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Aquaculture 264 (2007) 119–129

Aquaculture

www.elsevier.com/locate/aqua-online

Safety of electrolyzed seawater for use in aquaculture

Masahiko Katayose aa, Kyoichiro Yoshida bb, Nobuo Achiwa bb, Mitsuru Eguchi a,∗a,∗

aa Graduate School of Agriculture, Kinki University, Nara 631-8505, Japan

bb Hoshizaki Electric Co. Ltd., Toyoake, Aichi 470-1194, Japan

Received 16 June 2006; received in revised form 30 August 2006; accepted 30 August 2006

Abstract

The safety of electrolyzed seawater was evaluated by measuring the production rate of organic halogen compounds and the occurrence of reverse mutations. Aquaculture feedwater and wastewater were collected from a fish-culturing facility, and available chlorine of approximately 1.0 mg/L was generated to ensure a disinfectant effect. More than 90% of the generated organic halogen compounds were bromoform. The amount of bromoform was far less than the reference values for drinking water standards in Japan and the U.S., provided that the electrolyzation was performed within the range of normal use. The reverse mutation assay of electrolyzed seawater showed no mutagenicity. Electrolyzed seawater with available chlorine at an adequate level for disinfection can be used safely and effectively in various aspects of aquaculture.

© 2006 Elsevier B.V. All rights reserved.

Keywords: Electrolyzed seawater; Organic halogen compounds; Reverse mutation assay

1. Introduction

In fish hatcheries, ultraviolet light and chemicals are usually used to disinfect seawater in managing water quality and preventing the occurrence of fish diseases. However, there is a worldwide tendency in aquaculture to reduce the usage of chemicals. In Japan, the new Pharmaceutical Law issued in July 2003 strictly regulates the use of chemicals on cultured fish. Furthermore, consumers are becoming increasingly conscious of the safety of marine products. These recent developments require that aquaculture becomes free from the use of chemicals such as antibiotics. In addition to seafood safety, the non-chemical management of water quality is also expected in an eco-friendly aquaculture system. In managing water quality, aquaculture wastewater should be treated appropriately before being discharged from the production site into bodies of natural water. As legal regulations concerning the discharge of effluent from factories have become increasingly strict, workers in the aquaculture sector must also consider the treatment of their wastewater. Electrolyzation of seawater is one approach that can be used to respond to these requirements. From an economic viewpoint, the cost of electrolyzing seawater is reasonable compared to other treatments such as UV, O₃, and treatment with sodium hypochlorite.

Electrolyzed water has a strong disinfectant effect (Iwasawa and Nakamura, 1996; Venkitanarayanan et al., 1999). This characteristic of electrolyzed water has attracted a great deal of attention from the food, medical, and agricultural industries (Fujiwara et al., 2000; Shiba et al., 2000; Park et al., 2002a,b; Takahashi et al., 2002;

Achiwa et al. 2003, Achiwa et al. 2004, Achiwa et al. 2005, Yoshida et al. 2003). Even in the fisheries industry, the disinfectant effect of electrolyzed seawater has been closely observed (Kasai et al. 2000, Kasai et al. 2001a, Jorquera et al. 2002) and applied to the sanitation of marine products (Kasai and Yoshimizu, 2003), aquaculture (Watanabe and Yoshimizu, 2001), and the disinfection of waste seawater (Kasai et al. 2001b, Kasai et al. 2002). In the food, medical, and agricultural sectors, however, NaCl added to tap water is usually used for electrolyzation. As the safety of NaCl-added tap water has been confirmed and guaranteed, it can be legally used on foods in Japan and the U.S. When applied to aquaculture, natural seawater will be used to obtain electrolyzed water; however, the safety of electrolyzed natural seawater, especially its mutagenicity, has yet to be assessed or reported.

When seawater is electrolyzed, chloride ions are oxidized to chlorine and then hydrolyzed to hypochlorous acid. Hypochlorous acid itself has a disinfectant effect, as the hypochlorous acids react with organic substances and bromine ions to generate organic halogen compounds and hypobromous acid (Wong and Davidson, 1977). Hypobromous acid also has a strong disinfectant effect and can react with organic substances to generate organic halogen compounds. Among the organic halogen compounds, trihalomethane is carcinogenic, raising the possibility that electrolyzed seawater may be mutagenic. Needless to say, the electrolyzation of seawater must not give rise to any adverse effects on cultured fish or animals and plants that living within the area of the aquaculture site.

It has been proven that the electrolyzation of NaCl-added tap water is a useful tool for sanitary management. As seawater naturally contains NaCl, the electrolyzation of seawater will be increasingly used for sanitary management. Thus, it is critical that the safety of electrolyzed seawater be checked before it is used regularly. In this report, we evaluate the safety of electrolyzed seawater by quantifying the production of organic halogen compounds and their effects on animals and plants. For the latter purpose, a reverse mutation assay was performed. We also discuss the potential of seawater electrolyzation for the treatment of wastewater, including denitrification.

Materials and methods

Seawater electrolyzer

For all of our experiments, we used an electrolyzer provided with a running-water-type electrolytic cell without a membrane (prototype, Hoshizaki Electric Co. Ltd. Japan). Fig. 1 shows a schematic diagram of the electrolytic cell and reaction pattern. Seawater is electrolyzed by a certain amount of direct-current electricity. Chloride ions in the seawater are oxidized to chlorine at the anode before being hydrolyzed to hypochlorous acid. The hypochlorous acid then reacts with bromine ions to generate hypobromous acid. Metallic ions within seawater such as Na^{+} are attracted close to the cathode, and some of them react with hydroxide ions to generate hydroxide such as NaOH. The production flow rate was adjusted to 2 L/min in the present study. Electrolyzed seawaters in different concentrations of available chlorine (0--27.2 mg/L) were produced by varying the current.

Sample seawater

Aquaculture feedwater and wastewater from the Fish Nursery Center, Kinki University, Japan, were used as samples, with the wastewater being sourced from the culturing of Torafugu (blowfish). Filtered (5 A and 4 A, ADVANTEC) natural seawater collected from Nagoya Port in Aichi Prefecture, Japan, was also used for the experiments. We measured the pH, dissolved oxygen (DO), dissolved organic carbon (DOC), and salinity of the samples using a pH meter (F-13, Horiba Co. Ltd. Japan), DO meter (OM-51, Horiba Co. Ltd. Japan), total organic carbon analyzer (TOC-500, Shimadzu Co. Ltd. Japan), and salinity meter (AAQ1182-H, Alec Electronics Co. Ltd. Japan), respectively.

Determination of concentration of available chlorine

A pocket chlorimeter (type 58700-00, HACH Co. U.S.) was used to measure the concentration of available chlorine.

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Table 1 Organic halogen compounds determined using the headspace method and mass

Numbers (m/Z) used for SIM analysis.

chlorine according to the N,NN,N-diethyl-pp-phenylenediamine (DPD) method (Palin, 1967) and JIS K0102 (Japanese Industrial Standard).

2.4. Determination of inorganic nitrogen

Ammonium, nitrite nitrogen, and nitrate nitrogen were measured spectrophotometrically (DR/2000, HACH Co., U.S.) using the salicylic acid, diazotize, and cadmium reduction methods, respectively.

2.5. Determination of organic halogen concentration using the solvent extraction method

Thiosulfate (10 mg thiosulfate against 1 mg available chlorine) was added to electrolyzed seawater to neutralize available chlorine. Next, 10 mL of the neutralized sample and 2 mL of n-hexane were shaken for 10 min. The obtained n-hexane layer was injected into GC/MS (GCMS-QP2010, Shimadzu Co. Ltd., Japan) for organic halogen analysis. The analysis conditions were as follows: column: Rtx-624 (Restek); column temperature: 45 °C (7 min)–8 °C/min–220 °C (4 min); carrier gas: He controlled at 132.2 kPa; vaporizing chamber temperature: 240 °C; interface temperature: 240 °C; ion source temperature: 200 °C. The following seven compounds were quantified: Chloroform, 1,1,1-Trichloroethane, Trichloroethylene, Tetrachloroethylene, Bromodichloromethane, Dibromochloromethane, and Bromoform. To establish an analytical standard curve, we used an organohalides standard stock solution for the extraction method (Kanto Chemical).

While the official method described by JIS K0125 specifies ascorbic acid as the available chlorine neutralizer, most aquaculture sites regularly use sodium thiosulfate. As both ascorbic acid and sodium thiosulfate used for neutralizing available chlorine showed the same analytical results (data not shown), thiosulfate was used as a chlorine neutralizer in this study.

2.6. Determination of organic halogen concentration using the headspace method

Available chlorine was neutralized immediately after electrolysis using 10 mg of thiosulfate per 1 mg of available chlorine. A total of 10 mL of suspected fluid was poured into a vial container with 3 g of NaCl, sealed tightly with an aluminum cap, shaken vigorously, and warmed at 60 °C for 1 h. Next, 500 μL of gas phase sample in the headspace of the vial container was collected and injected into a GC/MS (GCMS-QP2010, Shimadzu Co. Ltd., Japan) using a gas-tight syringe. The analysis conditions were as follows: column: Rtx-624 (Restek); column temperature: 40 °C (7 min)–5 °C/min–180 °C–10 °C/min–230 °C (5 min); carrier gas: He controlled at 103.0 kPa; vaporizing chamber temperature: 150 °C; interface temperature: 220 °C; ion source temperature: 200 °C. We analyzed 15 compounds including halogen, out of 22 compounds specified in the water quality standards and watch list (Table 1). Table 1 shows the compound names and conditions of selected ion monitoring (SIM) analysis. Volatile organic compounds standard stock solution II (Kanto Chemical) was used to establish an analytical curve.

2.7. Reverse mutation assay (Ames test)

2.7.1. Strains

The following three strains were used for the Ames test: Salmonella typhimurium TA98 (NBRC 14193) and

Table 2 Quality parameters of the tested seawater

Wt: water temperature; DO: dissolved oxygen; DOC: dissolved organic carbon; PSU: practical salinity unit.

All measurements were repeated more than three times. As no statistically significant differences were observed among the three measurements, typical values are presented.

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Table 3 Properties of electrolyzed aquaculture feedwater and wastewater

All measurements were repeated more than three times. As no statistically significant differences were observed among the three measurements, typical values are presented. Flow rate was 2 L/min.

TA100 (NBRC 14194), which require histidine for growth, and Escherichia coli WP2uvrA (NBRC 14196), which requires tryptophan. The specific characteristics of these strains were confirmed prior to being used for the Ames test.

2.7.2. Culture conditions

The test strains were precultured in nutrient broth No. 2 (OXOID). The minimal glucose plate for the mutagenicity test was performed using Vogel-Bonner minimal media containing 2% of glucose and 1.5% of Bacto-Agar (Difco).

Amino acid solution, 0.5 mM of histidine-biotin for the S. typhimurium and 0.5 mM of tryptophan for the E. coli., was mixed with three types of top agar. The three types are as follows: Composition A: 0.6% of Bacto-Agar and 0.5% of NaCl solution were mixed with the amino acid solution at a ratio of 10:1 (v/v); Composition B: 0.8% of Bacto-Agar and 0.67% of NaCl solution were mixed with the amino acid solution at a ratio of 7.5:1 (v/v); Composition C: 1.2% of Bacto-Agar and 1.0% of NaCl solution were mixed with the amino acid solution at a ratio of 5:1 (v/v).

2.7.3. Assay procedure

The assay was performed basically according to the Guidelines for Toxicity Studies of Drugs (Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Japan, 1989) and the method described by Maron and Ames (1982). The reverse mutation assay was performed with (metabolic activation method) or without (direct method) metabolic activation (S9 mix, Oriental Yeast Co. Ltd.). Samples of electrolyzed seawater were collected and assayed within 30 min of electrolysis, without available chlorine neutralization and filter sterilization. The amount of each sample was determined basically by following the strong acidic water's reverse mutation assay (Inai et al., 1994), with a maximum of 1000 μL/plate and a common ratio of two to five amounts, with or without metabolic activation.

First, 62.5 to 1000 μL of the electrolyzed seawater sample, added to 500 μL of S9 mix for the metabolic activation method or 500 μL of sodium phosphate buffer for the direct method, and 100 μL of bacterial preculture in the late lag phase were mixed in a sterile tube and shaken at 37 °C for 20 min. Next, 2 mL of top agar of Composition A, 1.5 mL of Composition B, or 1.0 mL of Composition C were added to the sample amounts of 62.5 to 250 μL, 500 μL, and 1000 μL, respectively, mixed carefully without making bubbles, and spread onto the minimal glucose plate. The plates were incubated at 37 °C for 48 h. After incubation, we counted the revertant colonies that had appeared on the plates. When the number of revertants in the sample was at least twice the number in the positive control and a dose-dependency was observed, we treated the sample as positive mutagenicity.

3. Results

3.1. Experimental seawater

Table 2 shows profiles of the seawater samples used in the study. The temperature, pH, DO, DOC, and salinity were largely within the ranges of ordinary coastal seawater except for the salinity of samples from Nagoya Port, which was relatively low.

3.2. Properties of electrolyzed aquaculture feedwater and wastewater

The available chlorine concentration (both of free and combined chlorine), pH, and inorganic nitrogen concentration were determined for aquaculture feedwater and wastewater that were electrolyzed with electric currents in the range from 0.1 A to 5.0 A at a flow rate of 2 L/min (Table 3). The higher the electrolytic currents, the higher

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Fig. 2. Changes in concentrations of organic halogen compounds with different levels of available chlorine (free chlorine) (♦: 0.2 mg/L; ■: 2.0 mg/L; ▲: 27.2 mg/L) and reaction times. A: chloroform; B: trichloroethylene; C: 1,1,1-trichloroethane; D: tetrachloroethylene; E: bromodichloromethane; F: dibromochloromethane; G: bromoform: DWS: drinking water standards of the Ministry of Health, Labour and Welfare, Japan. A reaction time of 0 h indicates measurement immediately following electrolysis. Seawater samples were collected at Nagoya Port.

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Table 4 Concentrations of organic halogen compounds in electrolyzed aquaculture feedwater immediately following electrolysis (unit: ppb)

Values are mean ± standard deviation. –: unregulated. Control: no electrolysis. a Drinking Water Standards of the Ministry of Health, Labour and Welfare, Japan. b National Primary Drinking Water Standards of the U.S. Environmental Protection Agency.

the concentrations of available chlorine that were observed in both the feedwater and wastewater. The concentration of combined chlorine in the wastewater was higher than that in the feedwater.

3.3. Inorganic nitrogen

The results of measurements of inorganic nitrogen in electrolyzed feedwater and wastewater are shown in

Table 5 Concentrations of organic halogen compounds in electrolyzed aquaculture wastewater immediately following electrolysis (unit: ppb)

Values are mean ± standard deviation. –: unregulated. Control: no electrolysis. a Drinking Water Standards of the Ministry of Health, Labour and Welfare, Japan. b National Primary Drinking Water Standards of the U.S. Environmental Protection Agency.

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Table 6 Results of reverse mutation assays with electrolyzed aquaculture feedwater

AF2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; 2AA: 9-aminoacridine·HCl. a μg/plate.

Table 3. The concentrations of available chlorine (free chlorine) were 0.36–23.6 mg/L. The amount of ammonium nitrogen decreased with increasing available chlorine in the wastewater, which included ammonium nitrogen. No significant decreasing trends were observed in the nitrite and nitrate nitrogens.

3.4. Concentration of organic halogen

We measured the concentrations of organic halogen with varying levels of available chlorine (free chlorine) and reaction times using natural seawater collected from Nagoya Port. Following electrolysis at varying electric currents to generate different levels of available chlorine, the seawater samples were kept at room temperature without neutralization. At appropriate times (reaction times were 0, 1, and 6 h), subsamples were withdrawn from the electrolyzed samples. The concentrations of organic halogen in the subsamples were measured using the solvent extraction method (Fig. 2). Most of the gen

erated organic halogen compounds contained Br radicals in the halogen group. Compounds containing Cl radicals were also observed, but at lower levels. More than 90% of the generated organic halogen compounds were dominated by bromoform. The concentration of compounds bearing Br radicals increased in accordance with the increase in available chlorine concentration. Even for low concentrations of available chlorine such as 0.2 mg/L, when the electrolyzed natural seawater was left for a longer period without neutralization, the amount of organic halogen with Br radicals increased (Fig. 2-E, F, G). The small amount of organic halogen compounds with Cl radicals also increased with increasing concentration of available chlorine; however, in contrast to the organic halogen with Br, the concentration of halogen compounds decreased naturally and gradually after a given period of time, even without neutralization (Fig. 2-A, B). When electrolyzed seawater was neutralized, the concentration of organic halogen compounds decreased over time (data not shown).

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Table 7 Results of reverse mutation assays with electrolyzed aquaculture wastewater

AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; 2AA: 9-aminoacridine·HCl. a μg/plate.

The concentration of organic halogen generated following the electrolysis of feedwater and wastewater was estimated using the headspace method. After electrolysis at various electric currents, the seawater samples were immediately neutralized and subsamples were drawn from the electrolyzed samples. The detection sensitivity of the headspace method is lower than that of the solvent extraction method; however, the headspace method is able to detect a greater variety of compounds. Bromoform and dibromochloromethane were detected within the electrolyzed feedwater (Table 4), but chloroform and other compounds with two or three Cl radicals were not detected. Bromoform and dibromochloromethane were also detected in the electrolyzed wastewater (Table 5). As the concentration of organics in the wastewater was higher than that in the feedwater (Table 2), the amount of bromoform tended to increase with increasing concentrations of available chlorine. Despite this trend, all concentrations of organic halogen remained well below the values of water quality standards legally established in Japan and the U.S.

3.5. Reverse mutation assay

The results of the reverse mutation assay with electrolyzed feedwater and wastewater are shown in Tables 6 and 7, respectively. We considered two different concentrations of available chlorine (free chlorine), 0.5 mg/L and 3.0 mg/L, in both electrolyzed feedwater and wastewater. We did not consider a concentration of 30.0 mg/L because we had previously confirmed the inhibition of bacteria growth at this concentration (data not shown). For all experimental sections and dosages, no increase of the revertant number was observed in any of the mutation assays undertaken using the direct and metabolic activation methods with the three different strains. On the basis of these results, we conclude that electrolyzed seawater has no mutagenicity, at least under the tested conditions.

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4. Discussion

Electrolyzed seawater shows a strong disinfectant effect (Kasai et al., 2000, 2001a; Jorquera et al., 2002). Kasai et al. (2000) reported that the available chlorine level required to kill or inactivate 99.99% or more of fish pathogenic bacteria and viruses was 0.07–0.58 mg/L. We also tested various non-pathogenic marine bacterial strains and found that even stress-resistant marine bacteria such as Sphingomonas sp. (Eguchi et al., 1996, 2001) were killed in entirety at an available chlorine concentration of 1.0 mg/L (data not shown). The main disinfectant substance in electrolyzed seawater is regarded to be hypochlorous acid, which is described as residual chlorine (Kasai et al., 2000, 2001b) or available chlorine (Kasai et al., 2001a; Kasai and Yoshimizu, 2003). On the basis of previous results (Kasai et al., 2000, 2001a) and those of the present study (data not shown), it is apparent that electrolyzed salt-water kills microorganisms more quickly than a chemical product of hypochlorous acid dose, even at equivalent concentrations of available chlorine. It has also been noted that the strong disinfectant effect of electrolyzed saltwater is caused by other reaction products (Kasai et al., 2000). In addition to chlorine, seawater contains bromide, iodine, and other halogens, and can therefore produce residual oxidants such as hypochlorous acid that show a strong disinfectant effect.

Several types of oxidants are generated in electrolyzed seawater, and previous reports have described the oxidants generated in seawater by chlorine treatment and electrolysis. Mimura et al. (1998b) reported that most of the oxidants are hypobromous acids that occur below the level of 8 mg O₃/L. Wong and Davidson (1977) also reported that hypochlorous acid immediately reacts to hypobromous acid when sodium hypochlorite is added to seawater.

The following is a group of relevant reaction formula.

Cl2+H2O↔HCl+HClOCl2+H2O↔HCl+HClO

HClO↔OCl−+H+HClO↔OCl−+H+

HClO+Br−↔HBrO+Cl−HClO+Br−↔HBrO+Cl−

HBrO↔OBr−+H+HBrO↔OBr−+H+

Our results in terms of organic halogen concentrations (Tables 4 and 5) show that more than 90% consisted of bromoform with three Br groups as a halogen group, while the next most common product was dibromochloromethane, which has two Br groups

and one Cl group as a halogen group. For the reasons outlined above, most of the oxidants generated in the electrolyzed seawater were hypobromous acid. The DPD method employed in the present study to determine available chlorine is also sensitive to bromine and iodine (Palin, 1967). Accordingly, the levels of available chlorine reported in this study may also have included bromine and iodine in addition to chlorine. As current analysis techniques are unable to detect iodine oxide, we described the oxidants within electrolyzed seawater as available chlorine.

We evaluated the safety of the generated organic halogen compounds according to the drinking water standards described in the Water Works Law (Ministry of Health, Labour and Welfare Ordinance No. 101, Japan) and the National Primary Drinking Water Standards (U.S. Environmental Protection Agency; EPA) (see Tables 4 and 5 for reference values). The concentration of organic halogen compounds was lower than the reference values immediately after electrolysis (Fig. 2); however, in the case of bromoform, the concentrations exceeded the reference values in the case that the sample water was left for a longer period of time following the generation of excessive amounts of available chlorine (Fig. 2-G). Accordingly, electrolyzed seawater should be used within several hours of electrolysis and the concentration of available chlorine should be kept below 2.0 mg/L.

Electrolyzed seawater showed no mutagenicity under our test conditions (Tables 6 and 7). Thus, electrolyzed seawater can be used safely and effectively provided that the level of available chlorine of around 1.0 mg/L has a sufficient disinfectant effect and that it is used immediately following electrolysis.

The denitrification method using ozone or chlorine under the presence of bromide ions is commonly used (Yang et al., 1997). Hence, electrolyzed seawater may be useful not only for disinfection but also for wastewater treatment using denitrification. The electrolysis denitrification method employs the same principle as the denitrification method: the reaction between hypochlorous acid and ammonia. This is known as the break point reaction:

NH3+HBrO↔NH2Br+H2ONH3+HBrO↔NH2Br+H2O

NH2Br+HBrO↔NHBr2+H2ONH2Br+HBrO↔NHBr2+H2O

NHBr2+HBrO↔NBr3+H2ONHBr2+HBrO↔NBr3+H2O

2H2O+NHBr2+NBr3↔N2+3Br−+3H+2H2O+NHBr2+NBr3↔N2+3Br−+3H+

+2HBrO+2HBrO

M. Katayose et al. / Aquaculture 264 (2007) 119-129

Our results also indicate the possibility that denitrification occurred. Following electrolyzation, the NH₄–N concentration decreased (Table 3). In the case of nitrite nitrogen and nitrate nitrogen, however, no significant decreases were observed, as a decrease in nitrite nitrogen and nitrate nitrogen is not involved in the chemical reaction of the denitrification method using chlorine. It appears from the data in Table 3 that the denitrification reaction is complete; however, in the break point reaction between chlorine and ammonia, combined chlorine increases with increasing chlorine concentration before the amount of combined chlorine decreases rapidly to reach the break point. At this stage, the denitrification reaction is complete (Jensen and Johnson, 1989). As shown in Table 3, the amount of combined chlorine does not show a decrease. In this case, the denitrification reaction does not seem to have gone to completion. Consequently, more than 1.0 mg/L of available chlorine concentration is required to complete the denitrification reaction, while less than 1.0 mg/L is sufficient for sanitary purposes. Thus, the obvious question is whether the electrolyzation of seawater can provide both a sanitary effect and treat wastewater (including denitrification) without adversely influencing natural marine life; the answer is yes. Even if a high concentration of available chlorine, e.g., 27.2 mg/L (Fig. 2), is required for denitrification, the concentration of available chlorine and its by-products can be reduced to within safe levels by dilution with natural seawater, exposure to sunlight, and/or a combination of these treatments.

A variety of applications is available for electrolyzed seawater. As disinfectant seawater with available chlorine, it can be used for the disinfection of feedwater tanks, equipment, the surrounding environments, and feed. To ensure its safe application as feedwater, it should be generated with an available chlorine concentration of less than 2.0 mg/L and be used within several hours. To negate the risk of harmful effects on marine animals and plants, discharged electrolyzed seawater should be diluted with natural seawater. Neutralizing the available chlorine means that the electrolyzed seawater can be used as sterilizing seawater in aquaculture. Mimura et al. (1998a) reported that larvae are less resistant to available chlorine than juveniles and that the 24-h LD₅₀ value for flounder larvae is 0.05 mg O₃/L. Electrolyzed seawater with no available chlorine is suitable for use in the aquaculture of such species with extremely low resistance to available chlorine, and may be useful for probiotic fish culture following the addition of probiotic microorganisms. From the perspective of eco-friendly aquaculture, the treatment of wastewater is required to minimize adverse effects on the natural environment.

Electrolyzed seawater is useful in many different stages of aquaculture in ensuring control of the electrolyzation process.

Acknowledgments

The authors gratefully acknowledge Dr. Takayuki Nishio, Osaka City Institute of Public Health and Environmental Sciences, for helpful advice on the reverse mutation assay. We also gratefully acknowledge the assistance of Prof. Shigeru Miyashita and Dr. Yoshizumi Nakagawa, Kinki University Fish Nursery Center, Shirahama, in collecting samples of aquaculture feedwater and wastewater. This study was partly supported by the 21st Century COE Program “Center of Aquaculture Science and Technology for Bluefin Tuna and Other Cultivated Fish” funded by the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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