Abstract

Twelve channel catfish were sampled on two occasions from a commercial culture pond that contained fish chronically (>1-2 years) off-flavored and unmarketable. These fish were analyzed by a new method (microwave distillation) for 2-methylisoborneol (MIB) and geosmin. The fish were found to contain high concentrations (>100 mg/kg) of 2-methylisoborneol. The highest concentrations were isolated from skin tissue. The concentrations decreased in the order: skin (subepidermal fat) > abdominal fat > muscle liver. On-flavored fish dosed intravenously with MIB at a concentration of 1 mg/kg body weight showed a similar pattern of distribution into tissue components.

Introduction

The channel catfish industry is a rapidly growing enterprise that incorporates over 90,000 acres in commercial channel catfish aquaculture as of Feb. 1988. However, off-flavor is a severe problem limiting production and further market expansion. Off-flavor describes a problem in palatability in which channel catfish acquire a taste and odor that renders their flesh unmarketable. It is a problem that affects water supplies and fish populations worldwide.

Two off-flavor compounds have been repeatedly isolated in association with off-flavor episodes. These compounds are 2-methylisoborneol (MIB) and geosmin (1,2). These compounds are secondary metabolites of certain species of cyanobacteria and actinomycetes. They are tertiary alcohols that have threshold odor concentrations of 6 ug/kg (geosmin) and .07 ug/kg (MIB) in fish (3). They both impart a musty odor to fish flesh. other less common causes of off-flavor with as yet unknown chemical etiologies have been suggested based on taste-test analysis of distinctive odors such as sewage, rancid, metallic, etc. (4). Analytical isolation of some of these odorous compounds is occasionally successful as in the isolation of the herbicide molinate from off-flavor fish with a taste similar to this rice herbicide (Martin, unpublished results).

Off-flavor is a problem of multifactorial etiology associated with factors describing a eutrophic environment. It is more frequently associated with high feeding rates and acidic soils. The incidence of off-flavor is between 30 and 70% and the intensity and frequency of off-flavor is most severe in the summer months (Delta Pride Inc., Indianola, MS, pers. comm.). The correlation of off-flavor with blocms of cyanobacteria has been suggested. Recent studies indicate MIB is elaborated when algal cells become senescent and fragmentary (5).

It is believed that channel catfish acquire the off-flavor odor by absorption of these volatile compounds across the gill membrane. The absorption is rapid and an equilibrium is established for 2-methylisoborneol. Wen placed in water devoid of musty off-flavor compounds, fish become "on flavor" in 4-18 days (6). The metabolism of geosmin and methylisoborneol in fish is an area that has not been investigated.

Both geosmin and MIB have been isolated from off-flavor fish using low temperature, high vacuum distillation. The method requires 4-8 hours or preferably overnight for completion. In addition, a correlation of the geosmin off-flavor intensity with analytical concentrations determined by this method are inconsistent. We describe a new more rapid method for isolation of MIB, geosmin, and other volatile organic compounds from off-flavored fish.

In this study, microwave distillation of off-flavor fish flesh has been used to calculate the Concentration of MIB in channel catfish obtained from a commercial source as well as fish intravenously dosed with MIB. The method is repeatable and has the necessary sensitivity to detect geosmin, MIB and molinate at ug/kg concentration. In this study, the concentration of MIB in liver as well as skin (subepidermal fat), muscle and abdominal fat were determined by the microwave distillation method. A bioconcentration value for this compound in off-flavored fish was estimated.

Materials and Methods

Fish were caught by hook and line from a commercial catfish pond chronically affected with off-flavor. One of the fish was subjected to organoleptic analysis at the processing plant and the type of off-flavor described and categorized. The remaining fish were transported at 4°C, dissected and the tissues frozen until analysis.

Water samples were collected in I gallon amber bottles to which 40 mg HgCl2 had been added. The water sample was collected in duplicate and compared with an in-the--field MIB-spiked sample.

Alternately, 10 on-flavor channel catfish obtained from a local source were anesthetized in a I mg/ml. solution of methyl sulfonate hydrochloride and then dosed intravenously I mg/kg body weight with MIB. Tissue samples were collected from three fish each at 2, 24, and 96 hours after MIB administration. The tenth fish served as a control.

All fish were analyzed by microwave distillation. The tissues were thawed, minced, and added to a 250 ml round bottom flask. Hexane, at the rate of 10% by weight of the sample, was added to facilitate a more complete and smooth distillation. The distillation was purged with N2 and was continued until no further extract was collected. The condensate, collected at -80C was thawed, and the hexane fraction concentrated to 100 ul prior to analysis. A diagram of the apparatus is shown in Figure 1.

Water from the pond producing off-flavored fish and in-the-field MIB-spiked blanks were subjected to closed loop stripping in duplicate using the method developed by Grob as modified by Hwang et al., 1984.

Figure 1. Diagram for microwave distillation of contaminated fish flesh.

The carbon disulfide water extract and hexane fish extract were analyzed by gas chromatography under the following conditions; column: DB-5, (J & W Scientific) 30 M length, 0.32 u = Internal diameter; initial temperature 40C 6 initial hold 1 minute, ramp rate 10C /minute, final temperature 275C , injector temperature 275C , flame ionization detector 325C , split ratio 3:1. The microwave distillation method was evaluated for reproducibility of analysis. A linear response regression coefficient .998 was obtained for geosmin and methylisoborneol for .5-200 ng and molinate from 0.1-10 ng.

The rapidity (1-1.5 hours) and reproducibility of the microwave analysis (1) gives it a considerable advantage over more time-consuming methods such as soxhlet and vacuum distillation.

Results and Discussion

MIB was found to concentrate differentially in various tissue compartments of the off-flavored channel catfish obtained from a commercial pond. The highest concentrations were found in the skin (subepidermal fat). Lesser concentrations were found in abdominal fat. The lowest concentrations were found in the liver and muscle tissue (Table 1).

Appreciable fish-to-fish variability existed at each sampling period in the intravenously dosed catfish and considerable overlap occurred between tissue sampling times (Table 2). MIB was cleared from the plasma to less than 40 ug/ml in 2 to 6 hours. The compound distributed rapidly into muscle, subepidermal fat, kidney and mom slowly for abdominal fat. The Table 1.Concentrations of MIB in various tissue compartments of 12 off-flavored fish collected two weeks apart from commercial ponds.

*Means in the same column followed by the same letter are not significantly different at the 5% level by the Least Significant Difference Test.

Table 1.Concentrations of MIB in various tissue compartments of 12 off-flavored fish collected two weeks apart from commercial ponds.

Concentrations then decreased in all components other than abdominal fat which showed a maximum MIB concentration at the 24 hour sampling period (Table 2). The concentration of MIB in liver, kidney, and muscle was less than 10% of that in fatty tissue. This compares well with the results from off-flavored fish from the commercial pond indicating MIB is preferentially concentrated in abdominal and subepidermal fat.

Table 2. The concentration ng/g of MIB in each tissue compartment at each sampling time for intravenously dosed fish.

Microwave distillation has been shown to be a useful method for isolation of MIB, geosmin and molinate from off-flavored fish. In addition the method has enabled us to determine in what tissue compartment MIB is preferentially concentrated so that a sensitive analytical analysis can be accomplished. This study suggests that the subepidermal layers are the primary organ of concentration for the musty odor compound MIB and that objective flavor analysis of these fish should preferentially involve this tissue. Microwave distillation may also have broader application for volatile contaminants as has been demonstrated for molinate.

References

1.  Martin, J.F., C.P. McCoy, W.G. Greenleaf, and L.W. Bennett (1987). Analysis of 2-Methylisoborneol in Water, Mud, and Channel Catfish (Ictalurus punctatus) from Commercial Culture Ponds in Mississippi. Can. J. Fish. Aquat. Sci., 44, 909-912.

2.  R.T. (1986). Geosmin and Musty-muddy Flavors in Pond-raised Channel Catfish Ictalurus, punctatus. Trans. Am. Fish. Soc., 115, 485-489.

3.  Persson, P.E. (1980). Sensory Properties and Analysis of Two Muddy Odor Compounds, Geosmin and 2-Methylisobomeol, in Water and Fish. Water Res., 14, 1113-1118.

4.  Johnson, P.B., G.V. Civille, and J.R. Vercellotti (1987). A Lexicon of Pond-raised Catfish Flavor Descriptions. J. Sensory Studies, 2, 87-91.

5.  Martin, J.F., C.P. McCoy, C.S. Tucker, and L. Bennett (1987b). 2-Methylisoborneol Implicated as a Cause of Off-flavor in Channel Catfish Ictalurus punctatus (Rafinesque) from Commercial Culture Ponds in Mississippi. Aquacult. Fish. Mgmt. In press.

6.  Persson, P.E. (1984). Uptake and Release of Environmentally Occurring Odorous Compounds by Fish. Water Res., 18, 1263-1271.

7.  Hwang, C.J., S.W. Krasner, M.J. McGuire, M.S. Moylan, and M.S. Dale (1984). Determination of subnanogram per liter levels of earthy-musty odorants in water by the salted closed loop stripping method. Environ. Sci. Technol., 18, 535-539.