Periphyton Biofilms Influence Net Methylmercury Production in an

Sep 12, 2016 - Between-site differences in net methylation for samples collected from an ... Environmental Science & Technology 2018 52 (4), 2063-2070...
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Periphyton Biofilms Influence Net Methylmercury Production in an Industrially Contaminated System Todd A. Olsen,† Craig C. Brandt,‡ and Scott C. Brooks*,† †

Environmental Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008 MS 6038, Oak Ridge, Tennessee 37831-6038, United States ‡ Biosciences Division, Oak Ridge National Laboratory, P.O. Box 2008 MS 6038, Oak Ridge, Tennessee 37831-6038, United States S Supporting Information *

ABSTRACT: Mercury (Hg) methylation and methylmercury (MMHg) demethylation activity of periphyton biofilms from the industrially contaminated East Fork Poplar Creek, Tennessee (EFPC) were measured during 2014−2016 using stable Hg isotopic rate assays. 201HgII and MM202Hg were added to intact periphyton samples in ambient streamwater and the formation of MM201Hg and loss of MM202Hg were monitored over time and used to calculate first-order rate potentials for methylation and demethylation. The influences of location, temperature/season, light exposure and biofilm structure on methylation and demethylation potentials were examined. Between-site differences in net methylation for samples collected from an upstream versus downstream location were driven by differences in the demethylation rate potential (kd). In contrast, the within-site temperature-dependent difference in net methylation was driven by changes in the methylation rate potential (km). Samples incubated in the dark had lower net methylation due to lower km values than those incubated in the light. Disrupting the biofilm structure decreased km and resulted in lower net methylation. Overall, the measured rates resulted in a net excess of MMHg generated which could account for 3.71−7.88 mg d−1 MMHg flux in EFPC and suggests intact, actively photosynthesizing periphyton biofilms harbor zones of MMHg production.

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INTRODUCTION

Recently, attention has turned to periphyton biofilms as an alternative and under-studied source of MMHg in freshwater systems. Periphyton biofilm communities comprise a diverse range of organisms from eukaryotic algae (green algae and diatoms), to microinvertebrates, insect larvae, protozoa, Bacteria and Archaea. The latter two groups are particularly important as they include organisms capable of methylating Hg.2 Physiological studies using single bacterial strains and mixed communities have demonstrated significant differences between attached and unattached cells with respect to enzyme activity, cell size, reproductive rate, and exopolymer production.7−9 Importantly, Lin and Jay (2007) reported, on a per cell basis, the specific mercury methylation rate for Desulfovibrio desulf uricans was 10 times greater for biofilm cells compared to planktonic cells.10 Several scientists have reported Hg methylation by freshwater periphyton biofilms from a range of environments (Table S1).10−18 In some of these studies both Hg methylation and MMHg demethylation were measured and in each case net

Industrial use of metallic mercury (Hg) at the Y-12 National Security Complex (Y-12 NSC) on the United States Department of Energy Oak Ridge Reservation contaminated the soil, ground and surface water in and around East Fork Poplar Creek (EFPC) near Oak Ridge, TN.1 Human exposure to Hg pollution is governed by the formation and transport of the neurotoxin monomethylmercury (MMHg) in aquatic environments. Once formed, MMHg bioaccumulates in organisms and biomagnifies in aquatic food webs. Ultimately humans are exposed to MMHg via consumption of fish with elevated MMHg levels. It is generally accepted that MMHg is produced in anaerobic environments where there are iron and sulfate reducing bacteria, fermenting bacteria or methanogens.2−4 Much of the research on MMHg generation in stream ecosystems has focused on anaerobic environments in wetlands adjacent and hydrologically connected to the main channel and within the bottom sediments of the streams themselves.5 Watersheds with low wetland abundance hosting streams with elevated or variable MMHg levels suggest there are in-stream sources of MMHg in those systems.6 These in-stream sources could include bed sediments, hyporheic zones, and periphyton biofilms. © XXXX American Chemical Society

Received: March 28, 2016 Revised: September 9, 2016 Accepted: September 12, 2016

A

DOI: 10.1021/acs.est.6b01538 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology MMHg generation was reported.11,16,18 Acha (2011), Correia (2012), and Desrosiers (2006) reported significant positive correlations between sulfate-reducing bacterial activity and rates of Hg methylation.11,13,14 Methanogens present in biofilms have also been implicated in MMHg production by periphyton.13,16 Additionally, algal activity has been shown to influence Hg methylation rates and MMHg concentrations in periphyton.13,14,16,19,20 However, the importance of periphyton biofilm structure (i.e., physical integrity of the biofilm and the consequent anaerobic environments formed within them) on methylation and demethylation has not been investigated for a natural system, although there has been speculation that periphyton structure is important for Hg methylation.10,12−14,16,17 The experimental methods adopted among these periphyton methylation studies vary as do the data analysis methods. For the periphyton studies published to date, the final reported methylation and demethylation rate potentials were based on concentration changes measured at a single time point and assuming either zero- or first-order kinetics. The choice of assay length varies among and within studies (range from 1 h to 90 days) which may affect the reported rate potentials. In some studies, concentration changes were measured at multiple time points, however, due to nonfirst-order behavior of the data, a single time point was used for the calculations.10,14,16,17 Designing multiple time point methylation/demethylation rate assays is challenging because (1) there is often limited advance knowledge about the kinetics; (2) kinetics can change based on season, location and treatments; and (3) developing and analyzing samples is time-consuming and labor intensive. These factors make it difficult to measure enough time points on the right time scale to determine kinetics accurately. Continued research into Hg cycling by periphyton is needed in general and for systems with elevated Hg levels. We know of only one study that reported ambient MMHg concentrations in periphyton that are as high as those in EFPC.17 To date there is limited information about spatial differences in methylation and demethylation rates within study sites, for example, along the length of flowing streams. Additionally, there has been little effort to connect measured periphyton net methylation rates with observed field-scale changes (seasonal, spatial, diel, etc.) in ambient MMHg concentrations at study sites. In this work, our primary goal was to determine if periphyton in EFPC is a net source of MMHg by measuring both Hg methylation and MMHg demethylation rate potentials. A secondary goal was to relate the results from the methylation/demethylation rate assays to observed MMHg concentration dynamics in EFPC. We hypothesized that net MMHg generation would decrease with incubation temperature and for samples incubated in the dark. Additionally, we hypothesized that the three-dimensional organization of the periphyton was important in net MMHg generation. Samples collected from different locations in the creek to assess spatial variability across seasons were used in laboratory assays utilizing enriched stable isotopes of Hg to simultaneously determine Hg methylation, MMHg demethylation, and net MMHg production.

35.9662°N, 84.35817°W). The average total and dissolved Hg concentration decreased between the upstream site and downstream site from 211 ng L−1 to 110 ng L−1 and 89 ng L−1 to 15 ng L−1, respectively (average of monthly samples collected over the 12 month period beginning in May 2014). Conversely, the average total and dissolved MMHg concentrations increased along the same downstream transect from 0.15 ng L−1 to 0.43 ng L−1 and 0.10 ng L−1 to 0.29 ng L−1, respectively. A strong seasonal pattern was evident in the MMHg concentrations which are highest from midspring through autumn. Additional water chemistry and flow characteristics of the two sites can be found in the Supporting Information (Table S2). Assay Design. The effect of location along the creek, temperature, light condition during sample incubation, threedimensional organization of the periphyton, and growth substrate were examined (Table S3). Periphyton samples used to determine methylation and demethylation rate potentials in the laboratory studies were grown in situ on artificial growth structures (see Supporting Information for details). The structures consisted of polypropylene mesh, where the periphyton grew, attached to a PVC base (Figure S1). The structures were immersed in EFPC and left for at least 3 months for biofilm development prior to sampling. Samples were taken by cutting 3 cm diameter discs from the polypropylene mesh using a beveled aluminum pipe. Care was taken to minimize disruption of the periphyton structure during sample collection. In addition to the polypropylene mesh three other growth substrates were tested. For the January 2016 assay fritted glass discs (ROBU 30 mm diameter with 10.5 mm hole and 3.5 mm depth) were used. In the June 2016 assay perforated polypropylene sheets (McMaster-Carr 25 × 25 × 6 mm) and naturally occurring rocks from EFPC were used. Eleven discs were cut (3 cm diameter) from a clean mesh sheet and weighed to determine the average and standard error of the mesh mass (0.0829 ± 0.0003 g). This mass was then subtracted from the dried sample to determine the sample dry weight. The substrate samples were placed in 60 mL glass I-Chem jars, with PTFE lined caps, containing 20 mL of unfiltered EFPC water taken at the same time as periphyton sampling. The samples were immediately brought back to the lab to perform the rate assays. Once back in the lab stable Hg isotopic tracers were added to each sample: MM202Hg to monitor demethylation; and 201HgII to monitor methylation. The isotopes used in this research were supplied by the United States Department of Energy Office of Science by the Isotope Program in the Office of Nuclear Physics. The enrichment of these isotopes are certified as follows: 202Hg (0.13% 198, 1.41% 199, 0.93% 200, 0.68% 201, 95.86% 202, 0.94% 204; 201Hg (0.07% 198, 0.13% 199, 0.9% 200, 96.17% 201, 2.62% 202, 0.11% 204). Isotopes were received as HgO; working stocks were prepared by dissolving HgO in 2%HNO3 and further diluted in 0.5% BrCl. MM202Hg was synthesized from the 202Hg using a methylcobalamin method.24 Fifty to 200 μL of the stable isotopic tracers were added to samples and gently mixed so as not to disturb the periphyton structure. One to 2 days prior to the assay sampling events MMHg and total Hg (THg) concentrations were measured for the mesh-bound periphyton. Six 3 cm diameter discs were taken and freeze-dried to determine the average dry weight (dw). Three of these discs were then analyzed to determine MMHg and the remaining three analyzed to determine THg. These results informed the

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EXPERIMENTAL SECTION Study Site Description. The detailed history and characterization of EFPC have been previously published.1,21−23 Samples for this study were collected from two locations separated by approximately 17 creek kilometers (upstream site 36.00175°N, 84.24929°W and downstream site B

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III AFS. Water samples were prepared following United States Environmental Protection Agency (USEPA) method 1631.25 Sediment and periphyton samples were digested in aqua regia (0.1 g-dw into 4 mL) followed by dilution, SnCl2 reduction and then analysis on the MERX system. MMHg concentrations were measured using a Brooks Rand MERX Hg Speciation GC & Pyrolysis coupled to an ELAN DRC-e ICP-MS. Water samples were prepared following USEPA method 1630.26 Sediment and periphyton samples were extracted following a modified procedure from Bloom (1997).27 Briefly this method consisted of a nonaqueous phase extraction of MMHgCl followed by a back extraction into water and ethylation/purge and trap/GC. Because of the elevated THg levels in EFPC sediments and periphyton we found it necessary to follow the back extraction step with a distillation step to further reduce the amount of ambient inorganic Hg before analysis. MM200Hg was used as an internal standard for all MMHg analyses.28 The isotope pattern deconvolution calculations used in this work are outlined in Meija et al. (2006).29 Quality assurance/quality control metrics are provided in the Supporting Information. Data Analysis. Methylation and demethylation rate potentials were calculated using a first-order kinetics model. According to the model net methylation is written as30

tracer additions which were kept in the range of 10 to 400% of the total mass of ambient MMHg and THg (Table S1). These ambient THg and MMHg values were also used in the net methylation rate calculations described later. The assays were conducted in Conviron CMP 3244 environmental growth chambers. The temperature was held at 20 °C for autumn, spring and summer samples and at 10 °C for winter samples which were the approximate temperature of the creek when periphyton samples were taken (additional information on creek temperature for a period prior to conducting the assays has been included in Supporting Information Table S4). The chamber light sources, averaging 600 Lux total light intensity (HOBO Pendant Temperature/ Light Data Logger, UA-002-64), were on for the duration of the assays in order to isolate light’s impact on periphyton activity. To facilitate comparison with other published studies of Hg methylation by epi- or periphyton a single time point assay was conducted in which triplicate samples were collected after a 48h incubation. Additionally, a time series analysis was conducted in which triplicate samples were collected at predetermined times over the course of a 72 h incubation. Killed periphyton controls were autoclaved (30 min at 120 °C and 158.6 kPa) before adding stable Hg isotope tracers. Live streamwater controls consisted of 20 mL unfiltered creek water with the stable Hg isotopic tracers but without periphyton discs. In addition to addressing seasonal/temperature effects three other variables were also assessed between November 2014 and June 2016. The first compared methylation and demethylation rate potentials between an upstream and a downstream site which were approximately 17 creek km apart and represent the end members of MMHg concentration in EFPC. Second the effect of light exposure was examined by wrapping “dark” controls in aluminum foil to exclude light during the incubation. The third treatment tested the importance of periphyton structure on Hg cycling rates. To disturb periphyton structure 1 g of fine sand (Iota Standard quartz sand, grain size 150−250 μm, Unimin Corp; see Supporting Information) was added to periphyton samples which were then vigorously shaken for 1 h prior to adding the stable Hg isotope tracers. All other incubation conditions for the intact and disturbed samples were identical. After disruption the sample was a mixture of residual biofilm on the substrate, suspended cells and biofilm fragments. For all time points after incubation the samples were frozen (−30 °C freezer) until they were processed as described later. To supplement the assay results a study was done to determine the spatial and temporal variation of MMHg and THg concentrations in water (total and dissolved), periphyton and sediment in EFPC. Samples were taken once a month for one year starting in September 2014 and ending in August 2015. Water samples were collected in glycol-modified polyethylene terephthalate (PETG) bottles midstream at each sampling location. The dissolved fraction was operationally defined as the portion passing a 0.2 μm poly(ether sulfone) filter. Periphyton was scraped from large rocks using a plastic spatula and collected in polypropylene vials. Sediment was scooped from the creek bed into polypropylene vials. The periphyton and sediment samples were frozen (−30 °C freezer) after sampling until they were freeze-dried for long-term storage. The water samples were acid preserved using trace metal grade HCl to 0.5% and refrigerated (4 °C) until analysis. Analytical Methods. THg concentrations were measured using a Brooks Rand MERX Total-Hg Purge and Trap model

d[MMHg] = k m[Hg II] − kd[MMHg] dt

(1) −1

where k m = methylation rate potential (d ); k d = demethylation rate potential (d−1); [HgII] = concentration of inorganic Hg; and [MMHg] is the concentration of methylmercury. When using the stable isotopic tracers mentioned previously in the methylation/demethylation rate assays eq 1 can be simplified to eq 2 for the methylation of 201 HgII and eq 3 for the demethylation of MM202Hg assuming the resulting [MM201Hg] and [202HgII] are near zero for the relatively short duration of the experiment so the back reaction can be ignored. This simplification after integration results in the following equations for rate potentials

(

−ln 1 − km =

[MM201Hg]t [201Hg II]0

) (2)

t

⎛ [MM202Hg] ⎞ t ⎟/t kd = −ln⎜ 202 ⎝ [MM Hg]0 ⎠

(3)

where [201HgII]0 and [MM202Hg]0 are the initial concentration of the tracers added to each sample; t is the time length of the assay; and [MM201Hg]t and [MM202Hg]t are the concentration of those species at time t. For the single time point assays, concentrations were entered directly into eqs 2 and 3 using triplicate samples to estimate the rate potential and its standard error. For the time series assay eqs 4 and 5, integrated forms of eq 1, were fit to the data by adjusting the rate potentials using OriginPro 8.6. The OriginPro output included the rate potentials and their standard errors. We found it necessary to include an intercept value (y0) in eq 4 because blanks and control samples showed a small amount of MM201Hg (0.03% of ambient) which could be an indication of MM201Hg artifact formation during extraction and analysis or small levels of MM201Hg in the inorganic 201Hg tracer. [MM201Hg]t = [201Hg II]0 (1 − e−k mt ) + y0 C

(4)

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Environmental Science & Technology [MM202Hg]t = [MM202Hg]0 e−k dt

demethylation. These results suggest the observed methylation and demethylation were controlled by processes associated with live periphyton. Methylation and Demethylation Rate Potentials Estimated from Single Time Point Assays. The creek location comparisons were conducted in November 2014 at 20 °C in full light and in January 2016 at 10 °C in full light. The methylation rate potential (km) was similar between the two creek locations within each experiment (Figure 1A). However,

(5)

Using eq 1 the net methylation rate (ng g-dw−1 d−1) was calculated by applying the methylation and demethylation rate potentials obtained in the stable Hg tracer rate assays along with the ambient level concentrations of MMHg and THg measured in the periphyton. Since we do not have a direct measure of [HgII] and because MMHg concentrations were all less than 0.2% of the total Hg we used the [THg] as an approximation of [HgII] when performing the net methylation calculations. The detection limits for the newly formed MM201Hg and for the decrease in MM202Hg were determined as described in Hintelmann (1997). The equations used to determine if the concentration of newly generated MM201Hg from the 201HgII tracer (cnew) or the decrease in concentration of MM202Hg tracer (Δ) were detectable are31 cnew ≥ bg × A × 3 × RSD

(6)

Δ ≥ (A + y) × bg × 3 × RSD

(7)

where bg = background ambient concentration of MMHg; A = natural abundance of the tracer isotope used; RSD = relative standard deviation (RSD) of isotope ratio measurement (tracer isotope/reference isotope); and y = spike addition in multiples of the background species concentration. The RSDs were calculated using standards with 10 pg ambient MMHg and 11.1 pg MM200Hg. The standards (78 in total) came from analytical runs spanning several months. Statistical Analyses. Pairs of rate potentials from different treatments were compared using a standard t test in OriginPro version 8.6 (OriginLab Corp., Northampton, MA). Overall factor effects were assessed with the generalized linear model procedure (PROC GLM) in the SAS/STAT software, Version 9.3 of the SAS system for Windows, to analyze the methylation and demethylation rate potentials (SAS Institute Inc. 2011). Because samples from certain combinations of the treatment factors were not collected (e.g., dark exposure at 10 °C at the upstream site), a cell means approach was used to compare the rates of the sampled treatment factors. In this approach each combination of treatment factors was assigned a unique cell identifier, and the cell identifier was used as the independent variable in PROC GLM. Least square means were used in the intercell comparisons to account for the differing number of observations in each cell.

Figure 1. Effect of treatments on (A) methylation rate potentials, (B) demethylation rate potentials, (C) net methylation, (mean ± standard error) in periphyton samples from EFPC determined using single time point assay (t = 48 h). Dates represent the day samples were taken and assays began. Blue bars = upstream site; Red bars = downstream site; green bars = rocks from creek; Filled bars = intact, incubated in light; Open bars = intact, incubated in dark; Diagonal fill = disturbed, incubated in light.

the demethylation rate potential (kd) for the downstream site was significantly (p < 0.05) smaller than the upstream site (Figure 1B) yielding a larger net methylation rate downstream in both assays (Figure 1C: November 2014 upstream 0.223 ± 0.041 ng g-dw−1 d−1, downstream 0.404 ± 0.062 ng g-dw−1 d−1; January 2016 upstream 0.202 ± 0.067 ng g-dw−1 d−1, downstream 0.266 ± 0.040 ng g-dw−1 d−1). Comparing across all assays and treatments, the kd values for the downstream site were consistently lower than those estimated for the upstream site (Figure 1B). The underlying mechanism(s) for the difference in kd at the two locations are not readily apparent but may include differences in the microbial communities, geochemical conditions (e.g., redox gradients) or physical structure of the periphyton between the two sites. The seasonal/temperature comparisons were conducted in February 2015 at the downstream site and in January 2016 at both sites, in full light and at 10 °C. The kd values for the 10 °C assays were similar to those conducted at 20 °C at the same sites (Figure 1B). The km values, however, were significantly (p < 0.05) lower for the 10 °C winter assays when compared to the 20 °C assays (Figure 1A) with the exception of the May 2015 assay which had greater variability (Table S5).

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RESULTS AND DISCUSSION Periphyton was grown in EFPC on an artificial surface with standardized surface area so they could be brought back intact to the lab where stable Hg isotopes of both inorganic Hg and MMHg could be added and Hg methylation and MMHg demethylation monitored. A variety of treatments including creek location, season/temperature, light exposure and physical structure were used to determine under what conditions EFPC periphyton could be a net source of MMHg. A summary of the net methylation results can be found in Table S5. Other instream sources (e.g., bed sediments) can contribute to MMHg concentrations in EFPC but the focus of this work was on the role of periphyton. There was no detectable methylation or demethylation in the live creek water control or killed control samples. Conversely, each sample containing live periphyton in all treatments was above the calculated detection limit for both methylation and D

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methylation rate potential (eq 1).19 However, the method used to calculate km in this study cannot distinguish an increase in HgII bioavailability from an increase in km value. Other researchers have measured higher net methylation potential when incubating periphyton samples in the dark versus the light suggesting different communities may react differently when exposed to light.12,18 However, demethylation was not measured in those studies so the extent to which photodemethylation contributed to lower net methylation potential in the light remains unclear. The intact versus disturbed assays were conducted at the upstream site in September 2015 and June 2016, at 20 °C and in full light. To disrupt the in situ biofilm integrity, the disturbed samples were amended with clean sand and shaken vigorously just prior to starting the assay. The demethylation rate potential for the June 2016 intact assay was significantly larger than the November 2014 upstream assay (0.267 ± 0.009 d−1 versus 0.171 ± 0.014 d−1 respectively; p < 0.05; Figure 1B) demonstrating temporal variability within the same site and at the same temperature. For samples in which the periphyton structure was disrupted the km value was approximately 50% lower than samples in which the structure remained intact (Figure 1A). Demethylation rate potentials between the intact and disrupted samples were comparable (Figure 1B). Notably, for samples in which periphyton structure was disturbed there was a significant drop in net methylation when compared to the intact samples (Figure 1C). The September 2015 disturbed assay resulted in net demethylating conditions, 0.095 ± 0.014 ng g-dw−1 d−1. These results show the three-dimensional organization of the periphyton contributes to its ability to methylate Hg. The periphyton structure is one controlling factor in the development of anaerobic zones conducive for iron and sulfate reducing bacteria, fermenting bacteria or methanogens.34,35 Once this structure is compromised and reducing zones favoring the activity of methylating Bacteria and Archaea are disturbed, the Hg methylation rate is significantly diminished. Generation of MM201Hg in the disturbed samples may have been due, in part, to reaggregation of portions of the periphyton during the 48-h incubation period or by reducing microenvironments within suspended periphyton flocs.36 For the June 2016 assay methylation and demethylation rate potentials were measured for periphyton covered EFPC rocks in addition to the artificial substrate. The resulting km, kd, and net methylation for the natural substrate were all comparable to those of the artificial substrates (Figure 1A−C). Additionally, we have analyzed THg and MMHg for biofilms grown on natural surfaces from both sites (n = 8; 15.33 ± 2.54 ug-THg gdw−1, 4.50 ± 0.41 ng-MMHg g-dw−1) and the artificial surfaces (n = 8; 14.64 ± 1.13 ug-THg g-dw−1, 2.50 ± 0.24 ng-MMHg gdw−1). The combination of these results suggests similarity of function between biofilms grown on natural and artificial substrates. The overall results from the cell means analysis of the methylation and demethylation rate potentials are shown in Table S6. For km the cell means model had an R2 of 0.57 and an F-value of 2.88 (p = 0.012). In the case of kd, the cell means model had an R2 of 0.95 and an F-value of 44.47 (p < 0.0001). Cell pairwise tests results are shown in Table S7. Based on these results, a summary of significant differences between the cell means in given in Table S8. The group identifier denotes those cells whose means are statistically indistinguishable at p ≤ 0.05. The results for kd show two distinct groups with group A

Consequently, the net methylation rate in February 2015 (Figure 1C: 0.259 ± 0.064 ng g-dw−1 d−1) and January 2016 were lower than the other assays from the same site but held at 20 °C. This effect was more pronounced at the downstream site. These results indicate that the activity and/or abundance of methylating organisms in the periphyton are more sensitive to temperature or covarying seasonal parameters than is the activity of demethylating organisms. The decline in net methylation for the 10 °C winter assays could be attributed to the decreased activity within the periphyton, including microbial activity, at the lower temperature. These results, gathered using samples colonized by mixed communities in the field, are confounded by likely seasonal changes in relative abundance and members of the periphyton community. Nevertheless, in spring assays conducted at 20 °C, the km value rebounded to the value measured several months earlier (Figure 1A, downstream site: 11/24/2014 to 2/3/2015 to 5/12/2015 and upstream site: 9/15/2015 to 1/29/2016 to 6/9/2016) suggesting temperature is an important environmental determinant of net methylation rate via its influence on km. Desrosiers (2006) reported a similar impact where km decreased an order of magnitude when assay temperatures decreased from 20 to 15 °C.14 Nevertheless, few studies have addressed the effect of temperature on methylation, demethylation and net MMHg production. St. Pierre (2014) reported results similar to ours for incubations with marine sediments conducted over the temperature range 4−24 °C. Temperature had a significant effect on methylation potential but not on demethylation potential. Through the use of different inhibitors, those authors suggested that the greater temperature sensitivity of methylating sulfate-reducing bacteria was responsible for their results.32 The assays used to examine the impact light exposure has on Hg cycling were performed in May 2015 at the downstream site and June 2016 at the upstream site held at 20 °C, in full light. For the companion assay conducted in the dark, samples were wrapped in foil to shield them from light exposure during the assay. The kd values for the light and dark assays were not significantly different (May 2015, p = 0.86; June 2016, p = 0.39; Figure 1B) indicating the light source in the environmental growth chamber was not powerful enough to photodemethylate MMHg and further supports, along with the absence of demethylation activity in the killed controls, the idea that the demethylation observed in the lab incubations was due to microbial processes mediated by the periphyton. The km values for the light assays were similar to those from the same site and incubation temperature (Figure 1A). In contrast the dark assay km values were less than half of those from assays with the same site and temperature (Figure 1A). The contrast in km values for the light versus dark assays resulted in the net methylation rate in the light May 2015 assay being 10 times greater than the dark assay (Figure 1C: light 0.395 ± 0.229 ng g-dw−1 d−1; dark 0.035 ± 0.012 ng g-dw−1 d−1). Interestingly, the June 2016 dark assay resulted in net demethylating conditions whereas the light assay was net methylating (Figure 1C: light 0.278 ± 0.062 ng gdw−1 d−1; dark −0.224 ± 0.008 ng g-dw−1 d−1). The higher net methylation observed in the light assay may be due to the active algae supplying metabolites (e.g., labile organic carbon) to the rest of the methylating community enhancing their activity.33 Alternatively, as suggested by Leclerc et al., the release of low-molecular weight thiol ligands by algae could bind HgII increasing its bioavailability thereby increasing the methylation rate term (km[HgII]) without changing the E

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Environmental Science & Technology

estimated from the single time point analysis (Table 1; 0.158 ± 0.038 ng g-dw−1 d−1 versus 0.259 ± 0.064 ng g-dw−1 d−1). Calculating the net methylation rate using the single time point method at each time point in the February 2015 time series gave rise to a clear trend: shorter assay times resulted in larger km and kd values and a higher net methylation rate (Table 1). Using the methylation and demethylation rate potentials determined by the February 2015 time series and assumptions about creek bed area, creek bed periphyton coverage and Hg species bioavailability (see Supporting Information for details) we calculated that the periphyton in EFPC could have produced between 3.71 and 7.88 mg-MMHg that day. For comparison the flux out of EFPC that day was 14.8 mg-MMHg. Although large uncertainties are associated with estimates of methylating periphyton abundance in the creek, these results are consistent with our hypothesis that periphyton makes a substantial contribution to the MMHg levels in EFPC. Study Intercomparison. Variations in the design of experiments intending to measure Hg methylation by periphyton found in the Hg cycling literature could impact the rate results reported. These variations include whether both methylation and demethylation were measured, the length of the assay, the number of time points used in assays, the number of time points used in rate calculations, the amount of Hg tracer added relative to ambient Hg levels, and use of stable or radiolabeled tracers. Based on our February 2015 results it seems likely some of these factors greatly impact the rates being reported (Table 1). Further work is needed to standardize the methods used and determine how best to control these factors in order to facilitate comparisons within and between studies. For the reasons outlined previously, a direct comparison between the results of this study and other published studies is difficult. With that caveat in mind we make general comparison of our results to those of seven other periphyton or epiphyton studies (Table S1). Only 7 of 32 experiments in the seven papers conducted demethylation assays with a range of demethylation rate potentials from 0.068−0.232 d−1, very similar to the range of values estimated in this study (0.067− 0.273 d−1). The consistency of demethylation rate potentials is notable given the range of environments and experimental conditions encompassed by these studies. In contrast, the estimated methylation rate potentials reported in the other studies spans a much broader range (1.6 × 10−5 − 0.232 d−1) with most of the values at least 5 times larger than the highest methylation rate potential measured in this study. This comparison suggests that the estimation of Hg methylation rate potentials is subject to greater variability than is the estimation of MMHg demethylation rate potentials. The underlying causes of this variability merit closer attention (e.g., site-specific variability in governing environmental parameters, systematic errors due to procedural differences). Net methylation depends on the rate potentials and the amount of Hg and MMHg available for methylation and demethylation,

consisting of all downstream samples and the other group consisting of all upstream samples. The cell means for km are less statistically distinct. Nevertheless, these results suggest that light exposed samples held at 20 °C (cells 4, 5, 10, 11, 12 and 13) may have higher km than the light exposed samples held at 10 °C and the samples incubated in the dark and kept at 20 °C. Growth substrate (mesh, glass frits, polypropylene sheet, or native rocks from the creek) had no discernible effect on either methylation or demethylation rate potential. In summary, the observed difference in net methylation between the two creek locations was driven by differences in kd. In contrast, the within-site differences observed for different treatments (incubation temperatures, light exposure and structural integrity) in net methylation rate were controlled by the changes in km. During our controlled experiments periphyton had a higher net production of MMHg when exposed to light. Extrapolating these results to the field setting is difficult since the potential for photodemethylation is greater under natural sunlight as opposed to the artificial light of the growth chambers. Nevertheless, there was a clear enhancement in estimated km for samples incubated under light versus those incubated in the dark. Finally, disruption of the periphyton structure diminished its ability to methylate Hg. Methylation and Demethylation Rate Potentials from Time Series Assay. The methylation/demethylation rate assay results described above were based on a single 48-h time point. In addition to the 48 h time point during the February 2015 assay, samples were collected at 6, 24, and 72 h. For this time series experiment the km and kd values were calculated by fitting eqs 4 and 5 to the methylation and demethylation data, respectively (Figure 2). Calculating the rate potentials using the

Figure 2. February 2015 methylation/demethylation time series. Data points and error bars represent the mean and standard error of triplicate samples. The dashed line is a fit of eq 4 to the methylation data and the solid line is a fit of eq 5 to the demethylation data.

time series method resulted in significantly (p < 0.05) different results when compared with the 48 h single time point calculation yielding a net methylation rate 39% lower than

Table 1. Differences in Rate Potentials Dependent on Method Design: Single Time Point versus Time Seriesa km ( × 10−5; d−1) kd ( × 10−2; d−1) net methylation rate (ng g-dw−1 d−1)

6h

24 h

48 h

72 h

time series

12.5 ± 2.5 39.2 ± 4.8 0.789 ± 0.469

4.25 ± 0.92 8.92 ± 0.62 0.381 ± 0.138

3.02 ± 0.41 6.79 ± 0.64 0.259 ± 0.064

2.33 ± 0.15 4.60 ± 0.55 0.215 ± 0.013

1.42 ± 0.32 1.77 ± 0.63 0.158 ± 0.038

a Values are mean ± standard error. km values calculated using eqs 2 and (4) for single time point and time series respectively. kd values calculated using eqs 3 and (5) for single time point and time series respectively. Net methylation rates calculated using eq 1.

F

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Environmental Science & Technology respectively. Therefore, continuing efforts to identify and quantity these fractions of total Hg and MMHg will greatly facilitate the application of these rate assays to system-wide predictive understanding. Relating Laboratory Results to Field Observations. If the periphyton community is a source of MMHg production then one would expect environmental variables affecting the abundance and activity of the periphyton to have consequent impacts on MMHg concentration. We find evidence consistent with this hypothesis in our laboratory incubations and field data. It is impossible to isolate single factors in a field setting as many environmental parameters covary with, for example, season. For example, colder temperatures covary with shorter days and less intense sunlight through the winter months. Nevertheless, analogous results to our lab incubations are evident in the field observations. Our rate estimates suggest that periphyton is a net source of MMHg so one would expect to see elevated MMHg levels in periphyton when compared to other compartments. At both the upstream and downstream sampling locations the percent MMHg in periphyton was higher than in local bed sediment (Figure S2) suggesting either periphyton is a source of MMHg, as indicated in our lab incubations, bioaccumulates MMHg, or a combination of both. Interestingly, our lab estimates of higher net methylation for samples from the downstream site are paralleled by field observations of higher percent MMHg in periphyton and sediments and higher concentrations of MMHg in the water column at the downstream site (Figures S3, S4). The laboratory incubations demonstrate that net methylmercury production decreased by a factor of 2 with a 10 °C temperature decrease. This parallels field measurements where MMHg concentrations in the water column show a seasonal dependence with higher concentrations during the warmer spring and summer months. This pattern is stronger for the downstream location (Figure S3). Another laboratory result revealed net methylation decreases (in one case resulting in net demethylation conditions) for incubations conducted in the dark suggesting a photosynthetically active periphyton community enhances MMHg production. Similarly, we have sampled EFPC over several diel cycles across seasons and found dissolved MMHg is correlated to the daily photocycle during the summer with higher concentrations during the day (Riscassi et al., in prep; Brooks et al., in prep.). Mercury methylation and MMHg demethylation measurements using periphyton biofilm samples demonstrate that these complex assemblages support net MMHg production and that they could be responsible for a substantial fraction of MMHg flux in EFPC. Laboratory incubation results are consistent with field observations of seasonal and locational patterns in MMHg concentration in water. Our results suggest periphyton may be an important in-stream source of MMHg production and may regulate many of the observed concentration dynamics in EFPC.

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MMHg production by periphyton in EFPC. Figure S1: photographs of the periphyton growth structure. Figure S2: percent MMHg in periphyton and sediment. Figure S3: seasonal pattern in MMHg concentrations in EFPC. Figure S4: results of the periphyton net MMHg production calculations. Table S1: methylation and demethylation rate potentials from this work and those from other studies. Table S2: water chemistry of the upstream and downstream sampling locations. Table S3: matrix depicting the treatment factors for experiments. Table S4: water temperatures preceding sampling events for each assay. Table S5: summary results for methylation/demethylation rate assays. Table S6: analysis of variance results from the cell means model for km and kd. Table S7: Probabilities of pairwise cell differences. Table S8: results of the cell means analysis (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: 865-574-6398; fax: 865-576-8646; e-mail: brookssc@ ornl.gov. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the U.S. Department of Energy, Office of Science, Biological and Environmental Research, Subsurface Biogeochemical Research Program and is a product of the Science Focus Area (SFA) at ORNL. The isotopes used in this research were supplied by the United States Department of Energy Office of Science by the Isotope Program in the Office of Nuclear Physics. We thank Xiangping Yin for help with sample analysis and Kenneth Lowe for help with constructing the periphyton growth platforms, sample collection, and sample analysis. A. Riscassi and four anonymous reviewers provided comments on earlier versions of this manuscript that greatly improved the final version. This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paidup, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b01538. Quality assurance/quality control metrics are provided. Additional site description and details on the sand used to disturb the periphyton structure are provided. Explanation and results of the calculation of net G

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