Abstract

Intensive culture practices employed in the commercial growth of catfish in western Mississippi frequently result in poor water quality and increased stress to the fish. These eutrophic water conditions can result in an increased incidence of disease and mortality due to bacterial pathogens and in an increased prevalence of off-flavor. Few therapeutic compounds are currently available to improve water quality and decrease disease mortality. Preliminary results from field studies indicate that Pergenox-32G\ (Sodium carbonate peroxyhydrate), an oxidative alkali, is beneficial in the control of the 2-methylisoborneol (MIB) type of off-flavor. In addition laboratory results indicated that Pergenox-32G was an effective bacteriocidal agent against Edwardsiel a ictaluri the cause of enteric septicemia in catfish. A method is described wherein fiber glass cylinders (1.8 meters high by 3.8 m in diameter) are placed in commercial culture ponds experiencing off-flavor or natural outbreaks of enteric septicemia (ESC). Sufficient replicates can be obtained using this technology to determine the efficacy of this compound for off-flavor management and disease control.

Introduction

The commercial culture of catfish in western Mississippi is a rapidly expanding industry that encompasses 91,000 acres from which 291 million pounds of fish were processed in 1988. However, the future expansion of the industry is presently hindered by several problems related to water quality. Off-flavor in which fish acquire a taste and odor that makes them unpalatable and unmarketable, is regarded as the most serious economic problem facing producers today. Entcric septicemia in catfish (ESC) has reached epidemic proportions in fingerling production, is caused by the bacterium Edwardsiella ictaluri, and is considered the most serious bacterial pathogen in catfish production. Both Off-flavor and ESC are serious problems that limit profitability and production in the commercial catfish industry today.

Prevalent culture practices involving high stocking densities (5-8000 lbs/surface acre) and heavy feeding regimes (3% bodyweight) result in conditions best characterized as a eutrophic environment (Brown and Boyd, 1982). Off-flavor is the most devastating production problem and is associated with eutrophic conditions prevalent in commercial catfish culture. The problem results in an unpalatable and unmarketable product. The off-flavor condition afflicts 50-80% of fish subjected for taste testing during the summer growout season. Off-flavor has been associated with the production of 2­methylisoborneol (MIB) and geosmin (March et al, 1987; Lovell et al, 1986). Chronic of f-flavor has been associated with two dehydration products of MlB, 2methylenebornane and 2-methyl-2-bornene and off-flavor may persist for more than one year (Martin et al, 1 988a). Off-flavor compounds are excretion products of several species of Cyanobacteria (Slater and Blok, 1983; Izaguirre 1982) and actinomycetes (Sivonen, 1982; Gerber, 1983). They are believed to be produced in the isoprenoid pathway involved in carotenoid synthesis (Bentley and Meganathan, 1981). In addition, MIB off-flavor has been associated with senescent and fragmentary populations of Oscilla toria agardhii (Martin et al, 1988b). Die-offs of dense algal blooms result in rapid oxygen depletion and therefore both oxygen depletion and off-flavor may occur simultaneously (Boyd et al, 1982a).

There is currently no acceptable method for off-flavor control. Solricin 135, a selective bluegreen algicide, has been proven to be an ineffective treatment (Tucker and Lloyd, 1987). Tilapia polyculture has also been found to be ineffective (Martin and Tucker, 1987 unpublished results). Copper sulfate, although a potent algicide often exacerbates off-flavor problems due to the release of endogenous off-flavor compounds upon lysis of algal cells (Naes, 1985). In addition, copper can cause oxygen depletion due to its rapid algicidal activity.

Enteric septicemia of catfish (ESC) is considered the most serious bacterial disease in commercial catfish production. The disease is caused by a gram negative bacterium Edward siella ictaluri in the family Enterobacteriaceac. The bacteria is cytochrome oxidase negative and catalase positive (Former and McWhorter, 1984). The infection is apparently host specific for channel catfish and occurs within a narrow temperature range (22-28°C) in the late spring and tall (Hawke, 1979). The dependence of outbreaks may be related to motility as Edwardsiella ictaluri normally motile at 22-28°C has been reported to be non-notile at 30°C (Hawke, 1979). All sizes of catfish can be affected although the disease is more prevalent and a higher mortality is generally observed in fingerlings. Mortality may approach 50% for exposed, previously-naive populations of catfish.

Currently, the preferred treatment is by medicated feed (Terramycin, oxytetracycline) and early detection is extremely important. Therapeutic failure is often attributed to late diagnosis, poor feeding by affected fish, and therefore failure to get significant concentrations of oxytetracycline into the fish.

Pergenox-32G is an oxidative alkali with the chemical formula 2 Na2CO3 3H2O2. It is 32% hydrogen peroxide by weight which is degraded to oxygen and water via hydroxyl radical intermediates. It is bacteriocidal, algicidal and may significantly reduce off-flavor because of its selective action on Oscillatoria species (bluegreen alga) generally implicated in the MIB off-flavor (Kay et al, 1982). It is significantly less toxic to the green alga Chlorophyta generally associated with greater oxygen production and improved water quality (Kay et al, 1982). The active ingredient in Pergenox-32G has been used to improve water quality in eel farming and for odor control in water treatment facilities Pergenox- 32G is marketed in a solid granular form and readily dissolves in water providing distinct advantages in handling over hydrogen peroxide.

The present study was designed to gather preliminary data on the efficacy of Pergenox-32G to control Off-flavor due to MIB in field situations and to determine the toxicity of this compound to the bacterium Edwardsiella ictaluri, an important pathogen of channel catfish.

Experimental

Off-flavor

Two commercial catfish ponds were selected for treatment based on a history of MIB off-flavor of three to six months duration as determined by sensory analysis of catfish at the processing plant (Delta Pride Inc., Indianola, MS.) Water and fish samples were collected as previously described (Martin et al. 1988) three days prior to treatment; immediately prior to treatment, and on day three, six and nine after treatment. Both ponds were treated with Pergenox-32G al a rate of 80 lbs/surface acre (average depth 4.5 ft) by chemical treatment boat. Water quality parameters were measured using the DREL-2000 water quality laboratory (Hach Chemical Company, Loveland, CO). Off-flavor compounds in the water were analyzed and identified by closed loop stripping and gas chromatography using flame ionization and mass selective detectors as previously described (Martin et al., 1988).

Four catfish at each sampling were caught by hook and line and subjected to sensory analysis by an industrial taste panel (Delta Pride Inc., Indianola, MS) and a portion of each sample was transported on ice to the laboratory for chemical analysis. The fish were analyzed for MIB and its dehydration products using microwave steam distillation coupled with gas chromatography using flame ionization and mass selective detection as previously described (Martin et al. 1988).

Microbiology

Edwardsa ictaluri was inoculated into one liter of brain heart infusion agar (BHI) (Difco, Detroit, Ml.), and incubated for six hours to achieve a final concentration range of 20 - 200 x 108 bacteria/ml.

Minimum Bacteriocidal Concentration (MBC)

Bacteria from the stock solution were then diluted 1:10 in duplicate with 85% physiological saline into which 0 (control), 0.25, 0.5, 1.0, 2.5, 10, 20, 50, 500, or 1000 mg/L of Pergenox-32G had been previously added. After a 30 min exposure time each reaction flask was diluted 1:10 several times and each dilution plated on BHI agar to quantitate bacterial numbers using the pour plate method (Standard Methods, 1985). The experiment was then repeated to determine the average % bacteria remaining for each exposure concentration. Bacteria were counted after incubation at 25°C for 72 hours.

Minimum Inhibitory Concentration (MIC)

Bacteria from the stock solution of Edwardsiella ictaluri in BHI broth were diluted 1:10 in duplicate by inoculation into BHI broth dosed with Pergenox-32G as per the MBC experiment except that the 500 and 1000 mg/L concentrations were omitted. After a six hour exposure period each reaction flask was diluted 1:10 several times and each dilution plated on BHI agar to quantitate bacteria using the pour plate method (Standard Methods, 1985). The experiment was then repeated as for the MBC determination. Bacteria were counted after incubation at 25°C for 72 hours.

Sterility Check

For each experiment the saline or broth used for dilution was inoculated into pour plates to ensure no bacterial contamination had occurred.

Confirmation of Edwarsiella Ictaluri

For each experiment both before and after inoculation and exposure, isolated colonies were identified as Edwardsiella ictaluri using the API strip (API 20E, Sherwood Medical Inc., Plainsview, NY); motility test on motility media S (Disco' Detroit, Ml) with TTC 1% solution added (Difco, Detroit, MI) Triple Sugar Iron (TSI) slant (Dipco, Detroit, Ml), oxidase test and inoculation simultaneously at 25 and 37°C on 10% sheep's blood agar. In all cases Edwardsiella ictaluri was the organism identified.

Figure 1. The percent of viable bacteria remaining after exposure of Edwardsiella ictaluri to various dosages of Pergenox-32G.

Results and Discussion

The two ponds selected for treatment consistently produced off-flavored fish (3-6 months duration) and the off-flavor was characterized by an industrial taste panel several times as being intense and characteristic of 2-methylisoborneol (MIB). The water and fish samples collected 3 days before and immediately prior to treatment were essentially identical. Following treatment, no MIB was detected in the water and the fish were acceptable for processing (on-flavor) by day nine post treatment. In both ponds a decrease in chlorophyll a was observed and the difference in percent decrease may be due to the selective toxicity of Pergenox-32G to Oscillatoria species implicated in MIB production (Kay et al. 1982). MIB dehydration products were detected in fish samples and have been implicated as causes of off-flavor (Martin et al., 1988). These are unsaturated hydrocarbons produced in the water at low, usually undetectable (2ng/L) concentrations but presumably are highly bioconcentrated in channel catfish. These preliminary results indicate that further studies with suitable controls are warranted.

The toxicity of Pergenox-32G to Edwardsiella ictaluri, the causative organism of enteric septicemia in catfish (ESC) was clearly shown in both 30 min and 6 hr exposure times. Essentially all the bacteria were kithekilled with the 20 mg/L dose for the 6 hr exposure time. An approximate 50% reduction in bacteria was observed for both exposure times at the 0.5 mg/L concentration of Pergenox-32G.

Table 1. Water quality parameters and concentration of MIB and its dehydration products in water and fish. Three days prior and nine days after treatment. MDP=MIB dehydration products ; MIB = 2 - methylisoborneol

The results indicate that the active ingredient in Pergenox-32G, hydrogen peroxide may have some efficacy in reducing bacterial numbers and possibly mortality in ESC-infected ponds. Hydrogen peroxide has been reported to be an effective sterilant in numerous applications presumably decomposing via hydroxyl radials that cause the bacterial toxicity. Previous ureter treatments have been largely unsuccessful because the chemicals significantly stress the fish while removing the pathogen. This is largely because the range between toxic and therapeutic doses is very narrow. However, the therapeutic dose of Pergenox-32G is approximately 5% of the LD50 '36 hr dose reported for channel catfish and so this stress may be significantly reduced (Kay et al, 1982).

Ultimately, the efficacy of Pergenox-32G for taste and odor control and disease management in channel catfish culture will be determined by field studies that incorporate suitable replicates and controls. Polyethylene cylinders (1.8M in height and 3.8 M in diameter) have been developed to be placed in off-flavor ponds or ponds experiencing mortality due to ESC so that suitable replicates can be obtained. These cylinders also allow "field" trials to be conducted without contaminating the total fish population with the experimental compound.

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