High concentrations of methyl-mercury (CH₃Hg⁺), a substance toxic to both animals and humans, recently have been measured in a number of top predators (including panthers and game fish) native to the Florida Everglades.

As part of a larger research group investigating the mercury (Hg) cycle in south Florida, our work has focused on the microbiological and geochemical processes that control CH₃Hg⁺ degradation in Everglades peat-sediment. Field measurements of CH₃Hg⁺ degradation rates are currently being provided to ecosystem managers. From this and other field data, predictive Hg cycle models are being constructed to aid in making informed decisions regarding hydrologic and nutrient management strategies that may influence the Everglades Hg cycle.

PROJECT GOAL

The objective of this research is to provide ecosystem managers with CH₃Hg⁺ degradation rate data from a number of study sites that represent a diversity of hydrologic and nutrient regimes common to the Everglades. Further, our focus is on better understanding the microbial and geochemical controls regulating CH₃Hg⁺ degradation. At present, little is known regarding the specific factors influencing this process in natural systems.

BACKGROUND

The methylated form of Hg (i.e. CH₃Hg⁺) is the most readily bioaccumulated Hg species in the food chain as a result of its affinity for protein groups. The primary source of Hg to the Everglades is from particle bound atmospheric deposition. Particle bound inorganic Hg⁺² is transported to the sediment where it is methylated by sulfate reducing bacteria (SRB). In addition to methylating Hg, sediment bacteria can also demethylate CH₃Hg⁺, although much less is known about this reverse reaction. Both SRB and methane producing bacteria (MPB) are thought to be involved in this process. The sum of both the methylation and demethylation reactions determines if a particular location is a net source or sink of CH₃Hg⁺.

The microbial degradation of CH₃Hg⁺ may proceed by two known pathways, namely, methyl-cleavage (MC) (producing Hg⁺² and CH₄) or oxidative demethylation (OD) (producing Hg⁺² , CH₄ and CO₂). By measuring the carbon based end-products (i.e. CH₄ and CO₂), the fraction of CH₃Hg⁺ degradation attributable to each of these pathways may be assessed at a particular location and/or depth .

Bacteria that degrade CH₃Hg⁺ via MC may also posses the capacity to further reduce Hg⁺² to volatile Hg⁰. However, it is unknown if this reduction is associated with conditions favoring OD. Since the formation of volatile Hg⁰ potentially represents a permanent loss of Hg from the system, a clear understanding of the processes controlling both MC and OD is needed.

The Everglades Water Conservation Areas are large sections of wetlands currently being used as nutrient removal zones for water draining the Everglades Agricultural Area. To the extent that nutrients affect the complex microbial dynamics in water and sediments, their direct and indirect impact on bacteria involved in the Hg cycle is of primary interest.

RESEARCH PLAN

Our work is divided into both a field and laboratory component. One year of research has been completed. The specific areas of current and future investigations are outlined below.

1. Field Measurements
   
   At a number of select sampling sites, sediment cores are sectioned into three to five discrete 2 cm horizons, within hours of sample collection. Sub-samples from each horizon are transferred into crimp sealed serum vials, purged with N₂ gas, and injected with radiolabeled ¹⁴CH₃Hg⁺. After incubating for a number of days, end-products (¹⁴CH₄ and ¹⁴CO₂ ) are measured via gas proportional counting. The relative amounts of these compounds are a direct indication of ¹⁴CH₃Hg⁺ degradation via MC or OD, respectively. Degradation rates are assessed with respect to both sediment depth and site location.

   While high concentrations of ¹⁴CH₃Hg⁺ have been measured in some Everglades animal species, the concentrations in the sediment are extremely low (> 0.01 ng * cc wet sediment). Therefore, the amount of ¹⁴CH₃Hg⁺ needed to produce detectable end-products is well above these natural background levels. Hence, measurements to date represent upper estimates of potential rates. An alternative method for extrapolating to in-situ rates is currently being explored (see below).
   
   

2. Nutrients and Microbial Inhibitors
   
   The affect of nutrients (NO₃^-, PO₄^-3, NH₄⁺) and SO₄^-2 on the degradation of ¹⁴CH₃Hg⁺ is assessed by amending parallel sets of incubation samples with these substrates and processing as described above. Likewise, specific microbial inhibitors of both SRB and MPB are used to determine the relative contribution of these microbial groups to ¹⁴CH₃Hg⁺ degradation.
   
   
3. Bacterial Isolates
   
   In a series of laboratory experiments we will attempt to isolate specific bacteria (SRB and MPB), from Everglades sediment, capable of degrading ¹⁴CH₃Hg⁺. We will concentrate on isolating strains that degrade ¹⁴CH₃Hg⁺ by either MC or OD exclusively. We will then be able to explore the bacterial mechanisms by which degradation proceeds under each pathway.
   
   
4. The Fate of Hg
   
   In additional laboratory experiments the fate of Hg after ¹⁴CH₃Hg⁺ degradation will be explored. Vapor phase Hg⁰ is collected on gold traps by flushing the head-space of previously incubated samples. The concentration of Hg⁰ is then assayed by cold vapor atomic fluorescence spectroscopy. Preliminary work in this area using whole sediment was recently completed, while work with bacterial isolates is pending.
   
   
5. Kinetic Studies

   As previously indicated, experiments with radiolabeled ¹⁴CH₃Hg⁺ at or below natural concentrations (i.e. tracer experiments) are not currently feasible. However, an alternative approach for the assessment of in-situ rates is currently being explored. This approach entails measuring the degradation rate over a large range of ¹⁴CH₃Hg⁺ concentrations, and extrapolating results to predict in-situ degradation rates. Preliminary laboratory experiments with this method suggest that the rate of CH₃Hg⁺ degradation is a linear function of concentrations over two orders of magnitude (see graph). With an order for custom-made high specific activity ¹⁴CH₃Hg⁺ pending, we expect to repeat these types of measurements at all sites and decrease our lowest addition by another order of magnitude. Thus, this approach appears promising and will be used during upcoming field work.
   

Kinetic Experiment:14CH3Hg+ degradation rate versus 14CH3Hg+ spike concentration plotted on log-log axis. X-axis is extended to the range typical for in-situ 14CH3Hg+ concentrations. Dashed line represents linear least-squares fit. Resulting r2 = 0.988 [larger image]

Product Plans:

• yearly reports summarizing field and laboratory results
• submission of field rate data
• publication of findings in a relevent peer reviewed journal