The anti-oomycetic effects of sodium chloride and potassium permanganate and the toxicity of these compounds to tilapia (Oreochromis niloticus) eggs

The anti-oomycetic effects of sodium chloride and potassium permanganate on the vegetative and zoosporic stages of Achlya sp. BKKU 0502, Saprolegnia diclina BKKU 0506, Aphanomyces sp. BKKU 0508, Achlya ambisexualis BKKU 0615 and Achlya bisexualis BKKU 0616 were investigated at 25°C. The results showed that the exposure of the samples to 3.0 and 2.5% sodium chloride for 24 h was toxic to the vegetative and zoosporic stages, respectively. Moreover, 200 and 25 ppm of potassium permanganate were effective at killing the vegetative stage at 24 h and the zoosporic stage at 1 h, respectively. The toxicities of 2.0, 2.5 and 3.0% sodium chloride for controlling oomycete activity were determined using tilapia (Oreochromis niloticus) eyed eggs for both 1 and 24 h treatments. The results showed that salt concentrations at levels of 2.0% or higher reduced the hatching rate percentage of the treatment groups to a value significantly different from that of the control group (P<0.05). A noteworthy result was that the 3.0% sodium chloride treatment for 24 h produced a 0% hatching rate. The treatment of the eyed eggs with 100, 150 and 200 ppm potassium permanganate to control the oomycete activity had a highly toxic effect on the eggs: the results showed a 0% hatching rate for all of the treatment groups after 1 and 24 h exposures. Therefore, it is not possible to use sodium chloride or potassium permanganate to prevent the activity of oomycetes on tilapia eggs because these two chemicals decrease the hatching rate. Other chemicals that may be safe to use on other edible aquatic organisms will require further investigation.


INTRODUCTION
Aquatic oomycetes are ubiquitous in the natural water supplies of fish hatcheries and often cause serious disease problems for fish culturists (Schreck et al., 1993;Rach et al., 1997Rach et al., , 2005)).Outbreaks of oomycete infections on fish and fish eggs continue to cause a big problem in cultured fish.The Saprolegniales such as  Saprolegnia, Achlya and Aphanomyces are endemic to all freshwater habitats and they affected fish culture (Noga, 1996).This infection is one problem in brood stock husbandry and it can increase the mortality rate up to 80-100% of incubation eggs (Chukanhom and Hatai, 2004).In case of fish eggs, the infected eggs turned to be white and become dead.In fry, oomycetes start to infect on some part of body surface and then covered the entire body.It causes low productivity of fry and fish cultures (Paxton and Willoughby, 2000).In the past, this problem was solved with the extremely effective chemicals malachite green and formalin (Hansen, 2004).However, due to their toxicity and carcinogenicity to fish, fish eggs and the health of the farmers, the use of these two chemicals was discontinued for oomycete control in edible aquatic animals (Schreck et al., 1993).Many chemicals and drugs have been recommended for use in aquaculture for the prevention and treatment of diseases, and also for improving the water quality (Chinabut, 1997).
Standard treatments with sodium chloride (NaCl) and potassium permanganate (KMnO 4 ) are now effectively used in aquaculture to treat external parasites (Klinger and Francis-Floyd, 2002;Marecaux, 2006), and these compounds have also been reported to be anti-oomycete agents in fish culture (Schreck et al., 1993;Bruno and Wood, 1999).The aims of the present study were to examine the anti-oomycete effects of sodium chloride and potassium permanganate on (1) the vegetative and zoosporic stages of 3 genera, Achlya, Aphanomyces and Saprolegnia, and (2) their toxicities in the eyed stage of tilapia eggs.

Oomycetes and culture conditions
Five selected oomycete isolates (Table 1) were cultured on glucose yeast extract agar (GYA), incubated at 25°C to obtain the vegetative stage, and then sub-cultured every month.The actively growing hyphae of the 3 days colony of oomycete isolates were used as oomycete inoculums in all experiments.

Source of eggs and incubation water
Samples of healthy eyed-stage tilapia eggs (Figure 1) were obtained from Khon Kaen Inland Fisheries Research and

Oomycetestatic activity of NaCl and KMnO4
The minimum inhibitory concentrations (MIC) of NaCl and KMnO4 that inhibited the growth of the oomycetes were verified to determine the proper oomycetestatic dosages.The commercial grades of NaCl (Univar, Asia Pacific Specialty Chemicals Limited, Australia) and KMnO4 (EIS, Inter-education Supply Inc. Ltd., Bangkok, Thailand) were used in all the experiments, and prepared at various concentrations immediately before use.Approximately the same amount of hyphae was placed into 10 ml of various concentrations of NaCl (0.5, 2.5 and 5.0%) or KMnO4 (50, 100, 200 and 400 ppm); the hyphae for the control groups were placed in STW without NaCl and KMnO4.The vegetative growth of the treatment groups with NaCl and KMnO4 was compared with the control group, and was observed by the naked eye after 1, 2 and 5 days of inoculation at 25°C.If no growth appeared after 5 days, the hyphae were removed, washed with STW, and then placed on new GY agar plates for 2 days at 25°C to observe the oomycete viability.

Oomycetecidal activity of NaCl and KMnO4 against vegetative growth
The actively growing hyphae was placed into 10 ml of various concentrations of NaCl (1.5, 2.0, 2.5, 3.0%) or KMnO4 (25,50,100,150,200 ppm) for treatments of 30 min and 1, 2, 6 and 24 h.The hyphae of the control groups were placed in STW without NaCl (0%) or KMnO4 (0 ppm) for the same durations as the treatment groups.The mycelia were then removed, washed with STW, and placed on GY agar plates at 25°C.The activities of the specimens containing NaCl and KMnO4 were compared to the activities of the control groups to determine the oomycete viability within 48 h.

Oomycetecidal effects of NaCl and KMnO4 on zoospore germination
Zoospore suspensions of each isolate were adjusted to 1x10 3 spores/ml.A total of 1 ml of NaCl or KMnO4 solution with 10 times the desired final concentration was added to 9 ml of the zoospore suspension, and the mixture was kept at 25°C for 30 min and 1, 2, 6 and 24 h.At each time point, 120 µl aliquots of the mixture were inoculated onto GY agar.The viability of the zoospores was determined by observing the appearance of the colonies over a 2 day period with the naked eye.The control groups without NaCl and KMnO4 treatment were also observed.

Toxic effects of NaCl and KMnO4 on the hatching rate of tilapia eggs
The experiments were divided into 3 trials as follows: (1) The control group contained STW; (2) the experimental group I contained 2.0, 2.5, and 3.0% NaCl; and (3) the experimental group II contained 1.5, 2.0, and 2.5 ppm KMnO4.Fifty eggs of each group were maintained in 500 ml of solution with aeration at 27-29°C for 1 and 24 h, respectively.Subsequently, all of the eggs were washed and then transferred to different beakers of sterilized incubation water for seven days until they hatched.Three replicates of each experiment were performed.The hatching rates and percentage of corrected mortality of the eggs were calculated using Abbott's formula of Barnes et al. (1998).
Percentage of corrected mortality (Pt) = Po -Pc 100 -Pc x 100 Where, Po, Mortality rate of test group; Pc, mortality rate of control group.

Statistical analysis
The mean hatching and corrected mortality rates of the fish eggs in the control and treatment groups were analyzed using a one-way ANOVA (Zar, 1999).

Oomycetestatic activity of NaCl and KMnO 4
As shown in

Oomycetecidal effects of NaCl and KMnO 4 on vegetative growth
As shown in

Toxic effects of NaCl and KMnO 4 on the hatching rate of tilapia eggs
As shown in Table 5, the untreated control trial (0% NaCl) showed the highest mean percentage hatching rate, 96.6%, for both the 1 and 24 h exposures.The treatment groups exposed to 3 selected concentrations of NaCl (2.0, 2.5 and 3.0%) for 1 h showed a decreased mean percentage hatching rate: 86.6, 80.6 and 70.6%, respectively.In contrast, the treatment groups exposed to 2.0, 2.5 and 3.0% NaCl for 24 h showed a decreased mean percentage hatching rate: 71.7, 37.3 and 0%, respectively.
The results showed decreased mean percentage hatching rates corresponding to the increased salt concentrations, and the control group differed significantly from the treatment groups (P<0.05,one-way ANOVA).The results for the KMnO 4 treatments showed that 100, 150 and 200 ppm KMnO 4 caused a 0% hatching rate after exposures of 1 and 24 h (Table 6).The different letters in the same column means statistically significant difference between treatments (P<0.05,oneway ANOVA).
Table 6.Mean percentages of hatching and mortality rates of the eyed egg stages after exposed with various concentrations of KMnO4 at 27-29°C for 1 and 24 h.

DISCUSSION
Oomycete infections of eggs are prevalent in many fish species reared in hatcheries, and the management of oomycete infections has traditionally depended on the prophylactic or therapeutic administration of chemicals.Sodium chloride has been recognized as a safe treatment for Saprolegnia on the eggs of fall Chinook salmon (Oncorhynchus tshawytscha) (Waterstrat and Marking, 1995).In the present study, the oomycetecidal activity of NaCl on the cultured vegetative growth of five isolates was 3.0%, whereas concentrations of 2.5% NaCl had toxic effects on zoospore germination after 24 h.The reason for this difference may be that the zoosporic stage was more sensitive to chemicals than the vegetative stage (Muller-Breban et al., 1995).According to Kitancharoen et al. (1997), an exposure to 2.5% of NaCl for 1 h twice a week was the best method for oomycete control, without affecting the condition of the eggs.Marking et al. (1994) reported that the exposure to 3.0% salt for 1 h every other day inhibited oomycete infection and increased hatching rates, whereas concentrations greater than 3.0% were toxic to the eggs.
The results of this study show that, although oomycete control is more effective at higher salt concentrations, the salt solutions also produced egg death at levels of 2.0% NaCl or higher, with the hatching rate percentage of the treatment groups exposed to 2.0, 2.5 and 3.0% NaCl for 1 or 24 h differing significantly from that of the untreated control groups (P<0.05).A noteworthy result was that the exposure to 3.0% NaCl for 24 h produced a 0% hatching rate.Therefore, the hatching rate percentage of the tilapia eggs was strongly affected by the concentration of salt and the exposure time.This finding is similar to the results of Edgell et al. (1993) who reported that salt solutions may cause egg deaths at levels of 2.5% or higher.According to Martinez-Palacios et al. (2004), it is possible that high salinities have an inhibitory action on the movement of the fish embryo due to the high osmotic impact on the perivitelline layer.Yamagami (1988) stated that hatching success is affected primarily by the level of chorionase activity and embryo movement.In this study, a longer chemical exposure period (24 h) proved to be harmful for all of the eggs.A shorter chemical exposure period could reduce the stress on the egg and allow the safe application of the chemical (Rach et al., 1997(Rach et al., , 2000a(Rach et al., , 2000b)).
Potassium permanganate has been used to treat external pathogens, including fungi, bacteria and certain parasites (Noga, 1996;Francis-Floyd and Klinger, 2002).In this study, the oomycetecidal effects of KMnO 4 on the vegetative and zoosporic stages varied among the isolates and exposure times, with lower concentrations of KMnO 4 (25 ppm) and shorter chemical exposure periods (30 min and 1 h) having a stronger effect on the zoosporic stage than on the vegetative stage.The exposure to 200 ppm KMnO 4 for 24 h was effective in controlling both the vegetative and zoosporic stages of all of the oomycete isolates.However, this result does not agree with the findings of Yuasa et al. (2000) who reported that 200 ppm KMnO 4 was toxic to Achlya, Aphanomyces and Pythium at 6 h and toxic to the genus Saprolegnia after 12 h treatments.Marking et al. (1994) reported that KMnO 4 was an oomycetecide for the inhibition of a cultured oomycete (in vitro) at 50 ppm and was toxic to eggs at 150 ppm for 1 h exposures at 12°C.However, the treatment of eyed-stage eggs with 100, 150 and 200 ppm of KMnO 4 for both 1 and 24 h resulted in a 0% hatching rate.Prior to this study, the scientific community felt that it was not possible to use NaCl and KMnO 4 to prevent the invasion of oomycetes of tilapia eggs because it was believed that these 2 chemicals would decrease the hatching rates.From this result, it may be suggested that if the tilapia eggs are often bathed with these two chemicals at high concentrations, that is, 3.0% NaCl and 200 ppm KMnO 4 for 30 min or less than 1 h, may inhibit the growth of the aquatic oomycetes without harming the eggs, which require further investigation.

Conclusion
The MIC of NaCl against five zoosporic-stage oomycete isolates was 2.5% for 2 h of treatment, whereas a concentration of 3.0% NaCl was toxic to the vegetative stage after 24 h of treatment.This study found that 25 and 200 ppm KMnO 4 were effective in killing the zoosporic and vegetative stages at exposures after 30 min and 24 h, respectively.The toxicity of 2.0, 2.5 and 3.0% NaCl was high for the eyed eggs, decreasing the percentage hatching rate.In addition, 25, 50, 100, 150, and 200 ppm KMnO 4 had strong toxic effects on the eggs, resulting in a 0% hatching rate.These results are important because many hatcheries mistakenly assume that NaCl and KMnO 4 affect oomycete viability, yet these compounds are also toxic to tilapia eggs.

Figure 1 .
Figure 1.The healthy eyed stage of tilapia eggs used in this experiment (1 scale bar=1 mm).

Table 1 .
Oomycetes isolated from tilapia eggs and used in this study.

Table 2 .
Oomycetestatic effect of NaCl and KMnO4 dosages on vegetative oomycete isolates at 25°C.
Table 2, the treatments with various concentrations of NaCl at 25°C demonstrated that all of the isolates were able to grow in 0 and 0.5% NaCl.The MIC of NaCl was 2.5% for Achlya sp.isolate BKKU 0502, Saprolegnia diclina isolate BKKU 0506, Aphanomyces sp.isolate BKKU 0508, Achlya ambisexualis isolate BKKU 0615 and Achlya bisexualis isolate BKKU 0616.The results of the treatment with KMnO 4 at 25°C showed that the MIC values of each oomycete species were 50 ppm for S. diclina isolate BKKU 0506 and A.

Table 3 .
Oomycetecidal effect of dosages and exposure times of NaCl and KMnO4 on vegetative stage of oomycete isolates at 25°C.

Table 4 .
Oomycetecidal effect of dosages and exposure times of NaCl and KMnO4 on zoospore germination of oomycete isolates at 25°C.

Table 5 .
Mean percentages of hatching and mortality rates of the eyed egg stages after exposed with various concentrations of NaCl at 27-29°C for 1 and 24 h.