced by electrolysis of seawater (salt water) showed bactericidal and viricidal effects. This method
can easily treat large volumes of water, and is suitable for disinfecting wastewater before discharging.
KEY WORDS: disinfection, aquaculture, fish pathogen, UV, ozone, oxidant, electrolization
Water supplies for seed production and aquaculture
often provide an efficient means for the introduction
and spread of infectious diseases. A pathogen-free
water source is essential for success in aquaculture.
Surface waters commonly used in aquaculture come
from coastal waters or rivers and may contain some
fish pathogens and such open water supplies should
not be used without treatment. Disinfection of
wastewater before discharging is necessary to avoid
the pathogen contamination in the environment. In
this study, we examine the cidal effect of ultraviolet
(UV), oxidant produced by ozonization of seawater
and hypochlorite produced by electrolization of
seawater. Additionally the disinfectant effects of the
three methods for a hatchery water supply and
4 _____ Standard type U.V. [amp
10) HIRRV. LCOV, V. anguillarum
l ~ Q . o l ' n Q r : s : L _____ .
Fungi and Microparasites
Fig. I. U.V. susceptibility offish pathogens.
1 Vlasenko, M. I. (1969) 2 Hoffman, G. 1. (1974)
3 Normandeau, D. A. (1968)
wastewater were compared and the survival rate of
cultured fish that were reared using water treated with
these methods was assessed.
UV susceptibility of fish pathogens and the effects
of UV treatment on hatchery water
The disinfectant effects of UV irradiation were
examined on cell suspensions of 4 species of fish
pathogenic bacteria and Escherichia coli. using a
punched agar medium disk covered with 10 strains of
aquatic fungi and 14 strains of cell free fish
pathogenic viruses. Of the viable bacterial cells of
Gram negative bacteria and Gram positive bacteria.
99.9% or more were killed by UV irradiation at the
dose of 4.0 X 10'> and 2.0 X 10
respectively.4) The hyphae of aquatic fungi showed
relatively lower susceptibility to UV irradiation, levels
that inhibited the growth of hyphae were 1.5 X lOs to
2.5 X lOs 11 W' sec/cm2•S)
three fish herpesviruses and two fish iridovirus were
found to be sensitive to UV irradiation. The dose that
resulted in a 99 % or more infectivity decrease (lD99)
was observed at the dose of 1.0 X 103 to 3.0X 103
J.L W' sec/cm2• Susceptibility of two birnaviruses. a
fish reovirus and a fish nodavirus was found to be low.
ID99 measured 1.5 X lOs to 2.5 X lOs 11 W' sec/cm2
(Fig. 1 ).6)
The infectivity of infectious hematopoietic necrosis
virus (IHNV), in virus-contaminated river water and
pond water. as measured by the molecular filtration
method was 0.56 and 5.6 TClDsoII. respectively. UV
treatment of river water with 104 11 W' sec/cm2 UV
dose prevented an IHN outbreak.7) Furthermore, UV
treatment of the hatchery water supply also decreased
the viable bacterial counts and fungal infection rates
of salmonid eggs.8)
J.L W' sec/cm ,
Six fish rhabdoviruses.
Table 1. Effect oftota! residual oxidants (TROs) concentrations produced by ozonization of seawater on infectivities offISh pathogens
Yellowtail ascites virus (Y A V)
hirame rhabdovirus (iliRRV)
Infectious pancreatic necrosis virus (IPNV)
Infectious haematopoietic necrosis virus (IHNV)
Oncorhynchus masou virus (OMV)
Chum salmon virus (CSV)
lAM 1018 0.5
1 Initial viral infectivity (TCIDsolml). 2 Initial viable bacterial number (CFU/ml). 1 Initial viable number.
Disinf ectant effect of oxidant produced by
ozonization of sea water on flSh pathogens
Treatment of natural seawater with ozone produced
oxidant that showed a disinfectant effect. Total
residual oxidant (TROs) produced in seawater were
stable for 1 h or more. Disinfectant effect ofTROs
against flSh pathogenic organisms was observed at a
dose of 0.5 mg/I for 15 to 30 s or 0.1 mg/I for 60 s,
Ozonization Aeration Culture tank
~ 0 . 0 0 3 mg/I
~ 0 . 0 0 3 mg/I I
Fig. 2. Ozonization of water for aquaculture.
and killed more than 99.9 % of bacterial cells of
Vibrio anguillarum, Enterococcus
Aeromonas salmon icida, A. hydrophila and E. coli,
and inactivated 99 % or more of IHNV. hirame
rhabdovirus (HlRRV) and Oncorhynchus masou virus
(OMY). To inactivate or kill more than 99 % of
yellowtail ascites virus (YAV). infectious pancreatic
necrosis virus (lPNV). chum salmon virus (CSV), and
a Scuticociliatida (ciliata). higher doses of 0.5 to 1.0
mg/I for 1 min were required (Table 1).9.10)
TROs showed toxicity for fish. Barfin flounder
Verasper moseri and herring Clupea pa/lasii died after
16 and 2 h exposure to TROs of 0.1 and 0.5 mg//.
respectively. However, Japanese flounder could be
cultured in ozonized seawater after the TRO were
removed by charcoal (Fig.2), resulting in survival
rates similar to fish cultured in UV treated or
non-treated seawater. 11)
Disinfectant effects of electrolyzed salt water on
fish pathogenic bacteria and viruses
The bactericidal and viricidal effects of hypochlorite
produced by electrolysis of salt water were examined
against pathogenic bacteria and viruses of fish.
Table 2. The chlorine concentration produced by electrolysis of salt water and treatment time
required to 'reduce the viability of bacteria and the infectivity of viruses by 99.9 %
Aeromonas salmonicidc ATCC 14174
Yellowtail ascites virus (Y A V)
hirame rhabdovirus (HIRRV)
I Initial viable bacterial number (CFU/m/). 2 Initial viral infectivity (TCIDsolrnl).
Table 3. Effects oru.v. irradiation, ozonization and electrolyzation on the viability of bacteria in hatchery waste-seawater
Flow rate Treatment
1.3 x 10°
l.Ox 1051lW ·sec/cm!
TROs 0.5 mg/t. 1 min
Chlorine 0.6 mg/I, I min
Sodium chloride solutions, ranging from 0.5 to 3 %
were electrolyzed and the concentration of chlorine
produced was measured. Similar concentrations of
chlorine were produced when 1.0 % or h i ~ h e r NaCl
solution and seawater were electrolyzed. t
solution of sodium chloride containing pathogenic
bacteria or virus was electrolyzed and the organisms
were exposed to chlorine. Greater than 99.9 % of V.
anguil/arum and A. salmonicida cells were killed
when the bacteria were exposed to 0.1 mg/I chlorine
for I min. YAY and HIRRV were 99.9 % or greater
inactivated after treatment with 0.45 mg/I chlorine for
1 min (Table 2).13)
The bactericidal and viricidal effects of hypochlorite
produced by electrolysis (3.5 m3 Ih. 0.1 A) were
greater than that of the chemical reagent. The purity of
the sodium chloride used influenced the effects of
production of hypochlorite. Sodium chloride obtained
as a super grade chemical reagent was more effective
than food-grade sodium chloride.
sufficient disinfectant effect was
electrolyzed seawater. a treatment which may have an
application in aquaculture. To use electrolyzed
seawater for culture. the chlorine has to be removed
with charcoal because of its toxicity.
A 3 %
Disinfection of wastewater
The bactericidal effect of hypochlorite produced by a
continuous flow electrolyzer on hatchery wastewater
was investigated. The number of viable bacteria in the
wastewater was reduced more than 99 % when the
water was treated with chlorine at a concentration of
0.6 mg/I for 1 min, and over 99.9 % of the bacteria
cells were killed when treated with 1.28 mg/I for 1 min
(Table 3). In another experiment. 2.0 m3/min of
hatchery wastewater was electrolyzed and the
produced hypochlorite that was mixed with the
remaining, 16.5 m3/min wastewater. Viability of
bacteria was reduced greater than 99 % after treatment
with 0.5 mg/I of chlorite for 1 min. The bactericidal
effect of electrolysis was almost the same as that of
ultraviolet irradiation (1.0 X lOs Jl. W' sec/cm2) or
ozonization (TROs 0.5 mg/I, I min) of seawater.
ElectroIization can be used to treat a large volume of
wastewater compared with the ultraviolet irradiation
or ozonization. 14)
Disifection of water for hatchery water supplies
and survival rate of cultured fish
The effects of the three disinfection methods on
bacteria in the hatchery water supplies are shown in
Table 4. All methods resulted in a reduction of 96.6 to
99.8 % after the treatment. Survival rate of Japanese
flounder Paralichthys olivaceus and barfin flounder
Table 4. Viability of bacteria in hatchery water supply
after treattnent using different disinfection methods
Method Treatment Viable counts
U.V. irradiation 1
1 U.V. dose: 1.0 X lOS Jl W·seclcm2•
2TROs: 0.5 mg//. 3 Chlorine: 0.5 mg/I
1.1 x 10°
Table 5. Survival rate of Japanese flounder cultured in U.V. irradiated. ozonized, electrolyzed or non-treated seaw
Barfin flounderS NT 34.3
I U.V. dose: l.OX 104 JJ. W·seclcm2•
2 Ozonization: 1.0 mg/I TROs for 8.5 min for Japanese flounder. 0.5 mg/I for 5 min for Bartin flounder.
3 Electrolization: 0.5 mf!JI chlorine for 5 min.
4 Tank size: 0.5 1, Number offish: 2000, Duration: 49 days. Feeding: 1 time/day.
S Tank size: 0.5 1, Number of fish: 6500-7100, Duration: 30 days, Feeding: 2 times/day.
cultured in UV irradiated, ozonized and electrolyzed
seawater are sho\\n in Table 5. No statistically
significant differences in survival rates were found
between the three groups of fish cultured with treated
Ozonized and electrolyzed seawater have been
to be effective
equipment used in aquaculturel5•16) and ozonized
seawater is effective for disinfecting fertilized barfin
flounder eggs contaminated with viral nervous
electrolization of seawater seem to be effective
methods for disinfection of the water for fish culture.
Gram negative bacteria and fish rhabdoviruses,
herpesviruses and iridoviruses were killed when UV
irradiated at the dose of 104
inexpensive UV lamps can irradiate at that dosage and
may be suitable for hatcheries or culturing stations
that have problems caused by these microorganisms.
This would be the best method for disinfection of UV
susceptible pathogens. Water contaminated with
reoviruses, fish nodaviruses and aquatic fungi that
showed lower susceptibility should be disinfected
with ozonization, electrolization or high quality UV
Disinfection of wastewater is necessary to prevent
pathogenic contamination of the environments.
Electrolization is easy to scale up and can be used to
treat a large volume of water. thus making it a suitable
method for disinfecting wastewater.
JJ. W·sec/cm2• Standard,
fish bimaviruses, fish
This research was supported in part by Grant in Aid
for Scientific research (B)(2) No.09556041 and (B)(2)
13556027 under the Ministry of Education, Science,
Sports and Technology, Japan.
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