Formation and Emission of Volatile Polonium Compound by

The 3-dimethylsulfoniopropionate (DMSP) is the main biogenic precursor of DMS, many marine phytoplankton species (17) and intertidal macroalgae (18) ...
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Environ. Sci. Technol. 2001, 35, 2956-2960

Formation and Emission of Volatile Polonium Compound by Microbial Activity and Polonium Methylation with Methylcobalamin N O R I Y U K I M O M O S H I M A , * ,†,§ LI-XIANG SONG,† SUSUMU OSAKI,‡ AND YONEZO MAEDA† Department of Chemistry and Physics of Condensed Matter, Graduate School of Science, and Radioisotope Center, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan

We observed biologically mediated emission of Po from culture solution inoculated sea sediment extract and incubated under natural light/dark cycle condition or dark condition the emitted Po compound would be lipophilic because of effective collection in organic solvent. Sterilization of the culture medium with antibiotics or CuSO4 completely suppressed growth of microorganisms and resulted in no emission of Po, indicating biological activity of microorganisms is responsible for formation and emission of volatile Po compound. Po emission also occurred when seawater was used as a culture medium. Our finding indicates a possibility of biotic source for atmospheric Po in the environment, which has been believed to be originated from abiotic sources. We compared emission behavior of Po and S in the culture experiments, the elements belong to XVI group in the Periodical Table, and consider that their emission mechanisms involved would be different though the emission of both elements is supported by biological activity of microorganisms. One of the chemical forms of S emitted was confirmed to be dimethyl sulfide (DMS) but that of Po is not known. Methylation experiments of Po with methylcobalamin demonstrated a formation and emission of volatile Po compound. The methylation of Po with methylcobalamin might be related to the observed Po emission in the culture experiments.

to estimate a residence time of atmospheric aerosols and reported mostly 10-300 days of the residence time, which is significantly longer than that obtained from the activity ratio of 210Bi/210Po, showing mostly less than 10 days (1, 5, 6). The longer residence time on 210Po/210Pb has been explained by an additional input of 210Po and particles with secular equilibrium component of 210Pb-210Bi-210Po to the troposphere because a single source of radioactive decay of 222 Rn should give the same residence time for 210Po/210Pb and 210Bi/210Po. In addition to the above sources, biologically supported emission of Po is likely to occur but has not been proved with conclusive verification. Circumstantial evidence suggests the possibility of an unknown biological source of 210Po in the atmosphere: two volatility groups of 210Po compounds in the atmosphere (7), an excess of 210Po in airborne dust collected in seacoast (8) and an increase of dissolved 210Po concentration in seawater during the spring phytoplankton bloom. Phytoplankton and zooplankton in marine environment accumulate Po effectively (9), and bacteria in freshwater has accumulated Po at the laboratory culture experiment (10). Fractionation of 210Po accumulated in microorganisms revealed an association of Po with cellular organic compounds (11, 12). The bioaccumulation would suggest the participation of hydrophobic polonium compound having a high affinity for lipophilic cell structure, although the mechanisms involved are not well understood. Sulfur and selenium, which are in the same column of the Periodical Table of elements as Po, are released from seas and lakes by microbial activity as organic volatile compounds, dimethyl sulfide (DMS) (13) and dimethylselenide (DMSe) (14, 15), respectively. This fact speculates the possibility that Po might be involved in the same biological metabolism as S and Se, and metabolically formed volatile Po mighty be a methylated compound, if biological emission of Po truly occur. In the present work we hypothesized a formation of volatile Po compound in an aqueous phase by biological activity of microorganisms and a subsequent emission of this compound into atmosphere. The primary purpose of this study is to confirm biovolatilization of Po as a potential source of Po to the atmosphere by laboratory experiments using radioactive tracers and then to examine emission behavior of Po by comparing with that of S.

Material and Method Introduction Polonium-210 (half-life 138.4 days) is the member of 238U decay series and ubiquitously found in the environment. As far as 210Po observed in the troposphere researchers have considered several sources, among them radioactive decay of 222Rn that escaped from the earth’s surface would be the major contributor (1) and other sources considered are releases by volcanic eruption (2), savanna combustion (3), sea spray from oceanic surface layer (4), decent of old aerosol from stratosphere (5), and migration of soil dust from the ground (1). An activity ratio of 210Po/210Pb has been utilized * Corresponding author phone: (096)342-3469; fax (096)342-3469; e-mail: [email protected]. † Department of Chemistry and Physics of Condensed Matter, Graduate School of Science, Kyushu University. § Current address: Department of Environmental Science, Faculty of Science, Kumamoto University, Kurokami 2-39-1, Kumamoto, 8608555, Japan. ‡ Radioisotope Center, Kyushu University. 2956

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Culture Medium. 208Po (half-life 2.9 y, alpha emitter 5.11 MeV) was produced by nuclear reaction of 209Bi(p, 2n)208Po with a SF cyclotron at Tokyo University using Bi2O3 as a target material. The Bi2O3 irradiated was dissolved in 1 M HCl, and 208Po was separated from 209Bi by ion-exchange method. The 208Po fraction from the ion-exchange separation was almost dried up, and 208Po tracer solution at concentration of 1.0 × 10-10 M (463 Bq mL-1) was prepared by dissolving the residue in 1 M HCl. The chemical form of Po is PoCl4. Carrier free 35S (half-life 87.2 d, beta emitter 0.167 MeV, H SO ) was 2 4 purchased from Dupont NEN Research Products (USA) and diluted at concentration of ca. 57000 Bq mL-1 with 0.1 M HCl. Basal medium was prepared by dissolution of analytical grade reagents at concentrations of 0.51 M NaCl, 2.3 × 10-3 M MgCl2‚6H2O, 8.7 × 10-4 M Ca(NO3)2‚4H2O, 6.2 × 10-4 M KNO3, 2.5 × 10-4 M MgSO4‚7H2O, 1.3 × 10-4 M KH2PO4, 1.1 × 10-5 M Fe-EDTA, 5.8 × 10-7 M H3BO3, 9.5 × 10-8 M ZnSO4‚ 7H2O, 6.5 × 10-8 M NaMoO4‚2H2O, 4.0 × 10-8 M CuSO4‚ 5H2O, and 50 g L-1 tryptone in deionized water. Each 1 mL of 208Po and 35S tracer solution and 40 mL of the basal medium 10.1021/es001730+ CCC: $20.00

 2001 American Chemical Society Published on Web 06/07/2001

were taken in a 250 mL round flask, and pH of the solution was adjusted to 7 with NaOH. The concentrations of 208Po and S in the culture medium were ca. 2 × 10-12 M and 2.5 × 10-4 M, respectively, and the specific activities of 35S and 208 Po were ca. 1425 Bq mL-1 (ca. 178 Bq µg-1 S) and ca. 11 Bq mL-1 (no stable isotope in Po), respectively. After sterilization at 120 °C for 20 min, 0.5 mL of seashore sediment extract was added to the culture medium. The seashore sediment extract was prepared every time by mixing ca. 100 g portion of wet seashore sediment taken from a stock bottle with 10 mL of deionized water. The seashore sediment with seawater was collected at a tidal zone of Kashii, Fukuoka, on May 7, 1998, and preserved in the plastic bottle at 4 °C in a refrigerator and used throughout the experiments. Culture System. The culture was carried out by immersing the 250 mL round flask in a 30 °C water bath. Room air filtered with a 0.25 µm membrane filter was introduced to the culture medium at a flow rate of 1200 mL h-1 during the culture. The discharged air from the 250 mL round flask was passed through a cotton-filled testing tube (ca. 15 cm length) to remove mist in the air and introduced to three successive 20 mL glass-vial traps. Each vial contained 10 mL of scintillation cocktail, Ultima Gold AB (Packard Ltd.), which is specially prepared for alpha/beta separation measurement by liquid scintillation counting. Main ingredient of the scintillation cocktail is diisopropylnaphthalene, which would give high affinity for hydrophobic compound, such as DMS. Volatile Po and S compounds emitted from the culture medium were collected in the scintillation cocktail in the glass-vial traps. Teflon tubes were used to join up the lines, and no air leak in the culture system was confirmed. Incubation was carried out under natural light/dark cycle condition or dark condition. In the dark condition, the 250 mL round flask, the cotton-filled testing tube, the 20 mL glass-vials, and Teflon tubes were covered by aluminum foil. The control experiments without addition of the seashore sediment extract were carried out as the same manner as the culture experiments. Glycine Betaine, Antibiotics, and CuSO4 Cultures. Culture mediums were prepared as the same manner as mentioned above but added a growth or metabolic inhibitor of microorganisms to verify participation of biological activity of microorganisms in Po volatilization. One of the culture mediums prepared contained 50 mM glycine betaine, which inhibits DMSP degradation, DMSP is a precursor of DMS, and resulted in lower production of DMS (16). The emission behaviors of S and Po were compared in the culture medium containing glycinebetaine. Other culture mediums prepared contained antibiotics, chloramphenicol, and tetracycline at concentrations of 2.5 and 1.2 mg mL-1, respectively, or CuSO4 at concentration of 1000 ppm. Seawater Culture. Each 1 mL of 208Po and 35S tracer solution was taken in a 250 mL round flask and neutralized. Forty milliliters of unfiltered seawater, collected from the seashore of Hakozaki, Fukuoka, on February 19, 1999, was added to the flask. The flasks were incubated at 18 °C under the natural light/dark cycle condition. The culture system explained above was used. Po Methylation. Five milliliters of deionized water and 0.1 mL 208Po tracer solution were taken in a 20 mL glass-vial and sterilized at 120 °C for 20 min. One milliliter of 0.67 mM methylcobalamin, a vitamin B12 analogue (SIGMA, Steimheim, Germany), was added to the sterile solution and neutralized with NaOH. Dried N2 gas or room air passed through a 0.25 µm membrane filter was bubbled into the solution at a flow rate of 500 mL h-1. Discharged air from the vial was passed through a cotton-filled testing tube followed by two successive 20 mL glass-vial traps in which each 10 mL scintillator was contained. The vial was covered with aluminum foil to suppress decomposition of methylcobal-

FIGURE 1. Change in emission rates of S (solid lines) and Po (dashed lines) and microorganisms concentration (b) in the culture medium with time under the natural light/dark cycle condition. The errors for the emission rates were not shown due to the small counting errors on the measurements. The symbols (]) show the control experiment (S and Po). amin by light. The whole culture system was put in an incubator (SANYO MIR-153) controlled at 17 °C. The stability of methylcobalamin was investigated with an UV-vis-NIR scanning spectrophotometer (Shimadzu UV-3100PC) under light and dark conditions at room temperature. A similar culture experiment was carried out using methyl iodide, which is a methylation reagent. Measurements. Growth of microorganisms was followed by optical density measurement at 660 nm on a Hitachi UV spectrometer. Analyses of air in the culture flask were performed on a Shimazu 14A gas chromatograph equipped with a GS-Q column and FID. The radioactivity of 35S and 208Po was measured with a Packard TriCarb 2200 liquid scintillation counter for 100 min for each sample. Peaks of 208Po and 35S appeared completely different positions in the scintillation spectrum. At the optimum regions for 208Po and 35S in the spectrum, background counting rates were 0.13 and 0.047 cps for 35S and 208Po, respectively, and typical counting efficiencies were 68% and 100% for 35S and 208Po, respectively.

Results and Discussion Volatile Po compound as well as S compound was emitted from the culture medium to air in the experiments carried out under natural light/dark cycle and dark conditions with growth of microorganisms. No emission of S and Po and no growth of microorganisms was observed on the control experiments, which did not inoculate the seashore sediment extract. The typical results were shown in Figure 1 (natural light/dark cycle condition) and Figure 2 (dark condition); there is no large difference in the emission patterns of Po and S and in the microorganisms growth. The emission rates of Po and S increased in conjunction with the growth of microorganisms in both culture conditions albeit somewhat behind it. The microorganism concentration reached a maximum at 30-40 h after initiation of the culture, followed by a rapid decrease owing to coagulation and precipitation; brown insoluble material accumulated at the bottom of the flask. The maximum emission rates of Po and S occurred at later than 50 h, and the emission continued until 150-300 h, even after the microorganism’s concentration in the aqueous phase decreased by precipitation. It is evident that the biological activity of microorganisms is responsible for formation and emission of Po and S compounds. No emission was observed in the culture mediums that contained 1000 ppm CuSO4 or antibiotics, chloramphenicol and tetracycline, the culture mediums were clear even at the end of long culture, suggesting a complete inhibition of microorganism growth. The emission maximums of Po and S were usually VOL. 35, NO. 14, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Change in emission rates of S (solid lines) and Po (dashed lines) and microorganisms concentration (b) in the culture medium with time under the dark condition. The errors for the emission rates were not shown due to the small counting errors on the measurements. The symbols (]) show the control experiment (S and Po). observed at different times, mostly the emission of S preceded that of Po as if their emission mechanisms had been different. Emissions of DMS and methanethiol (MeSH) were confirmed by gas chromatographic analyses of the culture air at the same culture condition but without radioisotopes. This suggests that some sulfur elements (SO42-) in the culture medium were emitted in relation to DMS-sulfur cycle. The DMS-sulfur cycle is the common phenomenon in marine environment, and marine microorganisms are deeply concerned in emission (13). The 3-dimethylsulfoniopropionate (DMSP) is the main biogenic precursor of DMS, many marine phytoplankton species (17) and intertidal macroalgae (18) accumulate DMSP as an osmolyte and cryoprotectant, particularly when salinity is high. The formation of DMSP begins with the reduction of incorporated SO42- to methionine followed by a series of biosyntheses to DMSP (19) but little is known for Po metabolism. Cherrier et al. (12) compared biological metabolism of SO42- and Po4+ on bacteria, initial uptake patterns of S and Po were different, and the difference was explained on the basis of the element’s surface reactivity. Po was passively adsorbed onto the negatively charged bacterial cell surface that is preferable to adsorb positive species. The uptake mechanisms may differ; however, cellular distribution patterns of 35S and 208Po were similar, which might suggest similar mechanisms within cells. The metabolic decomposition of grazed DMSP by zooplankton (20) as well as the bacterial degradation (21) of DMSP contributes to the atmospheric emission of sulfur as DMS. A number of aerobic marine bacteria have been isolated that degrade DMSP by an initial cleavage to DMS and acrylate or by an initial demethylation to 3-S-methylmercaptopropionate (MMPA) followed by MeSH production or a further demethylation to 3-mercaptopropionate (MPA) (22). Larger amounts of S were emitted under the natural light/dark cycle condition than the dark condition as shown in Table 1. The percentage of emitted S under the natural light/dark cycle condition was 1 order of magnitude larger than that of the dark condition, while that of the Po was not so different for both conditions. This fact would suggest that phytoplankton species grown in the present culture under the natural light/ dark cycle condition were deeply concerned in S emission; phytoplankton multiplied and produced DMSP, and the DMSP was decomposed to DMS actively in the light period. Furthermore, this fact would suggest a bare possibility that the S metabolic process, DMS cycle, of the phytoplankton species is directly responsible for the Po emission. The volatile S production under the dark condition indicates that sulfate could be assimilated at night, if necessary, with supporting by the expenditure of carbohydrates (23). 2958

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The glycine betaine culture experiment maintained the possibility that the biological metabolism of S and Po are a result from different mechanisms on their volatile behaviors. The addition of glycine betaine to the culture greatly suppressed the S emission but did not suppress the Po emission so much. The released Po/S ratio of the glycine betaine experiment differed from that carried out under the natural light/dark cycle condition but was similar to that of the dark condition as shown in Table 1. The inhibition of DMSP degradation with glycine betaine, a functional analogue and a widely used compatible solute of DMSP, has reportedly substantially lowered production of both MeSH and DMS (16). The phytoplankton species grown in the culture might incorporate glycine betaine, resulting in reduction of DMS production. This fact would suggest that the metabolic processes of Po and S concerning the volatile compound formation are probably different though S and Po are in the same column of the Periodical Table of elements. The chemical form of the volatile Po is unknown but it, like DMS, should have low solubility in aqueous phase and high solubility in organic solvent because of the effective trapping in the scintillation cocktail. The most probable chemical form of the volatile Po compound in the case of our experiments may be alkyl Po with neutral charge, and biological activity accomplished the transformation of cationic Po4+ to volatile Po compound. We assumed that the formation of volatile Po compound occurs in relation to methylation mediated by biological activity of microorganisms such as reported on Hg (24, 25). To examine the possibility of Po methylation, we investigated methylcobalamin-mediated Po methylation. As shown in Figure 3, formation and emission of volatile Po compound was confirmed from the solution in which 208Po and methylcobalamin were contained but not from the solution contained only 208Po. The initial pH of the solution was critical to Po emission; emission occurred at pH 7 and pH 9 but not in acetate buffer solution with pH 4.6. The total amount of Po released (9.1%) is significantly larger compared to that in the culture experiments (Table 1). The emission rate of Po shown in Figure 3 increased during the first 150 h, seemed constant following 150-400 h, and then decreased gradually. The solution in the vial, ca. 6 mL at the beginning, was almost dried up at the end of experiment (900 h). An evaporation of 6.5 mL of water was expected to occur for 900 h judging from the vapor pressure at 17 °C and 500 mL h-1 flow rate of dried N2 gas. Methylcobalamin film was deposited on the inner surface of the glass vial, from the initial water level to the present water level reduced by evaporation, so we considered the change in Po emission rate was caused by water level change. Although the concentration of methylcobalamin would change with evaporation, we simply assumed that the Po emission rate was directly proportional to the water level (the solution volume). The change of Po emission rate corrected for evaporation, multiplied by the volume reduction factor (initial water level/reduced water level), was rather simple, showing an almost linear increase until 350 h and following plateau (Figure 3). The methylcobalamin molecules existed in large excess compared to Po atoms in the solution (108:1). As a rather long time was required to attain the steady-state emission rate of Po, the formation of volatile Po compound might not proceed by a single step reaction. Methylcobalamin is known to be unstable for light and heat (the reagent is sold in a brown bottle preserving below -20 °C), we examined stability for light. After 1-h irradiation of water dissolved methylcobalamin to light, the absorption spectrum showed a new absorption peak at 350 nm suggesting transformation of methylcobalamin to hydroxylcobalamin, and the transformation was completed within 1 h because further irradiation did not show any increase in

TABLE 1. Total Quantity of S and Po in the Culture Solution at the Beginning of the Experiments and Those Emitted during the Experimental Perioda quantity at the beginning culture condition light/dark

dark

glycine betaine antibiotics CuSO4 seawater

methylcobalamin

quantity emitted

S (µg)

Po (Bq)

S (µg)

Po (Bq)

320 (2.5 × 10-4 M) 320 (2.5 × 10-4 M) 320 (2.5 × 10-4 M) 320 (2.5 × 10-4 M) 320 (2.5 × 10-4 M) 320 (2.5 × 10-4 M) 320 (2.5 × 10-4 M) 320 (2.5 × 10-4 M) 31340 (2.4 × 10-2 M) 31340 (2.4 × 10-2 M)

463 (2.6 × 10-12 M) 463 (2.6 × 10-12 M) 345 (1.9 × 10-12 M) 338 (1.9 × 10-12 M) 338 (1.9 × 10-12 M) 318 (1.8 × 10-12 M) 318 (1.8 × 10-12 M) 463 (2.6 × 10-12 M) 324 (1.8 × 10-12 M) 324 (1.8 × 10-12 M) 27.4 (1.0 × 10-12 M)

0.40 ( 0.01 (0.13%) 0.96 ( 0.01 (0.30%) 0.81 ( 0.01 (0.25%) 0.21 ( 0.01 (0.07%) 0.22 ( 0.01 (0.07%) 0.05 ( 0.01 (0.02%) 0

4.0 ( 0.1 (0.9%) 8.1 ( 0.1 (1.7%) 4.2 ( 0.1 (1.2%) 6.4 ( 0.1 (1.9%) 6.8 ( 0.1 (2.0%) 1.6 ( 0.1 (0.5%) 0

0

0

0.08 ( 0.01 (3 × 10-4 %) 0.09 ( 0.01 (3 × 10-4 %)

0.92 ( 0.04 (0.3%) 0.34 ( 0.03 (0.1%) 2.5 ( 0.1 (9.1%)

Po/S emitted (mol/mol) (×10-8) 7.0 ( 0.3 6.0 ( 0.1 3.7 ( 0.1 22 ( 1 22 ( 1 26 ( 5

7.8 ( 1.1 2.8 ( 0.4

a The S and Po concentrations at the beginning and the percentages of emitted are shown in the parentheses. Errors are associated with counting (1σ).

CH3+ because Hg(IV) species formed are unstable. The oxidation state of the Po tracer is IV, which is the most stable oxidation state, suggesting methylation with CH3+ transfer to Po is unlikely to occur. We carried out methylation experiments using methyl iodide as the same manner as methylcobalamin; methyl iodide is a typical regent that performs CH3+ transfer reaction; however, no emission of Po was confirmed.

FIGURE 3. Change in emission rate of Po (solid lines) with time from the solution containing 208Po and methylcobalamin. The dashed lines represent that corrected for evaporation (see in the text). The errors for the emission rates were not shown due to the small counting errors on the measurements. intensity of the peak at 350 nm. The water-dissolved methylcobalamin, however, was quite stable under the dark condition, no change was observed on the absorption spectrum if it was shaded with aluminum foil. The irradiated methylcobalamin revealed no ability to form volatile Po compound. Oxidative decomposition of methylcobalamin by air oxygen was slow because significant difference was not observed on the emission of Po with either room air or N2 gas used as a bubbling gas in the experiments. The large emission of Po in this experimental condition also showed thermally induced decomposition would be slow at 17 °C. The B12 cofactor, with cobalt bonded to the methyl group, is utilized by B12-dependent enzymes of several important metabolic processes and is necessary for living organisms (26). The enzymes that bind methylcobalamin carry out methyl transfer reactions in which the methyl-cobalt bond is cleaved heterolytically, leaving both bonding electrons on the cobalt atom and formally transferring a methyl carbocation, CH3+, two electron reduction of Co(III) to Co(I). In this case, the transfer of CH3+ to nucleophiles occurs, and the nucleophiles are oxidized. Metals with the highest stable oxidation state such as Hg(II), therefore, cannot react with

The other heterolytic cleavage process of cobalt-carbon bond in methylcobalamin results in two-electron reduction of carbon and yields carbanion, CH3- (27). On this cleavage process attack of methylcobalamin by electrophiles such as mercury(II) produces trivalent oxidation state cobalt and methylated mercury, the charge of monomethylmercury becomes +1 as a whole, two times carbanion transfer yields Hg species with neutral charge. The same carbanion transfer process occurring on mercury(II) would be expected for Po(IV) and may be responsible for the Po emission in the methylcobalamin experiments. Methylated Po would be a most realistic candidate for volatile Po compound emitted in this experiment considering the very simple composition of the experimental solution and procedure. The extent of occurrence and concentration of vitamin B12 and its methyl derivative, methylcobalamin, in the aquatic environment is not well-known; however, methylcobalamin found in the environment would be originated from biotic processes. The methylation of Po observed in the present methylcobalamin experiments may be abiotic; however, we would like to say that methylation of Hg(II) and Po(IV) by methylcobalamin in the environment would be necessary to be supported by biological activity of microorganisms which continuously produce and supply methylcobalamin. Seawater culture experiments were carried out under the natural light/dark cycle condition using unfiltered coastal seawater in which only 208Po and 35S tracers were added. Emission of Po compound was observed after 400 h and lasted for more than 12 weeks, while no visible growth of microorganisms occurred throughout the culture period (Figure 4). The high concentration of sulfate ions (784 µg S mL-1) in the seawater decreased the specific activity of 35S VOL. 35, NO. 14, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Change in emission rates of S (b, 9) and Po (O, 0) from coastal seawater with time under the natural light/dark cycle condition. The arrows represents addition of methylcobalamin, 2 µg (V) and 4 µg (V), respectively. The counting errors (1σ) were represented as bars but usually smaller than the size of symbols. and obscured S emission. The Po emission rate decreased as the culture temperature lowered from 18 °C to 4 °C; however, it recovered again with the increase in temperature to 18 °C. LaRock et al. (10) observed a long adaptation time, 10 days, for bacterial mobilization of Po in laboratory culture experiments as we observed the Po emission after 400 h. Addition of methylcobalamin (2 µg) at 1100-1200 h seemed to promote the emission rate of Po temporarily but was not clear at 1600 h though 4 µg methylcobalamin was added. The amount of methylcobalamin added was not large compared with that of total organic carbon originally contained in the seawater, 84 µg at the beginning of the experiment. The emitted Po/S ratios in the seawater culture experiments were similar to that of the culture experiments carried out under the natural light/dark cycle condition (Table 1). The present research demonstrated that formation and emission of volatile Po compound certainly occurred in relation to biological activity of microorganisms in culture medium as well as in natural seawater. The Po methylation with methylcobalamin would offer insight into the key processes responsible for formation of volatile Po compound.

Acknowledgments The present research is partially supported by the REIMEI Research Resources of Japan Atomic Energy Research Institute and by the Grant-in-Aid for Exploratory Research (09874106), The Ministry of Education, Science and Culture, Japan.

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Received for review October 3, 2000. Revised manuscript received April 18, 2001. Accepted April 19, 2001. ES001730+