1. Environmental Controls on Methylmercury Production and Degradation in Everglades Sediments Environmental Controls on Methylmercury Production and Degradation in Everglades Sediments M. Marvin-DiPasquale, U.S. Geological Survey, Menlo Park, CA 94025 INTRODUCTION HYPOTHESIS I. II. III. OBJECTIVES I. II. III. INTRODUCTION The net production of toxic methylmercury (MeHg) by sediment bacteria is arguably the most critical step in the chain of events leading to mercury (Hg) contamination of Everglades wildlife. Our understanding of the microbial processes involved (e.g. Hg-methylation and MeHg degradation) has progressed over the last decade, although, much is still unknown. In a series of controlled laboratory experiments, this study attempts to directly quantify the effect of a suite of environmental parameters thought to be important in controlling microbial Hg-transformation rates. These parameters include: temperature, sulfur chemistry, organic matter, and oxidation/reduction (redox) conditions. The quantified response coefficients generated in this project may be used to further refine the Everglades Hg-model. HYPOTHESIS I. Key factors that impact net MeHg-production in Everglades sediment include: temperature, sulfur chemistry, organic matter content, and redox conditions. II. The opposing microbial processes of MeHg- production and degradation are impacted differently by the above factors. III. These differences lead to the spatial and temporal trends observed in net MeHg-production. OBJECTIVES I. Quantitatively investigate the influences of temperature, sulfur-chemistry, redox, and organic matter on microbial mercury cycling in Everglades sediments. II. Determine to what extent each environmental variable impacts the opposing processes of MeHg- production and degradation. III. Determine the relative influence of the above parameters among-sites along the eutrophication gradient. CONCLUSIONS I. net gross II. III. IV. V. VI. CONCLUSIONS I. MeHg-production was much more affected by changes in environmental variables than was MeHg- degradation. Thus, variations in net MeHg production are driven mainly by changes in gross MeHg production. II. MeHg-production increased as a function of temperature, and generally decreased as a function of reduced-S (solid phase FeS and porewater sulfide), sulfate and oxidizing conditions. III. The impact of these parameters on MeHg- production generally increased from north to south. Thus, MeHg-production at pristine Everglades sites are most susceptible to variations in environmental conditions. IV. Solid phase reduced-S (e.g. FeS) appeared to be more important than pore-water free sulfide (H 2 S) in inhibiting MeHg-production. V. MeHg-producing microbes did not generally appear carbon limited in this organic rich environment. Similar experiments with humic acids are pending. VI. The activation energies and parameter response values calculated as part of this study can be used to further refine the existing Everglades Hg-model. Oxygen (Air) Amendment In a third experiment, anoxic sediment slurries (3g sed per 1 ml anoxic DI water) were sequentially bubbled with air (O 2 ) while monitoring sediment redox (Eh). Sub-samples were taken at two oxidation levels above, and in addition to, the original anoxic conditions. Gas phase conditions were N 2 for the initial anoxic samples and room air (O 2 ) for the more oxidized samples. MeHg production and degradation samples were amended with 1.2  Ci 203 HgCl 2 (695 ng Hg*g wet sed -1 ) and 9 nCi 14 CH 3 HgI (11 ng Hg*g wet sed -1 ), respectively. Incubations were conducted at 20 0 C for 18-19 hours. MeHg production rates decreased with increasing oxidized sediment conditions, while MeHg degradation rates did not vary as a function of sediment redox. Further, the more pristine site 3A- 15 had a stronger response to changing sediment redox than did the eutrophied ENR site. Slope: [rate/E h ] -0.014 -0.012 -0.010 -0.008 -0.006 -0.004 -0.002 0 ENR-401 3A-15 * MeHg Production MeHg Degradation (not significant at P < 0.05). ** 0 1 2 3 4 5 0 102030 Potential Rates (ng*g wet sed -1 *d -1 ) 0 0.5 1.0 1.5 0 102030 F1U33A-15 MeHg-Production MeHg-Degradation ENR 401ENR 303 Temperature ( 0 C) -100 -80 -60 -40 -20 0 20 40 60 ENR 401 ENR 303 F1U3 3A-15 MeHg-Production % Change Relative to Anoxic Control -100 -80 -60 -40 -20 0 20 40 60 ENR 401 ENR 303 F1 U3 3A-15 MeHg-Degradation Air (Oxygen) Amendment Air (Oxygen) Amendment: 1 ml oxic water, air gas phase ENR 401 ENR 303 F1 U3 3A-15 -80 -60 -40 -20 0 20 40 60 -100 MeHg-Production % Change Relative to Anoxic Control -100 -80 -60 -40 -20 0 20 40 60 MeHg-Degradation ENR 401 ENR 303 F1U33A-15 Iron-Sulfide (FeS) Amendment Iron-Sulfide (FeS) Amendment: 10.6  mol S 2- *g slurry -1 1211581872 1211581872 -80 -60 -40 -20 0 20 40 60 -100 MeHg-Production % Change Relative to Anoxic Control -100 -80 -60 -40 -20 0 20 40 60 MeHg-Degradation ENR 401 ENR 303 F1 U3 3A-15 ENR 401 ENR 303 F1 U3 3A-15 Sulfide (H 2 S) Amendment Sulfide (H 2 S) Amendment: 1.4  mol S 2- *g slurry -1 1724 2582 2565 470 4045 1724 2582 2565 470 4045 -80 -60 -40 -20 0 20 40 60 -100 MeHg-Production % Change Relative to Anoxic Control -100 -80 -60 -40 -20 0 20 40 60 MeHg-Degradation ENR 401 ENR 303 F1U3 3A-15 ENR 401 ENR 303 F1 U33A-15 Sulfate (SO 4 2- ) Amendment Sulfate (SO 4 2- ) Amendment: 2.0  mol*g slurry -1 51 33 36 217 68 51 33 36 217 68 -80 -60 -40 -20 0 20 40 60 -100 MeHg-Production % Change Relative to Anoxic Control -100 -80 -60 -40 -20 0 20 40 60 MeHg-Degradation ENR 401 ENR 303 F1 U33A-15 ENR 401 ENR 303 F1 U3 3A-15 Acetate Amendment Acetate Amendment: 1.0  mol*g slurry -1 53834782125383478212 0 20 40 60 80 100 ENR 401 ENR 303 F1U33A-15 E a (kJ*mol -1 ) MeHg-ProductionMeHg-Degradation METHODS I. Sediment Collection II. Experimental (General) METHODS I. Sediment Collection  Surface sediment (0-4 cm) was collected (May 8-9,2000) into acid cleaned mason jars (5 quarts/site) and stored at 5 o C until further use.  The north-south sampling transect along the existing eutrophication gradient included 2 sites in the experimental Everglades Nutrient Removal (ENR) area, 2 sites in the transitional WCA-2A, and 1 site in the comparatively pristine WCA-3A (see Figure 1). II. Experimental (General)  All sample processing (except redox experiment) was conducted in an N 2 flushed glove bag to preserve anoxic conditions.  Sediment was homogenized for a single composite sample per site.  All samples contained 3 g sediment plus 1 ml aqueous amendment (treatment specific) in a 13 cm 3 serum bottle.  All treatments consisted of 3 live samples and 1 killed control.  MeHg-Production measured via the 203 Hg(II) methylation (CH 3 203 Hg + production) technique [Gilmour 1995], modified for bottle incubations.  MeHg-Degradation measured via the 14 CH 3 Hg + amendment ( 14 CH 4 & 14 CO 2 production) technique [Marvin-DiPasquale 1998].  Parallel sets of MeHg-production and degradation samples are amended and incubated (18-21 hours) simultaneously.  IMPORTANT: All MeHg production and degradation rates were calculated as potential rates (i.e. based on the amount of radiolabel added), and not as in-situ rates.  Additional experimental specifics given with results. REFERENCES REFERENCES Gilmour CC, Riedel GS (1995) Measurement of Hg methylation in sediments using high specific activity 203Hg and ambient incubation. Water Air Soil Pollut. 80:747-756 Gilmour CC, Riedel GS,Ederington MC, Bell JT, Benoit JM, Gill GA, Stordal MC (1998) Methylmercury concentrations and production rates across a trophic gradient in the northern Everglades. Biogeochemistry 40:327-345 Marvin-DiPasquale MC, Oremland RS (1998) Bacterial methylmercury degradation in Florida Everglades peat sediment. Environ. Sci. Tech. 32:2556-2563 Figure 3. Temperature Response Curves Simultaneous incubations were conducted at a range of temperatures (6, 15, 20, and 27 0 C) for 21 hours. All radiolabel amendments were as noted in Figure 2. Both MeHg-production and degradation rates increased with temperature at all sites, although,the pristine site 3A- 15 exhibited the most pronounced MeHg production response. The increase in MeHg degradation was over a more limited range (note difference in Y-axis). Figure 4. Calculation of Activation Energies To more directly quantify the site-specific temperature responses depicted in Figure 3, the data was fit to an Arrhenius model of the linear form: Ln(rate) = (-E a /R)/T + Ln(B) Where E a is the activation energy, R is the gas constant, T is temperature in kelvin units, and B is a pre-exponential factor. E a simply reflects the response of the microbial community to temperature; the larger the value, the more responsive. E a for MeHg-production generally increased going from eutrophied to pristine sites. No significant spatial trend in E a was observed for MeHg-degradation. These E a values can be incorporated into the existing Everglades Hg-model to better predict MeHg-production rates at variable temperatures. Figure 5. Multiple Factors – Single Level Amendments Radiolabel amendment levels and incubation conditions are given in Figure 2. Specific treatment amendment levels (ie. Aerobic, FeS, H 2 S, SO 4 2-, and acetate) are indicated below each graph. The factor by which each amendment exceeds measured background concentrations of that constituent is given as a red number. Sites are arranged from north to south. The y-axis reflects either the increase (positive bar) or decrease (negative bar) in MeHg production or degradation relative to the anoxic control set (Figure 2) for each site. MeHg production was generally more impacted by treatment amendments, than was MeHg degradation. Oxygen, solid-phase reduced sulfur (FeS), and sulfide had the strongest inhibitory affect on MeHg degradation. Increased sulfate likely increased reduced-S concentrations and subsequently decreased MeHg production. Acetate, a carbon source for bacteria, did not have a consistent impact on MeHg production, suggesting bacteria were generally not carbon limited. Calculated Final Conc. (  mol*g slurry -1 ) Measured Final Conc. (  mol*g slurry -1 ) Sulfate (SO 4 2- ) 0 10 20 30 40 010203040 1:1 Line ENR-401 ENR-303 F1 3A-15 Figure 6. Multiple Level Amendments - Sulfur Chemistry A second series of amendment experiments focused exclusively on sulfur chemistry at four of the five sites (U3 excluded). A four-point series of amendment concentrations was calculated for each site, which ranged from a No Amendment control to a maximum of 4X, 500X, and 1500X background concentrations of solid phase reduced-S, pore- water sulfate, and pore-water sulfide, respectively.Within an hour of the sulfur amendment, but prior to Hg- radiolabel addition, sub-samples were collected from each treatment level sediment batch for concentration verification. The results plotted above indicate that the calculated final sulfate concentration was the same as the measured value. In contrast, measured values of solid phase acid-reducible sulfur (FeS) were somewhat lower than calculated FeS additions at high amendment levels. Finally, pore-water sulfide was consistently much lower than the calculated additions. These results indicate that there were rapid abiotic changes in the reduced-S pools upon addition of both FeS and H 2 S to sediment. In the case of H 2 S, this likely reflects incorporation of the analyte into polysulfides.