Comparison of Digestion Media for Speciation of Mercury in the

Despite many studies of mercury speciation in marine sediments, fish, and waters, studies on the very important aquatic plants are rare. This paper de...
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Environ. Sci. Technol. 1997, 31, 3325-3329

Comparison of Digestion Media for Speciation of Mercury in the Seagrass Zostera marina L. Followed by Quantitation by Hydride Generation-Atomic Fluorescence Spectrometry MATTHEW A. MORRISON AND JAMES H. WEBER* Chemistry Department, Parsons Hall, University of New Hampshire, Durham, New Hampshire 03824

Despite many studies of mercury speciation in marine sediments, fish, and waters, studies on the very important aquatic plants are rare. This paper describes use of the same digestion regime to compare three reagents for their ability to quantitate inorganic mercury (Hg(II)) and monomethylmercury cation (MeHg+) in estuarine eelgrass. The study highlights excellent results with the new hydrophobic digestion reagent alkaline tetrabutylammonium bromide (KOH/Bu4NBr). Because scientists must be particularly careful in evaluating new speciation methods in the absence of a certified reference material, we confirmed the accuracy of the speciation results in several ways. The confirming results include high recoveries of spiked samples, finding only traces of mercury upon redigestion of solid residue from the speciation procedure with concentrated HNO3, excellent agreement between sums of Hg(II) plus MeHg+ concentrations from speciation experiments and total mercury (Hgtot) concentrations from digestions in concentrated HNO3, and good agreement between quantitation of MeHg+ by vacuum distillation and KOH/Bu4NBr digestion. Digestions in KOH/Bu4NBr yielded average eelgrass concentrations (dry weight) of 39.7 ng/g for Hg(II) and 2.78 ng/g for MeHg+. The 42.5 ng/g sum agreed well with Hgtot of 44.7 ng/g from HNO3 digestion.

Introduction Seagrasses support complex food webs based on their physical structure and primary production capacity and rank with mangroves and coral reefs as some of the most productive coastal habitats in the world (1). The seagrass Zostera marina L. (eelgrass), which ranges along both coasts of the United States, Alaska, Japan, and Europe (2), covers 46% of the area of the Great Bay Estuary, NH (3). Eelgrass grows in extensive, submerged meadows, with blades up to 2 m in length (2). It slows current flow, acts as a filter for suspended solids, and provides habitat for juvenile fish and invertebrates (3). In a comparison of three nursery habitats in a Cape Cod estuary, Heck et al. (4) found that eelgrass supported the greatest species richness and had the greatest estimated production of macroinvertebrates. In addition, eelgrass is an important primary producer in the detrital food chain in coastal waters (3). * Corresponding author e-mail: [email protected]; telephone: 603-862-2527; fax: 603-852-4278.

S0013-936X(97)00396-9 CCC: $14.00

 1997 American Chemical Society

Despite the importance of seagrasses and other marine plants, very little research exists on their role on cycling of mercury in coastal waters. In a preliminary study, Puk and Weber (5) speciated mercury in eelgrass and in the saltmarsh cord grass Spartina alterniflora. They found Hg(II) and Me2Hg in both species, but found MeHg+ only in eelgrass using methods of lower sensitivity than ones in the current paper. Several studies (6-12) determined Hgtot concentrations in S. alterniflora, but only three groups (5-7) sought MeHg+. In a study of mercury in a different seagrass, Pe´rez (13) determined Hgtot concentrations in leaves of Thalassia testudinum. For this paper, we developed a rapid and simple digestion procedure for mercury speciation in eelgrass that should be applicable to mercury speciation in a broad range of marine biota. Comparison of 4 M KOH/0.25 M tetrabutylammonium bromide (KOH/Bu4NBr), 4 M HCl, and 25%(w/v) KOH in methanol (KOH/MeOH) demonstrated that the new and hydrophobic reagent KOH/Bu4NBr gives the best results. The digests are amenable to direct mercury speciation using hydride generation cold vapor atomic fluorescence spectrometry (CVAFS). Because of the absence of a certified reference material for marine plants, we compared our results by the use of several cross-checks. Our finding that 6.5% of the Hgtot concentration in eelgrass was MeHg+ has important implications for cycling of mercury in coastal waters.

Experimental Methods Reagents, Glassware, and Plasticware. Doubly deionized, distilled water (Corning Mega-pure still) was used in all experiments except for initial rinsing of eelgrass samples. HCl was trace metal grade (Fisher Scientific), and other reagents were of analytical grade. Aqueous 12% NaBH4 (m/v) was prepared from 99% sodium tetrahydridoborate (Aldrich) as described previously (5). SnCl2 (20% w/v) in HCl (10% v/v) was prepared (14) by stirring SnCl2‚2H2O in concentrated HCl for 5 min as the solution was brought to ca. 75 °C or until the SnCl2 dissolved completely. The solution was cooled to room temperature and diluted to the appropriate volume with H2O. Calibration standards (50 ng of Hg/mL) for Hg(II)Cl2, MeHgCl, and Me2Hg were made by successive dilution of stock solutions as previously described (15). Calibration standards were prepared weekly. All glassware and plasticware was cleaned prior to use following the procedure of Horvat et al. (16). The first step, which was done once, consists of heating for 24 h in concentrated HNO3 (85 °C). The items were cooled and rinsed with H2O and were heated overnight in 1% HCl (70 °C). For general cleaning, items were soaked in 7% HNO3 (10% of concentrated) overnight and then heated in 1% HCl as above. As a final step, all items were rinsed with H2O and dried in an oven (70 °C). CVAFS Determination. The mercury content of samples was determined by cold vapor atomic fluorescence spectrometry (CVAFS) using the system and operating procedures described by Morrison and Weber (15). The method of standard additions was used for eelgrass digests because of matrix interferences that reduce the sensitivity. One sample for each set of replicate digests was used to make the standard addition curve that was used for quantitation of the entire set. Prior to reduction of acidic samples with NaBH4, a slight excess of OH- (as 4 M KOH) was added to make the solution in the hydride generation flask basic (pH 12-13). This was necessary to maintain the proper sensitivity for MeHg+ during these analyses. The same system and procedures were used for reduction with SnCl2 in 1.2 M HCl except that 0.2 mL of the reagent was added to 5 mL of H2O.

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Eelgrass Collection and Processing. Samples of eelgrass (Zostera marina L.) were collected offshore of Jackson Estuarine Laboratory from the Great Bay Estuary, NH. Whole plants were pulled, rinsed briefly in the saltwater, and placed in Ziploc bags for transport. Before laboratory processing, they were rinsed with reverse osmosis water until the rinse was clear and triple rinsed with distilled H2O. The blades and stems were separated from the roots and rhizomes, cut into roughly 1-cm pieces, mixed, and frozen in acid-cleaned containers until use. Prior to digestion, eelgrass (ca. 10 g wet) was frozen in liquid nitrogen and ground to a fine powder in a mortar and pestle. To obtain a large homogeneous sample of ground eelgrass, a beaker, kept cold with dry ice, was used for intermediate storage and mixing. Aliquots for digestion and dry/wet ratio were taken from the beaker and placed into preweighed vials. Those samples not digested immediately were frozen in the sealed digestion containers until use. The average dry/wet ratio for eelgrass was ca. 0.11 for the 10/ 29/96 collection and ca. 0.13 for the 1/13/97 collection. Spike Recovery Experiments. Mercury calibration standards (50 ng/mL) were added to the eelgrass samples or to the reagents in the digestion vials to test spike recovery. For recoveries from eelgrass, the samples were vortexed for 1 min after the addition of spikes and equilibrated at room temperature for 30 min prior to addition of the reagent(s) for digestion. For digestion in concentrated HNO3, spikes consisted of 7.5 ng of Hg(II) and 5.0 ng of MeHgCl; for vacuum distillation, the spike was 10.0 ng of MeHgCl; and for the serum vial headspace, it was 0.25 or 0.50 ng of Me2Hg. For the speciation methods, the spike was 10.0 ng each of Hg(II), MeHgCl, and Me2Hg. Digestion in Concentrated HNO3 for Hgtot Concentration. Wet eelgrass (ca. 2 g) was placed into a 25-mL glass volumetric flask, and 10 mL of concentrated HNO3 was added. Acidcleaned glass marbles were used to cap the volumetric flasks, allowing dissipation of brown NOx fumes generated during digestion. The eelgrass was digested 8 h (or overnight) at room temperature and then placed in a room temperature sandbath. The temperature was brought to 85 °C during the first hour, 100 °C during the second hour, and mildly refluxed at 120 °C for the final 2.5 h of digestion. Digestion continued until the headspace in the volumetric flasks was colorless, signifying complete removal of NOx. After the flasks cooled, the digests were diluted to 25 mL with H2O, and aliquots (500 µL) were taken for determination of Hgtot concentration by CVAFS using 0.2 mL of SnCl2 reductant. Vacuum Distillation for MeHg+. Vacuum distillation for the determination of MeHg+ in eelgrass was carried out as described previously (15), except for the following modifications. About 2 g of ground eelgrass was digested in 4 mL of a solution containing 2.5 M H2SO4, 0.5 M NaCl, and 0.01 M CuCl2. The distillate was rinsed from the vacuum line traps with ca. 20 mL of 0.01 M Na2S2O3 buffered with 0.1 M Na2B4O7‚10H2O. The distillate was rinsed into a 50-mL glass beaker and boiled down to ca. 10 mL in a 200 °C sandbath prior to determination by CVAFS (500 µL aliquot). This method improves the detection limit of the vacuum distillation by decreasing the volume of the distillate without loss of MeHg+. Serum Vial Headspace Analysis for Me2Hg. This method was modified from a technique used in our lab to purge the headspace over sediment slurries for Hg0 and Me2Hg determination (17). After grinding, ca. 2 g of wet eelgrass was transferred to a 20-mL glass serum vial. The vial was sealed with a Teflon-faced rubber septum (Supelco) and crimp cap. The vial was kept at room temperature for 2 h and then heated for 5 min in a 60 °C sandbath. Connection was made to the CVAFS system via inlet and outlet needles puncturing the septum. The vial was heated and purged (standard CVAFS

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TABLE 1. Detection Limits for Hg(II) in Digestion Methods of This Study

reagent

Hg content of the blank (pg)

detection limit (pg) of blank

blank as % of eelgrass signal

detection limita in eelgrass (ng/g dry)

HNO3 4 M HCl KOH/MeOH KOH/Bu4NBr

20 75 40 80

10 15 30 30

10-15 25 15 15-25

2.0 1.2 2.4 2.4

a Based on detection limit, the fraction of digest used, and dry weight of eelgrass digested.

operating procedure) for 7 min to remove volatile Hg compounds from the headspace of the vial. Standard Digestion Procedure for Speciation of Hg. About 2 g of wet, ground eelgrass was placed into a centrifuge tube. Glass centrifuge tubes (40 mL with Teflon-lined caps) were used for acid digestions, and FEP centrifuge tubes (Nalgene, 30 mL, Oak Ridge) were used for alkaline digestions. The reagent used for digestion (5 mL) was added to the eelgrass, the sealed centrifuge tube was vortexed (VWR Vortex II) for 1 min, and the eelgrass was digested at room temperature for 7 h. The samples were sonicated (Branson 2200 L) at 60 °C for 1 h, cooled, and centrifuged at 6000 rpm (International Clinical Centrifuge, Model CL) for 8 min. Aliquots (250 µL) for CVAFS determination were taken directly from the centrifuge tubes. This method was used for digestion of eelgrass in 4 M HCl and 25% (w/v) KOH/MeOH. A slight modification was needed for digestion in a solution containing 4 M KOH and 0.25 M Bu4NBr (KOH/Bu4NBr) due to phase separation of the reagent. Immediately before addition of the reagent (5 mL) to eelgrass, it was vortexed for 10 s to homogenize it. Following digestion, it was necessary to add 5 mL of H2O before centrifugation in order to get a good separation of the remaining plant material from the solution. This was likely due to disappearance of the phase separation in the diluted reagent. To obtain the same fraction of the digest as for the other speciation methods, 500 µL aliquots were taken for analysis by CVAFS. Redigestion of Residual Eelgrass Solids from the Speciation Methods. Following digestion of eelgrass using the speciation methods described above, the solution was removed with a pasteur pipet, and 5 mL of H2O was added. The centrifuge tube was vortexed 30 s to mix and centrifuged for 8 min. After removal of the solution, this step was repeated. The remaining plant material was rinsed into a 25-mL volumetric flask with 10 mL of concentrated HNO3 and digested using the procedure described earlier for Hgtot.

Results Calibration Data and Detection Limits. Morrison and Weber (15) discussed calibration data and detection limits for the hydride generation cold vapor atomic fluorescence spectrometry (HG-CVAFS) system in a recent paper. Correlation coefficients (R 2) for the slopes of calibration curves are generally greater than 0.99, and detection limits (3σ), while running samples, are 13 pg for Hg(II) and 0.3 pg for MeHg+ and Me2Hg. The detection limit for MeHg+ and Me2Hg in eelgrass is 24 pg of Hg/g. (All concentrations of mercury compounds in eelgrass in this paper are based on eelgrass dry weight.) The 80-fold increase occurs because the CVAFS aliquot is 1/20th of the digest volume and the mass of eelgrass digested is ca. 0.25 g dry weight. The detection limit for Hg(II) in eelgrass varies with digestion method because it is present in varying amounts in the procedural blanks due to the digestion mixtures. Table 1 lists the Hg(II) procedural blank, the detection limit (3σ) for replicate blank runs, the blank peak area as percent of total

TABLE 2. Procedural Spike Recoveries of Mercury Compounds (% ( 1σ) reagent recoveries digestion method concn HNO3 vacuum distillation serum vial headspace 4 M HCl KOH/MeOH KOH/Bu4NBr

Hg(II) 7.2a

98.0 ( na na 106 ( 1a 78.1 ( 4.2 83.0 ( 2.7

eelgrass recoveries Me2Hg

MeHg+ 0 97.5 ( 3.1b na 0 113 ( 3.7 105 ( 4.5

Hg(II)

nad

13a

94.6 ( na na 83.4 ( 5.7 90.3 ( 5.0 105 ( 3.2

na 91.0 ( 3.0c na 118 ( 7.0 54.3 ( 1.2

a Spike recovery is for Hg + tot from added Hg(II) and Hg(II) from decomposed MeHg . Weber et al. (17). d na, none added.

b

MeHg+

Me2Hg

0 85.3 ( 10.4 na 84.8 ( 17 85.2 ( 7.4 94.4 ( 6.5

na na 90.7 ( 6.5 na 34.7 ( 4.0 26.9 ( 0.3

Reagent recovery is from previous work (15). c Data from

TABLE 3. Concentrations of Mercury (ng of Hg/g Dry Weight ( 1σ)a in Eelgrass from Great Bay Estuary, NH digestion method

collection date

Hg(II)

MeHg+

Hgtotb

4 M HCl 4 M HCl KOH/MeOH KOH/MeOH KOH/MeOH 4 M KOH/Bu4NBr 4 M KOH/Bu4NBr concn HNO3 concn HNO3 vacuum distillation vacuum distillation vacuum distillation 4 M KOH

10/29/96 01/13/97 10/02/96 10/29/96 01/13/97 10/29/96 01/13/97 10/29/96 01/13/97 10/02/96 10/29/96 01/13/97 01/13/97

31.6 (( 1.2) 26.1 (( 1.1) 22.2 (( 1.0) 36.2 (( 4.0) 24.9 (( 2.2) 41.3 (( 1.5) 38.1 (( 1.5)

1.55 (( 0.03) 2.27 (( 0.55) 3.01 (( 0.68) 2.60 (( 0.26) 3.47 (( 0.81) 2.88 (( 0.34) 2.67 (( 0.10)

33.1 (( 1.2) 28.4 (( 1.2) 25.3 (( 0.3) 38.8 (( 4.0) 28.3 (( 2.3) 44.2 (( 1.5) 40.8 (( 1.5) 52.5 (( 1.7) 38.9 (( 1.5)

22.8 (( 2.3)

2.70 (( 0.27) 2.50 (( 0.84) 2.51 (( 0.34) 1.11 (( 0.36)

24.0 (( 2.3)

2 M KOH/Bu4NBr

01/13/97

22.8 (( 1.2)

2.19 (( 0.25)

21.2 (( 0.9)

a

All determinations are in triplicate except for those from 10/02/96 and the KOH/MeOH determinations from 10/29/96, which are in duplicate. b For all methods except concentrated HNO , the Hg + 3 tot concentration is by the addition of Hg(II) + MeHg concentrations.

peak area for a typical eelgrass sample, and the detection limit for Hg(II) in eelgrass. The detection limit in eelgrass, calculated as above, is based on the detection limit for the procedural blank, the fraction of the total digest analyzed in a sample run, and the dry weight of eelgrass digested. Although the Hg(II) content of the blank varies from 20 to 80 pg, and can be as much as 25% of the signal for an eelgrass sample run, this has little effect on the detection limit because of the high reproducibility of the CVAFS determinations. Procedural Spike Recoveries. We employed two types of spike and recovery experiments in this paper (Table 2). The first, a procedural spike recovery from the reagents only, represents the stability of each Hg species under the chemical and physical conditions of the digestion. The second is a procedural spike recovery in the presence of eelgrass that accounts for changes produced during its digestion. Recoveries of Hg(II) after digestion in concentrated HNO3 in the absence or presence of eelgrass are nearly 100%. These values include recoveries for added Hg(II) and added MeHg+ that decomposed to Hg(II) (eq 1).

CH3Hg+ + H+ f CH4 + Hg2+

(1)

These results and others discussed below confirm that HNO3 in our procedure converts MeHg+ quantitatively to Hg(II). Recoveries of MeHg+ from the alkaline speciation reagents and vacuum distillation range from 98 to 113% in the absence of eelgrass and from 85 to 94% in its presence. Quantitative decomposition of MeHg+ (eq 1) in 4 M HCl in the absence of eelgrass strongly suggests that it is inappropriate as a speciation reagent. A reason for the 84.8% recovery of MeHg+ from 4 M HCl in the presence of eelgrass is not obvious. Apparently chemicals formed during eelgrass digestion prevented demethylation of MeHg+.

Nearly complete recovery (91%) of Me2Hg occurred for the serum vial headspace method in the absence and presence of eelgrass. We confirmed decomposition of Me2Hg in 4 M HCl to MeHg+ (eq 2) and then to Hg(II) (eq 1) (data not in Table 2).

(CH3)2Hg + H+ f CH4 + CH3Hg+

(2)

Incomplete recoveries of Me2Hg from KOH/Bu4NBr and KOH/ MeOH in the absence and presence of eelgrass (three of four experiments) did not result from its decomposition of Me2Hg to MeHg+ or Hg(II). Apparently we lost Me2Hg due to its volatility (BP ) 95 °C) during our procedure. Concentrations of Mercury in Eelgrass. Table 3 gives the concentrations of Hg in eelgrass samples collected from the Great Bay Estuary, NH, from October 2, 1996, to January 13, 1997. We chose three reagents for the speciation of Hg. Hgtot concentration is the sum of the Hg(II) and MeHg+ concentrations for the speciation methods. Digestion in 4 M HCl gives Hgtot concentrations of 33.1 ng/g (10/29/96 sample) and 28.4 ng/g (1/13/97 sample). An average MeHg+ concentration of 1.92 ng/g for both collection dates is obtained. Digestion in KOH/MeOH yields Hgtot concentrations of 25.3 (10/2/96 sample), 38.8 ng/g (10/29/97 sample), and 28.3 ng/g (1/13/97 sample) and an average of 3.09 ng/g for MeHg+ over these three collection dates. The reagent KOH/Bu4NBr resulted in the highest speciation Hgtot concentrations of 44.2 ng/g (10/29/96 sample) and 40.8 ng/g (1/13/97 sample). The average concentration of MeHg+ for this digestion reagent is 2.78 ng/g for the above collection dates. We tested the above speciation results by two independent methods. We determined Hgtot concentration by eelgrass digestion in concentrated HNO3 and the concentration of its MeHg+ component by vacuum distillation (15). Hgtot concentration by digestion in HNO3 decreased from 52.5 ng/g

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(10/29/96 sample) to 38.9 ng/g (1/13/97 sample). The average concentration of MeHg+ determined by vacuum distillation was a constant 2.55 ng/g over the three collection dates. Results from the KOH/Bu4NBr digestion method best agreed with the two independent methods. Absence of Me2Hg in Eelgrass. In spite of favorable recoveries for the alkaline digestions and excellent recoveries for the serum vial headspace (90.7 ( 6.5%), we found no Me2Hg in the eelgrass samples for these collection dates. This is contrary to earlier work on eelgrass by Puk and Weber (5), who found 9.7 ng/g fresh weight for Me2Hg, but the effects of seasonal trends on the speciation of Hg could explain this discrepancy.

Discussion Application of ANOVA Calculations. We tested the statistical validity of observed differences in Hg concentrations for all comparisons in this discussion by two-way ANOVA using collection date and digestion method as variables and 95% confidence level. All p-values are from these tests unless otherwise indicated. Hgtot Concentration by Concentrated HNO3 Digestion. When digesting environmental samples for the determination of Hgtot concentration, many researchers use a combination of strong acid(s) and a strong oxidizing agent such as BrCl (18), H2O2 (10), or KMnO4 (7). The mixture helps ensure that the environmental sample completely releases Hg compounds and that all MeHg+ demethylates to Hg(II) (eq 1) (19). We tested digestion in concentrated HNO3 in the absence of an additional oxidizing agent for complete recovery of Hg(II) from added Hg(II) and MeHg+ in two ways. First, procedural spike recoveries of Hgtot as Hg(II) (Table 2) including demethylation of MeHgCl resulted in recoveries 98.0% in the absence of eelgrass and 94.6 % in its presence. Second, in a more sensitive experiment, we directly searched for MeHg+ in HNO3 digests by hydride derivatization with NaBH4. In early experiments, NaBH4 reduced most MeHg+ to Hg0 after we derivatized an aliquot of unmodified HNO3 digest. In contrast, after adjusting the HNO3 aliquot in the hydride-generating flask to pH 12-13, MeHgH formed quantitatively. We analyzed duplicate eelgrass digests and eelgrass spike recoveries and one reagent spike recovery with both NaBH4 derivatization and SnCl2 reduction. We found no MeHg+, and Hgtot concentrations obtained by these two methods were identical, except that the spike recoveries were ca. 5% higher with derivatization by NaBH4 than by SnCl2. Both of these results confirm demethylation of MeHg+ to Hg(II). Speciation of Hg by 4 M HCl Digestion. We obtained consistently lower values for Hgtot concentrations in eelgrass by digestion in 4 M HCl (p ) 1 × 10-7 vs HNO3 digestion) (Table 3) and redigestion of the residual plant material from the initial 4 M HCl digestion in concentrated HNO3. The residual eelgrass (1/13/97 collection date) contained 12.1 ( 1.9 ng/g (based on original sample). Addition of it to the speciation total of 28.4 ng/g (Table 3) increases Hgtot concentration to 40.5 ng/g, which is consistent with Hgtot concentration of 38.9 ng/g from concentrated HNO3 digestion. This confirms that the standard digestion procedure with 4 M HCl does not quantitatively release Hg from eelgrass. The MeHg+ concentration is not significantly different from the vacuum distillation value (p ) 0.09) due to the large standard deviation of the vacuum distillation values for 10/29/96, but is significantly lower than the MeHg+ concentration obtained by digestion in KOH/Bu4NBr (see below). Speciation of Hg by 25% (w/v) KOH/MeOH Digestion. We tested the digestion reagent KOH/MeOH because researchers often use it for the speciation of Hg in fish and sediments (16, 18, 20, 21) followed by derivatization by ethylation (18, 20) or hydride generation (21). Hgtot concentration in eelgrass with this method (Table 3) is lower

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than that from digestion in HNO3 (p ) 0.02), but redigestion of the remaining plant material leaves this discrepancy unexplained by providing a value of only 1.5 ( 0.8 ng/g. MeHg+ concentrations obtained by digestion in KOH/MeOH and vacuum distillation are not significantly different (p ) 0.82). Speciation of Hg by 4 M KOH/0.25 M Bu4NBr Digestion. Researchers have used 20% aqueous tetramethylammonium hydroxide for determinations of alkyllead compounds (22) and mercury compounds (23) in fish. We reasoned that the tetramethylammonium ion would be insufficiently hydrophobic (24) and decided to test KOH/Bu4NBr. The reason for use of Bu4NBr was that Francois and Weber (25) found that the addition of Bu4N+ resulted in effective hydride derivatization of tributyltin cation from eelgrass extracts. The new digestion reagent KOH/Bu4NBr gave the best results for speciation of Hg in eelgrass. This result occurs because tetraalkylammonium salts with large alkyl groups serve as phase transfer catalysts by transporting OH- into hydrophobic sites to increase alkaline hydrolysis of the plant material (24). The last two results in Table 3 confirm that neither 4 M KOH alone nor 0.25 M Bu4NBr in 2 M KOH are capable of quantitatively releasing Hg from eelgrass. It is the combination of the two that promotes alkaline hydrolysis that is necessary for the quantitative release of Hg from eelgrass. ANOVA results for Hgtot concentration by KOH/Bu4NBr digestion compared to HNO3 digestion over two sampling dates show a significant difference between the methods (p ) 0.01), but it is due entirely to the 10/29/96 sample. In contrast, Hgtot concentration from the two methods for the 1/13/97 eelgrass sample are statistically identical, and the KOH/Bu4NBr method gives an average of 95% of the Hgtot concentration by HNO3 digestion. Redigestion of the remaining plant material from the 1/13/97 sample yielded an additional 3.9 ( 2.1 ng/g and supported the conclusion that KOH/Bu4NBr releases almost all Hg from eelgrass. In addition MeHg+ concentrations for KOH/Bu4NBr digestion and vacuum distillation are not significantly different (p ) 0.35). Because digestion in KOH/Bu4NBr provided the best results of the speciation methods, we compared concentrations of Hgtot and MeHg+ to those from the other two speciation methods using two-way ANOVA. The Hgtot concentrations for the KOH/Bu4NBr digestion are higher than ones from digestion in 4 M HCl (p ) 6 × 10-7) and KOH/ MeOH (p ) 0.048), and the MeHg+ concentration is higher than that from digestion in 4 M HCl (p ) 0.002). There is no significant difference between MeHg+ concentrations obtained by KOH/Bu4NBr digestion and those obtained by KOH/ MeOH digestion (p ) 0.74). Comparison to Hg Concentrations in Other Marine Plants Studies. The concentration of Hgtot in Zostera marina determined in this study by KOH/Bu4NBr digestion ranges from 40.8 to 44.2 ng/g. This value for Hgtot concentration is within the range of other studies on marine plants. As cited in a review (26), Lyngby and Brix found a 10 ng/g Hgtot concentration in the leaves of Z. marina from the Limfjord in Denmark (27). Pe´rez (13), in studies of leaves from the seagrass Th. testudinum in Venezuela, found Hgtot concentration ranges from 4 to 6 ng/g for an unpolluted site and from 16 to 30 ng/g at a polluted site. Hgtot concentrations in Spartina alterniflora from salt marshes are typically 20-150 ng/g (5-12). In this study, the concentration of MeHg+ in eelgrass is 2.67-2.88 ng/g, which is 6.5% of Hgtot. Puk and Weber (5), in the only other published measurement of MeHg+ concentration in eelgrass, found a concentration of 13.7 ng/g (fresh weight). In the only observation of MeHg+ in S. alterniflora, Gardner et al. (6) found a concentration near their detection limit of 1-2 ng/g. The concentration of MeHg+ in eelgrass in this paper is similar to the 2.2-7.4 ng/g (dry weight) concentrations we

found in salt marsh sediments of a tributary to the Great Bay Estuary, NH, during the summer of 1996 (15). Z. marina has been named as a suitable candidate for the monitoring of heavy metal contamination in coastal areas (26, 27), and our eelgrass concentrations of MeHg+ may reflect the mean 4.7 ng/g (dry weight) sediment concentrations in the Great Bay Estuary (15). Implications. The 6.5% MeHg+ component of Hgtot determined in eelgrass from the Great Bay Estuary may provide a crucial intermediate in the bioaccumulation of MeHg+ by marine organisms. MeHg+ is typically less than 1% of the Hgtot in marine sediments (6, 28, 29), but for the primary consumers rises to 3-10% in snails (7); 0.1-35% in filter feeders such as mussels (6, 28); and 45-72% in fiddler crabs (7), which feed on plant detritus. The percent MeHg+ rises again to ca. 100% in most fish, especially at higher trophic levels (6, 18, 28), and is 54-100% in the muscle tissue of birds (6). Despite considerable recent effort to determine Hg speciation in shellfish, fish, and sediments (19), seagrasses are little studied. This study and the work of other researchers confirm that seagrasses, as primary producers, merit considerable effort in studies of the cycling of Hg in marine systems.

Acknowledgments We thank Ryan Davis of Jackson Estuarine Laboratory for the collection of eelgrass samples from the Great Bay Estuary. We thank Chris Bauer for help with statistics. Grant BCS9224717 from the National Science Foundation partially supported this work.

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Received for review May 5, 1997. Revised manuscript received July 29, 1997. Accepted July 31, 1997.X ES970396N X

Abstract published in Advance ACS Abstracts, September 15, 1997.

VOL. 31, NO. 11, 1997 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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