AN EVALUATION OF METHYL MERCURY AS AN ENDOCRINE DISRUPTOR IN LARGEMOUTH BASS

Fynn-Aikins, K ³, Gallagher, E.², Ruessler, S.¹, Wiebe, J.^1,2 and Gross, T.S.^1,2

²USGS - Florida Integrated Science Center, Gainesville, FL
²Department of Physiological Sciences, University of Florida, Gainesville, FL
 ³USFWS, Vero Beach, FL

Abstract

Previous studies have primarily focused on the fate and transport of mercury in aquatic species, however, sublethal effects have not been routinely documented. Methyl mercury has been routinely listed as a potential endocrine disruptor in wildlife with little or no supporting data. Adult largemouth bass (n-240) were randomly distributed into four treatments: 0, 1.56, 3.12 and 6.25 mg methyl mercury chloride per kilogram of diet (30 male and 30 female per treatment). Prior to dosing, 10 fish (5 male and 5 female) were collected from each treatment tank for collection of blood and tissue samples to determine baseline levels of mercury. Additional fish (n=10, 5 male and 5 female) were collected from each treatment bi-weekly. Blood samples were utilized for the analysis of sex steroid hormones (estradiol and 11-keto-testosterone) and vitellogenin. Gonadal tissues were collected for histological evaluation of sex and reproductive status. Liver tissues were collected for glutathione transferase and mRNA analyses. Tissues were collected from each fish for methyl mercury analyses. Plasma estradiol and 11-ketotestosterone concentrations were not altered by the low dose (1.56 mg methyl mercury chloride/kg diet) treatment regardless of sex. However, plasma 11-ketotestosterone concentrations were decreased in male largemouth bass exposed to the 3.12 and 6.25 mg methyl mercury diets. Similarly, plasma estradiol concentrations in female bass were decreased following exposure to the 3.12 and 6.25 mg methyl mercury diets. Certain GST catalytic activities were also inhibited by high dose methyl mercury exposure. These results indicate that dietary exposure to methyl mercury affects hormone concentrations in largemouth bass, and that methyl mercury can act as a endocrine disruptor and potentially impair reproduction in fish.

Introduction

Considerable evidence has accumulated over the past several years that a number of compounds present in the environment can disrupt reproductive endocrine function in exposed animal populations.  Natural and synthetic estrogens (17ß-estradiol, estrone, and ethinyl estradiol) have been identified in sewage effluent (Desbrow, et al., 1996), chicken manure (Shore and Hall, 1997) and have been used in aquaculture to alter the sex of entire populations of fish (Arcand-Hoy and Benson, 1998). DDE and its metabolites have been associated with gonadal abnormalities and skewed sex hormone concentrations in Lake Apopka alligators (Heinz et al. 1991, Guillette et al. 1994).  Insecticides such as toxaphene, chlordane, and methoxychlor, all known endocrine disruptors (Keith 1997) are found in agricultural runoff. These substances have the potential to affect all life stages of fish producing aberrant sexual differentiation in larval stages and affecting gonadal and gamete development in adults.

Another environmental problem that has emerged as a global concern is contamination of aquatic systems by mercury.  While such contamination may sometimes be due to point source discharges, many contaminated locations lack such obvious causes. Atmospheric transport and deposition of anthropogenic mercury is now considered to be an important source of mercury to many systems (Mason et al. 1994; Fitzgerald et al. 1998). Much atmospheric mercury occurs in inorganic forms, while methyl mercury is the form that tends to accumulate in biota (Bloom 1992; Mason et al. 1995). Therefore, conditions that enhance mercury methylation and transport are often associated with high mercury burdens in biota (Driscoll et al. 1994; St. Louis et al. 1994; Rudd 1995; Gilmour et al. 1998).

Facemire et al. (1995) have argued that mercury pollution of waters may be the most important environmental problem in the southeastern United States. An area of particular concern is the Everglades of Florida, which has well-documented mercury problems (Ware et al. 1990; Sundlof et al. 1994; Stober et al. 1995).  Recent work in this area strongly suggests major roles for atmospheric transport and enhanced methylation in the mercury contamination of this system (Rood et al. 1995; Cleckner et al. 1998; Dvonch et al. 1998; Gilmour et al. 1998; Guentzel et al. 1998).

Much of the work on mercury in fish has focused on fish as vectors of contaminant exposure to wildlife and humans. However, there are relatively few studies of effects of mercury on the fish themselves at environmentally realistic levels.  Many older studies involved exposure of fish to dissolved mercury in water and at concentrations far exceeding those that might encounter in nature. This exposure scenario is unrealistic because fish accumulate most methyl mercury via the diet (Weiner and Spry 1996). Most recently, mercury has been proposed as a potential endocrine disruptor (Colburn and Clement 1992).  While reproductive toxicity effects have been demonstrated for mercury at high environmental concentrations, few research efforts have attempted to examine mercury as a potential endocrine disruptor in wildlife.  The current study was conducted to examine the potential endocrine disrupting effects of mercury in largemouth bass at eco-relevant exposures and doses.

Objectives

• To determine if largemouth bass would develop a significant, dose dependent, body burden of mercury by the ingestion of a methylmercury laden diet.
• To determine potential endocrine disrupting effects of methyl mercury in largemouth bass at eco-relevant exposures and doses.

Materials and Methods

Largemouth bass were exposed to methyl mercury through ingestion.  Commercially available fish diets were purchased, the pellets disintegrated, laced with methyl mercury to desired concentrations, then reshaped into pellets for consumption by the fish.  Fish diets were manufactured at the Harbor Branch Oceanic Institute. Treatments consisted of three concentrations of methyl mercury and a control.  Nominal methyl mercury concentrations, or test concentrations, were as follows: 0, 1.56, 3.12, and 6.25 mg/kg (ppm).

The experiment was conducted in four 400 gallon fiberglass, circular tanks, measuring 6 feet in diameter. Well water was continually flowing at equal rates throughout all tanks.  Well water temperature was 22 ± 2 ^oC. Effluent/outflow from all tanks was collected and pumped under 75 psi of pressure through a series of two particle filters with pore sizes of 300 and 60 mm respectively. Water then was forced through a series of four activated carbon beds, each having a volume of 0.5 ft³ to remove any methyl mercury not held by the fish or in the particle filters (See Photo and diagram below). Water samples were also taken from the cleanup system discharge and analyzed for methyl mercury not retained in the carbon beds.

Sixty fish (30 male, 30 female) were placed in each tank and fed three times a week with floating methyl mercury laden food until feeding activity ceased.  Fish (n=20) were sampled/sacrificed biweekly for 8 weeks. Blood and tissues samples were collected from each fish. Blood was analyzed for estradiol and

11-ketotestosterone, using standard radioimmunoassay procedures. Plasma vitellogenin concentrations were determined by Dr. Nancy Denslow at the University of Florida. Fish length and weight were also recorded at time of sampling.

Fish fillet samples were taken, wrapped in foil, and placed on ice and analyzed by Florida DEP for mercury concentrations.  Water samples from each tank were also taken for mercury analysis to document any mercury loss into the water column.

Results

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Gonadal Histopathology

For each fish, samples of gonadal tissues were fixed in Notox, embedded in paraffin and sectioned to 5mm for histological analyses. Ovaries were classified into four stages of sexual maturation: undeveloped; previtellogenic; early vitellogenic; and late vitellogenic.  Testes were classified into three stages of sexual maturation: low spermatogenic activity; moderate spermatogenic activity; and high spermatogenic activity.

For male largemouth bass in this study, gonadal histology indicated high spermatogenic activity and no effects of methyl mercury treatments.  All males were at a similar stage of reproductive development.

For female largemouth bass in this study, gonadal histology indicated previtellogenic ovarian activity and no effects of methyl mercury treatments.  All females were at a similar stage of reproductive development.

Discussion

This study demonstrated that fish in all methyl mercury treatments did develop tissue body burdens in as little as two weeks after test initiation. Fillet tissue mercury concentrations continued to rise throughout the entire study.  The medium and high treatment fish developed body burdens of 250-300 ppb after eight weeks of exposure. Twenty-five percent of the mosquitofish captured in south Florida marshes have a body burden that equals or exceeds these concentrations which indicates that we have developed environmentally relevant concentrations in this study (EPA Report 904-R-96-008).

For male largemouth bass, 3.12 and 6.25 ppm methyl mercury treatments decreased plasma 11-ketotestosterone concentrations after four weeks with little or no change in plasma vitellogenin and  estradiol concentrations.  11-ketotestosterone is the primary hormone for spermatogenesis in largemouth bass, however, we did not observe any significant decrease in spermatogenic activity, as determined by gonadal histopathology, for male bass regardless of treatment.

For female largemouth bass the medium and high methyl mercury treatments decreased plasma estradiol and vitellogenin concentrations while not affecting plasma 11-ketotestosterone. Estradiol and the resulting production of vitellogenin by the liver, are critical to egg production and maturation in fish.  However, we did not observe any significant changes in gonadal histopathology for female largemouth bass in this study regardless of treatment.

Overall, these results indicate the potential for methyl mercury to disrupt the endocrine system and alter reproduction in ways that could effectively decrease egg or sperm production, egg size, or general gonadal development. Indeed, this experiment demonstrates endocrine modulation in largemouth bass exposed to methyl mercury body burdens of greater than 200 ppb. Future efforts should involve the treatment of largemouth bass during later stages of seasonal reproduction and include measures of reproductive success (i.e. fecundity, egg hatchability and fertility etc.).