Bioaccumulation of Methyl Mercury by the Freshwater Mussel, Elliptio buckleyi

Kernaghan, N.J.^1,2*, Ruessler, D.S.¹, Miles, C.J.³, Gross, T.S.<sup>1,2

 1</sup>USGS - Florida Integrated Science Center, Gainesville, Florida

 ²College of Veterinary Medicine, Department of Physiological Sciences
University of Florida, Gainesville, Florida

³College of Agriculture, Department of Food Science & Human Nutrition
University of Florida, Gainesville, Florida

 

Abstract

Considerable evidence has indicated that a number of chemical contaminants present in the environment can negatively impact exposed wildlife populations.  Mercury pollution may be one of the most important environmental problems in the aquatic ecosystems of the southeastern United States.  Recent data from our laboratory has suggested endocrine disrupting effects of methyl mercury in fish.  Indeed, much of the work on methyl mercury accumulation and effects have focused on fish as vectors of exposure; however, freshwater mussels might be a more appropriate early indicator of environmental mercury exposure and effects. The current study examined freshwater mussels as potential vectors of acute methyl mercury exposure and bioaccumulation. Elliptio buckleyi were exposed to methyl mercury in the water column using a proportional diluter system and to methyl mercury via diet in a recirculating system. Water column exposures were 5.0 and 10 ng/L, whereas, dietary exposures utilized algae (Selenastrum capricornutum) which had been experimentally exposed at 1.0 and 10 ng/L. Mussels exposed in the water column were analyzed for methyl mercury at days 7, 21 and 32, while mussels with dietary exposure were analyzed at days 30, 60, and 90.  Results indicate that E. buckleyi did not significantly bioaccumulate methyl mercury following water column exposure; however, dietary exposure to algae treated at 10 ng/L resulted in significant bioaccumulation. Little or no mortality was observed regardless of exposure dose or source. An analysis of mantle sex steroid concentrations indicated a significant increase in testosterone concentrations following dietary exposures. These data indicate dietary sources for mercury exposure of freshwater mussels and suggest their potential utility as an ecological receptor to assess both exposure and effects.

Introduction

Mercury, a highly toxic heavy metal, is of special concern in the south Florida ecosystem. Mercury from urban and industrial pollution enters ecosystems primarily from atmospheric deposition. Average mercury emissions for south Florida are 3 times higher than the state average. Mercury at high levels can be toxic to organisms. However, lower, non-toxic levels of mercury have been known to cause reproductive rates to decline in wildlife.  Mercury levels measured in some areas of Florida are among the highest ever recorded in North America.  U.S. Food and Drug Administration has imposed a 1 part per million limit on tissues for safe consumption. Largemouth bass in some areas of the Everglades, including the Everglades National Park, and Water Conservation Areas II and III, contain mercury levels above 1.5 ppm.  Inorganic mercury must be converted to methyl mercury by microorganisms before it can enter the food chain, where it bioaccumulates in tissues and organs in various animal species. Methylation of mercury occurs primarily by periphyton which occur mostly as algal mats. When eutrophication occurs, algal colonies bloom resulting in increased levels of methyl mercury. It is generally accepted that freshwater mussels require algae as a primary food source.  This suggests that freshwater mussels have a high potential for exposure and bioaccumulation of methyl mercury, which could result in toxicity or reduced reproductive function. This study focuses on determining the potential of mussels to bioaccumulate methyl mercury from aqueous as well as dietary exposures. In addition, these effects provide the first preliminary assessment of potential reproductive effects in freshwater mussels.

Objective

Assess the feasibility of freshwater mussels to serve as indicators of methyl mercury bioaccumulation and potential reproductive effects.

Materials and Methods

Adult Elliptio buckleyi were collected from an experimental pond located at the Fisheries Department at the University of Florida in Gainesville, Florida. All mussels collected ranged in size from 32 to 56 mm in length, with an average length of 44.7 mm. All experiments were conducted under 16:8 Light Dark Cycle. Although the sex of the mussels was not determined prior to test initiation, previous research has indicated that this species is sexually dimorphic.

Experiment I
MeHg Water Exposure

Mussels were exposed to methyl mercury using a proportional diluter system, test concentrations were 0, 5, and 10 ng/L MeHg with two replicates per concentration. Dilution water was well water (hardness = 260 mg/L). Mussels were not fed during this experiment. At 7, 21, and 32 days, mussels were removed from each tank and analyzed for tissue concentrations of MeHg.

Experiment II
MeHg Diet Exposure

Mussels were exposed to methyl mercury via algae that contained concentrations of  0, 1, and 10 ng/L MeHg.  Dilution water was of moderate hardness (hardness = 80mg/L).  Selenastrum capricornutum was cultured and fed to mussels at a concentration of 3 x 10⁷ cells/mL.  Algae was inoculated with methyl mercury just prior to test renewal and allowed to bind to cell walls before being introduced into test chambers. At 30, 60 and 90 days ten mussels were removed from each tank. All mussels were dissected and a wet weight was taken on the shell and soft tissues. A body condition index (total wet weight as a percent of total wet weight) and a soft tissue index (soft tissue wet weight as a percent of total wet weight) were calculated for each mussel. Five mussels from each replicate were analyzed for tissue concentrations of MeHg. The remaining five mussels were dissected, mantle wet weights were recorded and mantle tissues were analyzed for glycogen and sex steriod concentrations. Glycogen concentrations were determined using a microtized version of Naimo et. al., 1998.

Samples were analyzed for the following sex steroids: estradiol and testosterone, using standard radioimmunoassay (RIA) procedures. Tissues were diluted with 3x volume of 30% KOH and incubated for 20 minutes in a 60°C water bath to facilitate tissue digestion. Tissue digests (150µl) were extracted twice with 4 ml diethyl ether prior to RIA analysis. Each sample was analyzed in duplicate for each of the sex steroids.  Standard curves were prepared in buffer with known amounts of radioinert sex steroid (1, 5, 10, 25, 50, 100, 250, 500 and 1000 pg). Cross-reactivities of the estradiol-17ß antiserum with other steroids were: 11.2 % for estrone; 1.7% for estriol; <1.0 % for estradiol-17alpha and androstenedione; and <0.1% for all other steroids examined. Cross-reactivities of the testosterone antiserum with other steroids were: 18.5% for dihydrotestosterone; 3% for androstenediol; <1.0% for androstenedione, androstanedion, estradiol and progesterone; and <0.01% for all other steroids examined. Results were calculated as concentrations: pg/g tissue. A potential sex specific index: estradiol:testosterone ratio was also calculated as an index of sex specific sex steroid pattern.

A cross section of the visceral mass was collected from each mussel and fixed in Notox®. Samples were paraffin embedded, sectioned (6 µm) and stained with hematoxylin and eosin. Slides were evaluated for gonadal tissues to identify sex and reproductive stage.

Results

Mussels in Experiment I, did not show any significant accumulation of methyl mercury in any treatment as compared to controls regardless of treatment duration. Mussels in Experiment II showed significant accumulation of methyl mercury in the 10 ng/L treatment at day 30 as well as day 90 compared to the control levels of methyl mercury.

There were no significant changes in body condition index or soft tissue index. Glycogen concentrations were significantly increased in all three treatments at day 90.

The only significant change in sex steroids over time was an increase in testosterone in males at 1ng/L. However, when compared to the controls at each separate time point, there were several significant differences observed. Estradiol was significantly reduced in females at 10 ng/L on days 30 and 90. Testosterone was significantly increased in males at 1 and 10 ng/L on day 90 and significantly reduced in females at 10ng/L. Estradiol/Testosterone ratios were significantly reduced in females in 1 ng/L and 10 ng/L, but no significant differences were obeserved for E/T ratios for males.

Conclusions

Concentrations of methyl mercury following dietary exposure was similar to levels observed in mussels collected in South Florida (Ruessler et. al., SETAC Conference, 1999) and indicates that freshwater mussels may, in fact bioaccumulate methyl mercury in their tissues.

The only physiological/health parameter to show a significant response to exposure to methyl mercury was glycogen. The observed increase in glycogen could be considered a response of the mussels to stress, in an attempt to store additional energy reserves. This response could also be due to the increased metabolic activity of the mussels to remove the increased load of methyl mercury in their tissues.

Analysis of sex steroids in this preliminary study indicate a general reduction in tissue estrogen for females exposed to the high mercury dose. In addition, testosterone is decreased in female mussels when exposed to the high dose for 90 days or greater. There alterations in endocrine parameters for female mussels also result in a decreased estrogen/testosterone ratio in response to the high mercury dose. In contrast, for male mussels, the only observed effect was a general increase in testosterone at longer exposures. These effects are similar to those reported for natural exposures in S. Florida (Ruessler et. al., 1999) which further validates the use of freshwater mussels as eco-relevant indicators of methyl mercury effects and exposure.

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