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Auxiliary Material for Paper 2011GB004202RR
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Mercury isotopes in a forested ecosystem: implications for air-surface exchange dynamics
and the global mercury cycle.
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Jason D. Demers
(University of Michigan, Department of Earth and Environmental Sciences, Ann Arbor, MI
48109, USA)
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Joel D. Blum
(University of Michigan, Department of Earth and Environmental Sciences, Ann Arbor, MI
48109, USA)
Donald R. Zak
(University of Michigan, School of Natural Resources and Environment, Ann Arbor, MI 48109,
USA)
Demers, J.D., J.D. Blum, D.R. Zak. (2012). Mercury isotopes in a forested ecosystem: new
insights into biogeochemical cycling and the global mercury cycle. Submitted to Global
Biogeochemical Cycles.
Supplemental Information: Text
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2. METHODS (Unabridged)
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2.1. Site Description
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The Rhinelander Free-air CO2 and O3 Enrichment (FACE) site is located in northeastern
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Wisconsin, USA (49º, 40.5’N; 89º, 37.5’E; 490 m elevation). The Rhinelander FACE study was
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designed to test the response of a northern hardwood ecosystem to elevated CO2, O3, and the
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interaction of elevated CO2 and O3, during regeneration from seedlings to mature trees (Dickson
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et al. 2000). The site consists of twelve rings (each 30 m diameter), spaced 100 m apart to
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minimize treatment effects between rings, in a full-factorial design with three ambient control
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rings, three elevated CO2 rings, three elevated O3 rings, and three elevated CO2 + O3 rings
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distributed among 3 blocks from north to south. Blocks were based on slight differences in soils
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across the study area (Dickson et al. 2000). Target concentration for the CO2 treatment was 560
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µmol/mol (~200 µmol/mol above ambient 1996 levels); O3 treatment consisted of daily episodic
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exposure following a diurnal profile, the maximum of which was adjusted based on daily
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meteorological conditions and ranged from 50-100 nmol/mol (Dickson et al. 2000, and
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references therein). Detailed information regarding the Rhinelander FACE study design and
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execution has been published previously (Dickson et al. 2000).
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The Rhinelander FACE rings were established on coarse sandy loam soils (mixed, frigid,
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coarse loamy Alfic Haplorthod) that were under prior agricultural management. The sandy loam
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surface soil (~15 cm) grades into an argillic horizon (Bt horizon; clay loam from ~15 to 45 cm
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depth) that is underlain by a C horizon composed of sandy loam, stratified sand, and gravel. Soil
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properties differed little across the site (see Dickson et al. 2000 for a complete analysis of soil
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within each ring). In June 1997, each Rhinelander FACE ring was divided into three sections
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and planted with three tree species common to northern hardwood forests of the Great Lakes
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region: trembling aspen (Populous tremuloides Michx), paper birch (Betula papyrifera Marsh.),
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and sugar maple (Acer saccharum Marsh.). Half of each ring was planted with 5 different aspen
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genotypes (8L, 43E, 216, 259, 271) with varying tolerance to O3 (Karnosky et al. 1996b) and
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varying response to CO2 enrichment (Kubiske et al. 1998); the remaining two ring quarters were
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planted with an aspen and sugar maple community, or an aspen and paper birch community.
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Stem density (10,000 stems/ha) was within the range of naturally occurring ~10-year-old aspen
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stands (Fraser et al. 2006, Mulak et al. 2006). Canopy closure was complete in all aspen-only
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ring sections beginning in 2003 (Zak et al. 2007).
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2.2. Sample Collection and Processing
In this study, we focused on foliage, forest floor, mineral soil, and soil evasion THg(g)
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measurements from the aspen-only community, with foliage mercury isotopes characterized for
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only the aspen 216 clone, which displays a moderate response to CO2 and O3 (Karnosky et al.
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1996a, Karnosky et al. 1996b). Atmospheric THg(g) samples were taken at the top of the canopy
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at plot center within ambient treatment rings, and precipitation samples were obtained in an open
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field nearby.
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2.2.1. Foliage, Forest Floor, and Mineral Soils - Foliage, forest floor, and mineral soil
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samples were collected during August 2008. Foliage was collected by hand with pole pruners
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along profiles through the canopy (top, >75% of total canopy height; upper-middle, 50-75%;
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lower-middle, 25-50%; bottom, < 25%) in each ambient and treatment ring section; canopy
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heights were ~8-10 m. Forest floor samples (4 per ring, 26 cm x 26 cm quadrats, 0.0676 m2)
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were collected randomly from each ambient and treatment ring section. After removal of the
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forest floor, mineral soil cores (0-20cm) from each ring section were composited into a single
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sample. Additional mineral soil samples collected in 2007 at depths of 0-5 cm and 5-10 cm were
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also analyzed to assess differences in mercury concentration and isotopic composition along the
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soil depth profile in ambient rings. All foliage, forest floor, and mineral soil samples were
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immediately stored in plastic bags and then frozen until processed, vinyl or nitrile gloves were
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worn during sampling and subsequent processing. Foliage consisted of new leaves, including
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petioles, and was dried at 60ºC. Mineral soil samples (0-20 cm cores) were also dried at 60ºC,
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whereas mineral soil samples from 2007 (0-5 cm, 5-10 cm cores) were freeze-dried. Forest floor
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samples were also freeze-dried. All samples were ground in an alumina grinding cylinder in a
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mixer mill; grinding blanks using Ottawa sand (baked at 750ºC) before and between forest floor
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sample sets showed no contamination associated with the alumina grinding cylinders nor carry-
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over from previous samples. To ensure accurate dry-weight concentrations subsequent to
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grinding, sub-samples of all materials were re-dried in a freeze-drier, and stored in glass screw-
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top vials in plastic bags in a dessicator prior to analysis. Organic matter content (%OM) of forest
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floor and mineral soil samples was determined by loss on ignition (LOI) in a muffle furnace at
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650ºC for 6 hours.
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2.2.2. Atmospheric THg(g) - Total gaseous mercury (THg(g) = GEM + RGM) was
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collected from the atmosphere at the top of the canopy in ambient rings simultaneously with
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THg(g) evading from the forest floor in the ambient, elevated CO2, and elevated O3 rings during
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June 2009, well after the forest canopy had fully developed. At the time of THg(g) sampling, the
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forest floor predominantly consisted of the foliage cohort sampled one year prior. In order to
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collect enough mercury for isotopic analysis of atmospheric THg(g), air was drawn through eight
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gold-coated bead traps deployed in parallel via a PVC manifold at a target rate of 1.8 L/min per
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trap for 24-72 hours (timing varied due to weather, samplers were not operated during
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precipitation events) (modified from Gratz et al. 2010, Sherman et al. 2010). For each manifold,
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flow was drawn from a single electrically powered pump, and total flow was recorded with a dry
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test meter (DTM). In order to remove particulates from the sample stream, the inlet to each trap
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utilized a pre-baked quartz-fiber filter (Whatman QMA 47mm disks, 0.7 µm nominal poresize)
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mounted in a teflon filter housing (Savillex Corp.). To prevent condensation within the gold-
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coated bead traps, traps were maintained at 55ºC. The performance of gold-coated bead traps
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was verified with spikes of 10 ng Hg (NIST SRM 3133), blanked, and then blank-checked prior
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to use. Mean recovery of spikes was 91.1% (n = 23, 5.7%, 1SD); maximum composite blank-
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check per set of 8 traps was ≤ 0.3 ng. Upon completion of sampling, traps were retrieved from
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the manifold sampler, capped, wrapped with Teflon tape, triple bagged, and sealed in a clean
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cooler. All desorptions and analyses of mercury collected on gold-coated bead traps was
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completed in the Biogeochemistry and Environmental Isotope Geochemistry Laboratory
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(BEIGL) at the University of Michigan.
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2.2.3. Soil Evasion THg(g) - Soil evasion THg(g) samples were collected in the same
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manner as atmospheric THg(g) samples, except that each sample collection trap was connected to
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the outlet of a flux chamber. Because the primary goal of our soil evasion collection was to
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determine the isotopic composition of emitted mercury (rather than quantify fluxes) it was
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necessary to isolate the evasion flux from ambient atmospheric THg(g); thus, our flux chambers
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differed in design from dynamic flux chambers typically used for evasion flux measurements.
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Flux chambers consisted of sturdy polycarbonate boxes (50 cm x 29 cm x 14.5 cm), with a
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footprint surface area of 0.15 m2. Although polycarbonate is less transmissive of UV light than
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Teflon (in particular, UV-B light <320 nm wavelength) and some studies have shown reduced
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soil mercury emissions with chambers made of polycarbonate materials (e.g., Carpi et al. 2007),
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numerous studies have also shown no consistent difference or that responses to natural light are
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muted but not eliminated (e.g., Gustin et al. 1999, Wallschlager et al. 1999, Engle et al. 2001,
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Lindberg et al. 2002). The inlet to the flux chamber consisted of a nylon ¼ inch fitting mounted
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in one end-wall of the chamber, 3.5 cm above the ground surface, attached to a gold-coated bead
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trap that acted as an ambient atmospheric THg(g) filter. The outlet from the flux chamber
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consisted of a teflon ¼ inch fitting mounted in the end-wall opposite the inlet, 8 cm above the
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ground surface and 3 cm from the top of the chamber, and was connected by Teflon tubing to the
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quartz-fiber pre-filter of the sample collection trap. Chambers were installed over intact forest
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floor by setting each chamber in place, carefully cutting around the perimeter, and then pushing
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the chamber 3.5 cm into the soil. As the chamber was pushed into place, the inward sloping
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walls promoted a firm connection with undisturbed soil. Additionally, chambers were pegged
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into place and the forest floor and soil around the outer perimeter was firmly pressed back into
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place around each installed chamber. The flushing rate based on DTM measurements was 2.2
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L/min on average, with a chamber volume turnover rate of 6.6 minutes (calculated from data in
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Supplemental Table S1), both of which are within the range used for dynamic flux chambers in
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other studies (e.g., Carpi and Lindberg 1998, Lindberg et al. 1999, Wallschlager et al. 1999,
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Gustin et al. 2002, Lindberg et al. 2002, Ericksen et al. 2006, Gustin et al. 2006, Kuiken et al.
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2008) and are similar to the ideal high flushing rates suggested by Lindberg et al. (2002).
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Polycarbonate chambers were cleaned by submersion in 5% trace metal grade nitric acid at room
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temperature for 24 hours, rinsed copiously with Hg-free de-ionized water, dried in a Class-100
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laminar flow hood in a clean room, and triple bagged in large clear plastic bags for transport to
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the field. In block 1 and block 2, soil evasion measurements were made as described; for
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comparison, in block 3, the effect of soil evasion on isotopic composition of ambient
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atmospheric THg(g) at the forest floor was assessed by placing the inlet tubes, without chambers,
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within 1cm of forest floor.
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2.2.4. Precipitation - Precipitation samples were collected on an event basis during late
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summer and fall 2010 using manual collection methods developed by the University of Michigan
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Air Quality Laboratory (Landis and Keeler 1997), hereafter referred to as manual event
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precipitation. In short, clean manual single-event sampling trains were deployed prior to
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forecasted precipitation and retrieved upon completion of an event (after Landis and Keeler
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1997). The sample train consisted of a borosilicate glass funnel (collection area 191 ± 9 cm2)
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connected to a borosilicate glass s-tube vapor-trap housed within a Teflon adapter that secured
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the funnel to a fluorinated polyethylene collection bottle (similar to Landis and Keeler 1997,
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Gratz et al. 2010). In order to obtain enough sample for mercury isotopic analysis, three
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completely separate sampling units were deployed approximately 2 meters above the ground on
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a freestanding tripod in an open field. All parts of the sampling train were cleaned by
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submersion in a 10% HNO3 bath at 75ºC for at least 12 hours; subsequently, sample bottles were
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filled with 5% BrCl and allowed to soak for at least 24 hours. All materials were rinsed
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copiously with Hg-free de-ionized water after each cleaning step, and ultimately set to dry in a
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Class-100 laminar flow hood in a clean room. All sample bottles were deployed with a 20ml
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aliquot of 1% HCl (trace metal grade, verified to be Hg-free) in order to preserve samples in the
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field and prevent volatile losses (Landis and Keeler 1997). After collection, samples were
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brought to 0.5% HCl by volume, further oxidized with 1% BrCl by volume (verified to be Hg-
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free), and allowed to react in a dark cold-room at 4ºC for at least 1 month prior to analysis for
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mercury concentration.
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2.3. Mercury Concentration Analysis, Calculations, and QA/QC
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Foliage, forest floor, and mineral soil sub-samples (~ 50 mg) were analyzed for total
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mercury concentration by combustion at 850ºC and analysis by cold-vapor atomic absorption
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spectrometry (CVAAS; MA-2000, Nippon Instruments). For foliage samples, linear standard
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curves had coefficient of variation (r) ≥ 0.9995; standards recovered 95-110% across all
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analyses; the reporting limit was 0.5 ng Hg. The second source standard recovered 92-102%;
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continuing calibration verification (CCV) standards recovered 93-106%; continuing calibration
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blanks (CCB) were all below detection limit (0.05 ng Hg). Reference materials were recovered
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within 103-109% (Apple Leaves, NIST SRM 1515). Duplicate sample analyses agreed to within
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a relative percent difference (RPD) of 4.2%. For forest floor samples, linear standard curves had
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coefficient of variation (r) ≥ 0.9999; standards recovered 96-105% across all analyses, with the
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lowest standard at the reporting limit of 0.5 ng recovering at 98-112%. The second source
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standard recovered 97.1 – 99.4%; continuing calibration verification (CCV) standards recovered
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92-103%; continuing calibration blanks (CCB) were mostly below detection limit (0.05 ng Hg),
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with maximum CCB of 0.1 ng. Reference materials were recovered within 95-100% (Olive
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Leaves, BCR 062). Triplicate analyses agreed to within 1.1% RSD (relative standard deviation).
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For mineral soil samples, the linear standard curve had a coefficient of variation (r) ≥ 0.9999;
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standards recovered 100-101%, with a reporting limit of 0.25 ng. The second source standard
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recovered at 89%; continuing calibration verification (CCV) standards recovered 95-106%;
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continuing calibration blanks (CCB) were all below detection limit (0.05 ng Hg). Reference
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materials were recovered within 97-99% (Apple Leaves, NIST 1515) and 97-100% (NRCC
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MESS-3). Duplicate analyses agreed to within a relative percent difference (RPD) of 5.2%.
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The concentration of total mercury (THg, all forms of mercury) was determined for
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manual event precipitation samples following a method modified from EPA Method 1631
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(USEPA 1998) using an automated CVAAS (MA-2000 with liquid delivery system; Nippon
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Instruments). Samples were analyzed with quality control that included independent primary
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standard (NIST SRM 3133) and secondary source (Inorganic Ventures MSHG-10ppm), as well
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as continuing calibration verification standards and continuing calibration blanks run before and
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after every sample. The linear standard curve had a coefficient of variation (r) ≥ 0.9999, and all
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standards recovered within 100-110%. The second source standard recovered at 98-101%; CCV
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standards recovered within 97-105%; CCBs were all below detection limit; on a mass basis, the
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method detection limit was 2.5 pg per 5 ml sample (i.e., 0.5 ng/L); the level of quantification was
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5 pg per 5 ml sample (i.e., 1 ng/L). Matrix spikes were recovered within 98 – 103%. Duplicate
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analysis of separately UV-treated sample aliquots ranged from 2.7 – 19% RPD (10.2 ± 7.9%,
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1SD, n = 5); duplicate analysis of individual UV-treated sample aliquots ranged from 0.1 -16.3%
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RPD (5.2 ± 4.8%, 1SD, n = 9); note that entire precipitation samples could not be UV-treated
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because of effects on mercury isotopic composition. The three funnel replicates for each event
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were analyzed individually before compositing by event date for isotopic analysis; field
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triplicates agreed within 3.1 – 8.1% RSD. Bottle blanks, and funnel blanks with complete
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sample train and bottle installed, were all below detection limit (0.5 ng/L Hg, n = 4 each).
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Atmospheric and evasion THg(g) samples collected on gold-coated bead traps could not
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be sub-sampled prior to preparation for isotope analysis. After the mercury on each set of eight
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gold-coated bead traps had been desorbed into a 1% KMnO4 trapping solution (see Methods:
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Sample Preparation for Isotopic Analysis), a small aliquot was analyzed for concentration and
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the total quantity of mercury collected was calculated. Average atmospheric and soil evasion
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THg(g) concentration (ng/m3) for each sample was determined by dividing total mercury mass
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(ng) by total volume of air sampled (m3), as recorded using DTMs.
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2.4. Sample Preparation for Isotope Analysis
2.4.1. Offline Combustion of Foliage, Forest Floor, and Soils - Foliage and soils were
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prepared by two-stage combustion with inline trapping of released mercury in order to remove
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matrix interferences and concentrate mercury for isotopic analysis. This apparatus and method
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was based on a previous study (Smith et al. 2005) that was modified for lower Hg concentration
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samples by Biswas et al. (2008). In this study, we further modified the procedure to allow for
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slow, controlled, complete combustion of organic matrices (i.e., vegetation) while preventing the
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generation of condensation of organic combustion residues upstream of the trapping media.
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Two tube furnaces (Lindberg Blue M) were placed in series and housed a custom-made
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continuous linear quartz glass combustion tube. Dry samples of foliage, forest floor, or soil were
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weighed into ceramic boats (up to 3.5 g for foliage and forest floor, up to 5.0 g for mineral soil),
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encased in sodium carbonate, calcium hydroxide, and alumina powders, all of which were
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cleaned by pre-combustion at 750ºC for 6 hours in a muffle furnace (Nippon Instruments Inc.).
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Sample boats were placed within the first furnace for combustion, and a 5 cm loosely packed
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plug of quartz wool was placed within the second furnace to create turbulent flow, retard passage
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of particulates, and aid in complete decomposition of gases released during combustion. The
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combustion furnace was heated from 25ºC to 750ºC over 7 hours during a series of ramp and
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dwell periods (Supplemental Table S8), and the decomposition furnace was held at 1000ºC for
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the duration of the procedure. A flow of ~12 ml/min O2 was injected into the inlet of the
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combustion tube prior to the combustion furnace, and a flow of ~6 ml/min O2 was injected into
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the midpoint of the combustion tube prior to the decomposition furnace. The right-angle outlet
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of the combustion tube was connected to a borosilicate sparger with medium frit that was placed
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into a 24g solution of 1% KMnO4 (m/m) in 10% sulfuric acid (v/v) (hereafter, referred to as 1%
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KMnO4) that oxidized elemental mercury released during combustion to Hg(II). Importantly, all
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exposed portions of the combustion tube were wrapped in heat tape and aluminum foil and
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maintained at or above 100ºC for the duration of the combustion. Upon completion of the
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combustion program, trap and sparger were removed from the combustion unit, and the sparger
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was rinsed into the 1% KMnO4 trap with 1.5% hydroxylamine hydrochloride (v/v) (USEPA
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1998), which completely removed residues from the sparger tip while maintaining oxidizing
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conditions within the trapping solution to retain Hg(II). To quantify offline combustion
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recovery, the 1% KMnO4 trap was reduced with hydroxylamine hydrochloride, and a small
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aliquot was diluted with 1% BrCl for analysis by AAS using methods similar to precipitation
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concentration measurements (see Methods: Mercury Concentration Analysis; Nippon
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Instruments Inc.). Reduced combustion trap solutions were concentrated into secondary traps (as
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small as 8g of 1% KMnO4), both to achieve the required 1-5 ng/g concentration required for
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isotopic analysis and to remove matrix interferences within the combustion trap solution.
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Finally, the mercury concentration of a small aliquot of transfer trap solution was measured in
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order to determine full procedural recovery, as well as to allow matching of standard and sample
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concentrations for isotope analysis.
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Offline combustion performance was monitored with procedural blanks, combustion of
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reference materials, and by the percent recovery of samples based on independent sample
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mercury concentration analyses (see Methods: Mercury Concentration Analysis, Calculations,
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and QCQA). Procedural blanks consisted of ceramic boats packed only with combustion
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powders, and were performed at a frequency of at least every 8 samples; average procedural
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blanks were 0.009 ng/g (± 0.008, 1SD, n = 9) prior to transfer and 0.027 ng/g (± 0.013, 1SD, n =
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9) subsequent to transfer. We combusted NIST SRM 1515 (Apple Leaves, 44 ± 4 ng/g) with
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recovery ranging from 88.5 to 99.3% (96.6 ± 3.6%, 1SD, n = 9); we also combusted several
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aliquots of BCR 062 (Olive Leaves 280 ± 20 ng/g) with recovery ranging from 95.1 to 105.7%
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(102.9 ± 6.9%, 1SD, n = 3; Supplemental Table S2). Mean percent recovery for samples of
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foliage was 92.7% ( ± 3.2%, 1SD, n = 12) for forest floor was 97.9% ( ± 5.9%, 1SD, n = 18) and
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for mineral soils was 94.3% ( ± 2.7%, 1SD, n = 9).
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2.4.2. Desorption of Atmospheric and Evasion THg(g). Atmospheric and soil evasion
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THg(g) samples collected on gold-coated bead traps in the field were concentrated into a 1%
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KMnO4 solution for isotope analysis through a multi-step process, modified from the procedure
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used by Gratz et al. (2010) in order to increase efficiency, and decrease the procedural blank.
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First, each gold-coated bead trap was dried for 1 hour in a gold-filtered stream of Argon gas at
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0.5 L/min. Next, each set of eight gold-coated bead traps was flash heated to ~500ºC with a
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nichrome wire and released mercury was re-trapped on a secondary analytical gold-coated
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sorbent trap by modifying the flowpath of the AAS used for concentration measurements (MA-
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2000, Nippon Instruments Inc.), with back-up trap in place, and while monitoring for
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breakthrough of mercury, which was negligible. Desorptions onto the secondary analytical trap
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were performed with gold-filtered ambient clean room air, at a flow rate of ~ 0.5 L/min. The
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secondary analytical trap was then thermally desorbed into a 24 g 1% KMnO4 trap solution by
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slowly heating to 550ºC using a nichrome wire connected to a Variac transformer and a
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programmable temperature controller (Temperature Controller R/S, Barnant Company) over 3.5
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hours in a gold-filtered Argon gas-stream (8 mL/min). The 1% KMnO4 trap solution was then
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measured for mercury concentration, and subsequently transferred and concentrated into a
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secondary 1% KMnO4 trap solution resulting in final concentrations ranging from 2.9 – 4.4 ng/g
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for isotope analysis, following the same procedure as outlined for the offline combustion trap
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solutions (see Methods:Offline-Combustion of Foliage, Forest Floor, and Soils). The
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performance of the desorption procedure was monitored with procedural blanks and procedural
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standards analyzed in the same manner as samples. Procedural blanks and procedural standards
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began with sets of eight blanked gold-coated bead traps; traps for the procedural standards were
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loaded with 30 and 80 ng Hg (NIST SRM 3133) per set to span the range of THg(g) collected.
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Average procedural blanks were 0.55 ng (± 0.40, 1SD, n = 3). Recovery of procedural standards
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using NIST SRM 3133 ranged from 89.2 – 94.2% (91.1 ± 1.9%, 1SD, n = 6), and were not
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significantly fractionated relative to NIST SRM 3133 bracketing standards (Supplemental Table
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S2).
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2.4.3. Purging and Trapping of Precipitation Samples - Mercury in manual event
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precipitation samples was purged and trapped into 1% KMnO4 solution for isotope analysis. Up
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to 1 L of previously acidified and oxidized precipitation was brought to room temperature and
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then weighed into a clean 2 L borosilicate glass media bottle, combining precipitation samples
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from all three collection funnels for each event. Samples of less than 1 L were diluted up to 1 L
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with similarly acidified and oxidized Hg-free de-ionized water, and thoroughly mixed. Samples
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were treated with 300 µl of 30% hydroxylamine hydrochloride (USEPA 1998), capped tightly,
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and allowed to react for 1 hour. We verified that all solutions turned clear, indicating complete
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destruction of free halogens, before initiating the purge and trap procedure. Once reaction was
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complete, a clean Teflon stir bar was added to the 2 L media bottle, and a three-port Teflon
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transfer cap was securely attached. The first port was reserved for the delivery of 100 ml of 5%
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SnCl2 via peristaltic pump without compromising the closed system. The second port was used
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to draw gold-filtered clean-room air into the sample solution through a sparger with a medium
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frit in order to purge reduced mercury into the headspace of the 2 L media bottle. The third port
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was an outlet tube that connected to another sparger and medium frit within a narrow elongated
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borosilicate glass trap (~2 cm diameter, ~30 cm height) containing 5.5 g of 1% KMnO4 trapping
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solution. The sidearm of the transfer cap of the 1% KMnO4 trap was connected in series to an
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inline soda lime trap to neutralize acid fumes, a flow meter, and a diaphragm vacuum pump.
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Once the purge and trap system was assembled, moderately vigorous stirring was initiated with a
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magnetic stir plate, and vacuum was applied to the system, achieving a flow through the system
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of ~ 0.7 L/min. After flows were established, the 5% SnCl2 was dripped into the sample solution
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at a rate of ~10 ml/min, and sample solutions were purged for 3 hours. If there was more than 1
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L of sample for a single precipitation event, the sample was split into 2 separate purge and trap
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bottles, and then purged sequentially into a single 1% KMnO4 trap; one precipitation event
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sample had to be split into 3 separate bottles. All connections and tubing in contact with liquid
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sample or part of the gaseous sample flow path were made of Teflon or borosilicate glass, and
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were cleaned in 10% HNO3, rinsed copiously between samples, and dried completely before use.
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Media bottles were cleaned by submersion in 10% HNO3 at 75ºC for at least 8 hours, rinsed
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copiously with Hg-free de-ionized water, soaked in 5% BrCl for at least 12 hours at room
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temperature, rinsed again, and set to dry in a Class-100 laminar flow hood in a clean room.
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Upon completion of the purge and trap procedure, the sparger in the 1% KMnO4 trap was rinsed
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into the solution following the same procedure as outlined for the offline combustion trap
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solutions (see Methods:Offline-Combustion of Foliage, Forest Floor, and Soils).
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To quantify purge and trap recovery, mercury concentration of the 1% KMnO4 trap
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solution was determined as outlined for the offline combustion trap solutions (see Methods:
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Offline-Combustion of Foliage, Forest Floor, and Soils), scaled to total precipitation volume,
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and compared to total mass of mercury within the three collection funnel samples from each
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event. The final concentration of Hg in precipitation samples in 1%KMnO4 for isotope analysis
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was 1.1 - 1.7 ng/g. We monitored the performance of the purge and trap system with procedural
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blanks (0.012 ± 0.005 ppb, 1SD, n = 3) and procedural standards. Procedural standards consisted
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of 12 ng of Hg (NIST SRM 3133) split between one, two, or three 2 L media bottles, and diluted
326
to 1 L with Hg-free de-ionized water acidified and oxidized the same as sample solutions.
327
Procedural standards were then purged, trapped, and analyzed in the same manner as samples.
328
Procedural standard recovery was 90.2 ± 2.0% (1SD, n = 3); there was no apparent difference
329
among samples purged singly, or in multiple sequential aliquots; purge and trap procedural
13
330
standards using NIST SRM 3133 were not significantly fractionated relative to NIST SRM 3133
331
bracketing standards (Supplemental Table S2).
332
333
334
2.5. Mercury Isotope Analysis
2.5.1. MC-ICP-MS - Mercury from natural environmental samples was concentrated
335
into a 1% KMnO4 solution and analyzed for isotopic composition with a multiple collector
336
inductively coupled plasma mass spectrometer (MC-ICP-MS; Nu Instruments) using continuous
337
flow cold vapor generation with Sn(II) reduction (Lauretta et al. 2001, Blum and Bergquist
338
2007). We employed an arrangement of faraday cups that allows for simultaneous collection of
339
masses 196, 198, 199, 200, 201, 202, 203, 204, 205, and 206. Instrumental bias was corrected
340
using an internal standard (NIST SRM 997, 205Tl/203Tl ratio of 2.38714) and strict sample-
341
standard bracketing with NIST SRM 3133 Hg standard. Isobaric interferences from 204Pb were
342
monitored to allow correction using mass 206; however, interferences from 204Pb were always
343
negligible. On-peak zero corrections were applied to all masses.
344
Samples stabilized in 1% KMnO4 were pre-treated with an aliquot of 30% hydroxylamine
345
hydrochloride equivalent to 2% by weight, and then diluted to 1-5 ppb Hg, with standard
346
concentrations matched to within 5% of sample concentrations. Mercury in sample solutions
347
was reduced inline with 2% SnCl2 (USEPA 1998), and the mixed solution was delivered to a
348
phase separator. Reduced mercury was stripped from solution with a counter-flow of Ar into
349
which a dry Tl aerosol was introduced using a desolvating nebulizer (Aridus, Cetac).
350
351
We report isotopic compositions as permil (‰) deviations from the average of NIST
SRM 3133 bracketing standards using delta notation:
352
353
(Eq 1) δxxxHg (‰) = ([xxxHg/198Hg)unknown / (xxxHg/198Hg)NIST SRM 3133] – 1) * 1000
354
355
where xxx is the mass of each mercury isotope between 199Hg and 204Hg. We use δ202Hg to
356
assess MDF. Whereas 204Hg has a larger mass difference relative to 198Hg, 202Hg has a greater
14
357
natural abundance and no isobaric interferences (Blum and Bergquist 2007). MIF is reported as
358
the deviation of the isotope ratio from the theoretically predicted values based on the kinetic
359
mass-dependent fractionation law and measured δ202Hg (Blum and Bergquist 2007). MIF is
360
reported in “capital delta” notation (∆xxxHg) in units of permil (‰) (Equation 2), and is well
361
approximated for small ranges in delta values (≤ 10 ‰) by Equation 3:
362
363
(Eq 2) ∆xxxHg (‰) = 1000 x ({ln[(δxxxHg/1000) + 1]} – β x {ln[(δ202Hg/1000) +1]})
364
365
(Eq 3) ∆xxxHg (‰) ≈ δxxxHg – (δ202Hg x β)
366
367
where xxx is the mass of each mercury isotope 199, 200, 201, 204 and β is a constant (0.252,
368
0.502. 0.752, 1.492, respectively; Blum and Bergquist 2007).
369
2.5.2. Reporting Analytical Uncertainty - We characterized the uncertainty of mercury
370
isotope measurements using a secondary standard that is widely distributed (UM-Almaden), as
371
well as with procedural standards for each natural environmental matrix (Blum and Bergquist
372
2007). To characterize the reproducibility of the mass spectrometry, we measured the isotopic
373
composition of UM-Almaden several times (3-7x) at each analytical concentration within each
374
analytical session. The 2SD uncertainty was then generated from the average of session
375
averages for each delta value; there was no difference in reproducibility for concentrations
376
ranging from 1-5 ng/g during the analytical sessions reported in this study. We do not report
377
internal precision (i.e., the uncertainty on the 24 ratio measurements made during a single pass of
378
a single sample or standard preparation) because it was typically much lower than the external
379
reproducibility of the method (Blum and Bergquist 2007). For procedural standards, we
380
calculated uncertainty as 2SE of the average of session averages. A separate estimate of
381
uncertainty was made for each sample matrix: for foliage, forest floor, and soil samples, the
382
procedural standard was generated by offline combustion of NIST SRM 1515 (Apple Leaves);
383
for THg(g) samples, the procedural standard was generated by loading NIST SRM 3133 onto
15
384
gold-coated bead traps; and for precipitation samples, the procedural standard was generated via
385
purge and trap of NIST SRM 3133; all as described in Methods: Sample Preparation for Isotope
386
Analysis. We represent the uncertainty of samples with the uncertainty (2SE) of associated
387
procedural standards; however, where the 2SE of procedural standards was less than the 2SD of
388
UM-Almaden, we instead represent sample uncertainty using UM-Almaden. The uncertainty
389
associated with the isotopic composition of UM-Almaden, procedural standards and reference
390
materials, and environmental samples are presented in Supplemental Tables S2-S7.
391
392
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