Environ. Sci. Technol. 1994, 28,514-520
Degradation of Methyl Bromide in Anaerobic Sediments Ronald S. Oremland,' Laurence G. Miller, and Frances E. Strohmaler
U S . Geological Survey, Mailstop 465,
345 Middlefield Road,
Methyl bromide (MeBr) was anaerobically degraded in saltmarsh sediments after reaction with sulfide. The product of this nucleophilic substitution reaction was methanethiol, which underwent further chemical and bacterial reactions to form dimethyl sulfide. These two gases appeared transiently during sediment incubations because they were metabolized by methanogenic and sulfate-reducing bacteria. A second, less significant reaction of MeBr was the exchange with chloride, forming methyl chloride, which was also susceptible to attack by sulfide. Incubation of l4C-1abeled methyl iodide as an analogue of MeBr resulted in the formation of 14CH4and 14C02and also indicated that sulfate-reducing bacteria as well as methanogens metabolized the methylated sulfur intermediates. These results suggest that exposed sediments with abundant free sulfide, such as coastal saltmarshes, may constitute a sink for atmospheric MeBr. Methyl bromide (MeBr), an agricultural fumigant, is a trace constituent of the troposphere (1-3). Concern exists with regard to its transport from the troposphere to the stratosphere. At these high altitudes, MeBr photolysis to Br causes ozone destruction, and on a molar basis, Br is as much as 100-foldmore efficient at scavagingozone than is Cl(4). Methyl bromide and its ozone oxidation product BrO have been detected in the stratosphere (5-9). In addition to anthropogenic sources, MeBr is produced biologically (10-13), as are other volatile alkylated bromine compounds (14-18). Halons (bromine-containing halocarbons used in fire extinguishers) can constitute an additional anthropogenic source of stratospheric Br (19, 20). Therefore, the importance of MeBr to the stratospheric Br pool and the quantitative extent of the anthropogenic MeBr source are uncertain. The atmospheric burden of MeBr is thought to be about 200 X lo6 kg, and based on kinetic models of its reaction with hydroxyl radicals, an atmospheric residence time of about 2 years is calculated. This yields an annual global flux of 100 X lo6kg. It is currently believed that natural sources comprise about two-thirds of this flux, but there is considerable uncertainty in these estimates (21-23). Adding to these uncertainties is the lack of information about other sinks for MeBr beside atmospheric hydroxyl radicals. Nucleophilic reactions of MeBr in water include its hydrolysis to methanol and its exchange with other halides (24, 25). Nonetheless, the oceans are supersaturated with respect to MeBr and constitute a net source to the troposphere (1). Soils, however,potentially have both chemical and microbiological MeBr consumption reactions, which include hydrolysis (26)and bacterial oxidation (27, 28). In the course of experiments with methaneoxidizing soils, bacterial consumption of MeBr under anaerobic conditions was observed (28). We report that, in anoxic sediments, MeBr can undergo nucleophilic substitution reactions with sulfide to form methylated
-
* A u t h o r to whom a l l correspondence should b e addressed. (415) 329-4463. 514
Environ. Sci. Technol., Vol. 28, No. 3, 1994
FAX:
Menlo Park, California
94025
sulfur gases, which in turn are metabolized by methanogenic and sulfate-reducing bacteria. Hence, MeBr removal is ultimately under biological control. We also observed that MeBr undergoes a less significant exchange reaction with chloride to form methyl chloride, which is also susceptible to attack by sulfide. Experimental Section Sediment Slurries. Reducing sediments (containing 1-2 mM free sulfide) from a saltmarsh located in Palo Alto, CA (29,301 were homogenized in a Waring blender under N2 with artificial San Francisco Bay water (31)and dispensed in 20-mL aliquots into serum bottles (57 mL) which were crimp-sealed under Nz.The final ratio of sedimenkwater was 1:4. Controls were autoclaved at 120 OC and 250 kPa for 60 min and allowed to cool to room temperature before a final headspace flushing with Nz. Sodium molybdate (- 10mM) and 2-bromoethanesulfonic acid (BES; 20 mM) were added to selected live slurries to inhibit sulfate-reducing and methanogenic bacteria, respectively (32). Triplicate sets of slurries were prepared for each of the experimental variables. Methyl bromide (10-12 pmol) was added to bottles by syringe injection, and the slurries were incubated in the dark at -20 "C with constant rotary shaking (200 rpm). To study the chemical reaction of sulfide with MeBr, cooled and Nzflushed bottles containing autoclaved slurries (see above) were injected with a sulfide solution (20 pmol mL-'; 1mM final concentration) and placed on a shaker for several hours. Another set of autoclaved slurries were sealed with 10pmol of MeBr and received 0,2.5,5, and 10-pmolsulfide additions. They were shaken for 20 h (200 rpm), after which time their residual MeBr content was analyzed. The consumption of MeBr and the formation of product gases were monitored by gas chromatographic analysis (see below) of syringe-sampled bottle headspaces, The longevity of MeBr exposed to autoclaved sediment slurries having less chemical reactivity was also investigated. Agricultural soil (28) was homogenized with deionized water (1:l) and allowed to stand for 12 h to remove exchangeable ions. The preparation was decanted, and fresh deionized water was added to restore the original volume. Aliquots (10 mL) of the preparation were added to serum bottles containing an additional 10 mE of deionized water, sealed under Nz, autoclaved, and injected with MeBr as described above. Radioisotope Experiments. Anaerobic saltmarsh sediment slurries were injected with 1.3 pCi of 14C-labeled methyl iodide (sp act. = 59 mCi/mmol; purity = 96.4%: Amersham Inc., Chicago,IL) dissolved in 100pL of acetone. Formation of 14CH4 and 14C02were measured by gas chromatography in conjunction with gas proportional counting (33). Experiments with Static Chambers. To measure the ability to undisturbed saltmarsh sediments to metabolize MeBr, laboratory experiments were conducted with sealed cores which functioned as both surface sediment collection devices and incubation vessels (381"
-
This article not subject to U.S. Copyright. Published 1994 by ?he American Chemical Socle?]/
The upper 7-8 cm of saltmarsh sediments were collected intact with circular Plexiglass cores (wall thickness, 0.6 cm; height, 16 cm; inner diameter, 7.6 cm), which were molded to a circular piece of Plexiglassat the top (diameter, 8.8 cm). The bottom sides of the chambers were tapered to facilitate placement into the sediment, and two holes existed (at the top and halfway up the side) to allow the displaced air to escape. A large rubber stopper (size 14) was maneuvered up underneath the captured material to seal it into the chamber. The holes were plugged with serum stoppers, and the sediment was removed intact from the saltmarsh. Immediately after recovery, the large rubber bung was taped in place, and the stoppers were removed from the holes. Approximately 330 mL of sediment was collected in each chamber, leaving a roughly equivalent volume of gas phase. Chambers were transported back to the laboratory, were stoppered shut, and were either flushed with N2 or sealed under air. Airincubated chambers received frequent injections of 02 (usually 320-400 pmol daily) in an attempt to retain an oxic atmosphere. Methyl bromide (120 pmol) was injected into the chambers at the start of the experiment, and its disappearance and the accumulation of product gases were followed by gas chromatography (see below) by syringe removal of headspace samples through the stoppered port at the top of the chamber. Selected controls did not receive MeBr and were incubated under N2 or air. Chambers were incubated statically in the dark at -20 “C. Experiments with Methanogenic Bacteria. The dimethyl sulfide- (DMS) utilizing methylotrophic methanogen “GS-16” was grown in crimp-seal test tubes as described previously (34). Methyl bromide (10pmol) was added to selected tubes incubated with or without DMS as a substrate (-5 mM). Controls consisted of MeBr additions to tubes with sterile medium (without DMS) or deionized water. A growth tube of methanogens inoculated into DMS medium without MeBr was run for comparison. Analysis of Volatiles. Gas chromatography was employed to measure headspace concentrations of MeBr, methyl chloride (MeCl), CH4, DMS, methanol, and methanethiol (MeSH). A flame ionization detector and a poropak column were used for CH4, methanol, and high concentrations of MeBr (30). Methanol in the liquid phase of slurries was determined (after centrifugation on a microfuge to remove particles) by direct injection of 10 pL of supernatant into a glass-wool-lined injection sleeve (to retain salts), which preceded the column. A 63Ni-electron capture detector equipped with a krytox column was employed for analysis of MeBr and MeCl(28,31). A flame photometric detector (Hewlett-Packard Series 5730) and a Super Q (Alltech ASSOC.,San Jose, CA) Teflon column (0.32 cm X 45.7 cm) was used for analysis of MeSH and DMS. The column was ramped from 30 to 210 “C (40 OC m i d ) to elute the sulfur gases. We were unable to achieve a chromatographic separation between MeBr and MeSH on any of our columns. Because the 63Nidetector responds to both MeSH and MeBr (it is about 25-fold more sensitive to MeBr than MeSH), unless indicated otherwise, we corrected the MeBr obtained on the electron capture instrument by subtracting the MeSH values obtained with the flame photometric detector. Pure gases (MeBr, MeCl, and MeSH) in lecture bottles were obtained from Matheson Gas Co., NJ, and the appropriate dilutions were made up as standards in N2-containing serum bottles. The reagent-grade volatile liquids DMS and dimethyl disulfide (DMDS) were obtained from Aldrich Chemical Co. (Mil-
waukee, WI), and standards were prepared by injecting microliter quantities of the compounds (usually 5-10 pL) into N2-sealed serum bottles (57mL). After hand-warming the bottles, the liquids were entirely vaporized. Standards for sulfur gases were run immediately, and new standards were prepared whenever required. The quantity of MeBr, MeC1, MeSH, and DMS dissolved in the liquid phase of the slurries (volume = 18 mL) was calculated by determining the fraction of the total present in the headspace by applying Henry’s Law constant (KH)to the equation:
where Fgis the fraction present in the gaseous phase, and Vwand V, are the volumes of the water and gas phases, respectively. The K H values used for MeBr, MeSH, MeC1, and DMS were 0.24, 0.055, 0.48, and 0.125, respectively (35,36).These calculations indicated that the amount of MeBr, MeSH, MeC1, and DMS present in the headspace represented 33,10,49, and 20%,respectively, of the total quantity present. No corrections were made for the “salting out” effect of salinity or for the binding of these compounds to sediment surfaces, and therefore final quantities calculated to be present in the sealed bottles were slightly overestimated. We did not calculate the dissolved fraction of these compounds in the chamber experiments because we did not have accurate estimates of the liquid volumes present, and results are given for only the gas phase.
Results Sediment Slurries. A typical time course incubation of sediment slurries is shown in Figure 1(the experiment was replicated several times). In all cases, near-complete consumption of MeBr occurred in a few days. Methyl bromide consumption was slightly more rapid in live estuarine slurries (Figure 1A) than in autoclaved ones (Figure 1B). In contrast, autoclaved deionized soils did not show evident consumption of MeBr over a 6-day incubation period (Figure 1B). Live slurries had transient accumulations of MeC1, MeSH, and DMS, all of which were consumed to below detectable levels by 10-day incubation, which also coincided with the period of peak methane evolution. In contrast, autoclaved slurries had a persistent level of MeC1, higher and more prolonged abundances of DMS, and did not form CHI. In quantitative terms, MeSH was of greatest importance as an intermediate, followed by DMS, while MeCl had the least significance (Figure 1; Table 1). Slurries in which sulfate reducers were inhibited with molybdate or methanogens were inhibited with BES differed from the uninhibited controls in that they both had higher levels of MeSH at day 8 of the incubation (Table 1). Levels of MeSH declined rapidly after day 8 in the experimental slurries (Figure 1A) as well as in the BESand molybdate-inhibited controls (not shown). This indicated that both sulfate-reducing bacteria and methanogens were involved in MeSH consumption. The products of MeBr degradation at 8 days (MeSH, MeC1, DMS, and CHI) roughly balanced the amount of MeBr consumed in the unamended slurries and autoclaved controls, but the inhibited controls exceeded the quantity of MeBr added by 2.2-2.6-fold, mainly due to the high accumulation of MeSH (Table 1). Molybdate stimulated methanogenesis (26 pmol of CH4 by day 17) while BES caused its inhibition (0.15pmolof CH4 by day 17). Controls Environ. Sci. Technol., Vol. 28, No. 3, 1994
515
Table 1. Products of MeBr Degradation Present in Sediment Slurries after 8-Day Incubations 15
2 0
10
umnl
1
I
V
'
4,
z
addition none MoodBES killed noMeBr
CHI
MeSH
DMS
MeCl
%b
1.54 (0.22) 0.32 (0.07) 0.15 (0) 0.15 (0.04) 0.32 (0.01)
12.0 (1.3) 22.4 (2.1) 28.3 (1.9) 0 (0)
1.3 (0.05) 1.9 (0.4) 1.0 (0) 7.0 (5.0) 0
0.46 (0.12) 0.41 (0.02) 0.55 (0.05) 0.77 (0.04) 0
138 224 258 124
0
Results represent the mean of three samples and parentheses indicate 1 SD. bPercent of MeBr (12 pmol added) recovered as products:
i 0
z vi
% recovered =
a
n
MeCl + MeSH + 2DMS + [CH4+MeBr- CHPMeBr]X 100 MeBr consumed
0.0 0
3
8
9
12
18
15
Tlrne (days)
100000
7
.~
1
1 10000
E t
k 1
- 1.5
-
0)
l5
c w
autoclaved sol1
0
T
I*
.* T
0
10.5 I2
, _-J .
1
0
12
0
16
20
Days
vi
I n
i0.0 Time (days)
Flgure 1. Consumption of MeBr (A) (12 pmol added) and the accumulation of MeCl (+), MeSH (V), DMS (A), and CH4 (0) durlng incubation of anoxic sediment slurries. Symbols represent the mean values of three slurries (bars Indicate f l Sd). The absence of bars indicates that the error is smaller than the symbol. (A) Live saltmarsh slurries. (B) Autoclaved saltmarsh slurries and autoclaved deionized soils; (A)MeBr (soil).
incubated without MeBr produced only 0.23 pmol of CH4 after 17-day incubation (not shown), which was about 26fold less than slurries incubated with MeBr (Figure 1A). Methane recovered in the unamended slurries (6 pmol; Figure 1A) could account for about half of the MeBr removed, with the remainder presumably in the C02 fraction (see below). However, we always observed that MeBr initially inhibited methanogenesis before resulting in stimulation, and a typical result from another experiment is shown as a semilog plot (Figure 2). These observations mean that methanogenesis occurred at the expense of the methylated sulfur intermediates, rather than direct attack of the MeBr. In all the experiments, controls with MeBr had trivial (or undetectable) levels of MeCl (52.5 nmol), MeSH (I0.4 nmol), or DMS (I0.15 nmol) present during the incubation (Table 1;time course not shown). We did not detect the presence of significant methanol either in the vapor (I10 nmol) or liquid phases in any of our incubations (with or without MeBr), including autoclaved controls. 516
4
Environ. Sci. Technol., Voi. 28, No. 3, 1994
Flgure 2. Production of methane In saltmarsh slurries incubatedwlthout MeBr (D),with MeBr (A), with MeBr and BES (A),and autoclaved with Symbols represent the mean values of three experlmental MeBr (0). samples, and bars Indicate f 1 SD. The absence of bars indlcates that the error is smaller than the symbol.
Considerable differences were evident between experiments with regard to the behavior and extent of production of the methylated sulfur gases. For example, in one experiment, DMS in molybdate-inhibited slurries steadily increased to 1.15 pmol by 10 days, after which time it declined and was undetectable by 17 days (Figure 3). In contrast, autoclaved slurries had 0.15-0.3 pmol throughout the incubation, while levels in the uninhibited and BES-inhibited slurries were about 2-fold higher. Consumption of DMS was observed in the uninhibited slurries after 10 days (Figure 3), which coincided with the period when methanogenesis became pronounced (not shown). Addition of 14C-labeledmethyl iodide (MeI) to slurries resulted in the production of 14CH4and 14C02(Figure 4). Production of 14CH4and CH4 in uninhibited slurries was similar, and formation of both the labeled and unlabeled gases were totally inhibited by BES (Figure 4A). Evolution of l4CO2 and COz differed from methane in that a maximum occurred at 5-day incubation, after which levels declined to near zero (Figure 4B). We were unable to detect 14C02in the uninhibited samples upon acidification with 2 mL of 6 N HCl at the end of the experiment. Hence we could not account for the dissolved portion of the 14C02. BES slightly inhibited l4COZproduction (-7 % at day 5) and retarded the decline of both 14C02and C02 after 10-day incubation. A molybdate control demon-
60
300
la* 6 0!
50 -
T
1.20 -
T
A
k
A
-
40 -
E,
,
u)
0
0.00
30
c
m
rn
5
z5
n
i
0.40
0.00
I/ 0
4
0
16
12
20
a, C
m
1
5
2
I
s I
-
20
I
l-
lode 00
4
8
12
18
Days
Figure 3. Accumulation of DMS during incubationof saltmarsh slurries. Symbols: (H) without MeBr, (0)autoclaved wlth MeBr, (A)live with MeBr, (A)BES with MeBr, (V)molybdate with MeBr. Results represent the mean of three experimental samples, and bars indicate f l SD. The absence of bars indicate that the error Is smaller than the symbols.
100
200
1150 m
strated -83% inhibition of 14CO2 and -56% inhibition of COz production at day 5 relative to uninhibited slurries (not shown). This supported the idea that sulfate reducers could oxidize the I4C-labeledintermediates formed from 1%-labeled methyl iodide. Autoclaved slurries amended with 20 pmol of sulfide exhibited a rapid loss of MeBr, resulting in the quantitative formation of MeSH (Figure 5)with lesser amounts (-0.056 pmol) of DMS (not shown). The two methylated sulfur gases accounted for -95% of the observed MeBr reacted. A small amount of MeCl (0.06 pmol) was present at the start of the experiment, but was removed within 0.5 h after the injection of sulfide (not shown). Autoclaved slurries incubated for 20 h with 0, 2.5, 5, and 10 pmol of added sulfide lost 19% ,47 % ,88%, and 97 % of the initially enclosed MeBr (not shown). Metabolic Chambers. MeBr was totally consumed after 6-day static incubation of anaerobic sediments (Figure 6A). Anaerobic chambers produced MeSH (Figure 6A), MeC1, and DMS (Figure 6B) as transient intermediates, and a small quantity of DMDS (-20 nmol) accumulated by the end of the experiment. Controls without MeBr (aerobic and anaerobic) did not form detectable levels of MeSH, DMS, MeC1, or DMDS (not shown). Methane increased during the course of the incubation, exhibiting an accelerated rate after 12 days (Figure 6A). At the end of the experiment, methane concentrations were on average 3.6-fold higher than the amount of MeBr added initially. An anaerobic control without MeBr formed more CH4 by the end of the experiment than those with MeBr (825 vs 442 pmol), indicating that MeBr caused a partial inhibition (46 % ) of methanogenesis. Much less methane ( 140 pmol) was formed in a control which received 0 2 , while sediments which received both MeBr and 02 had similar mean methane levels, but considerable variability around the mean (350 f 300 pmol; not shown). The chambers which had MeBr and received 02 all consumed MeBr, and produced MeSH and DMS. They differed from their anaerobic counterparts in the amount of DMDS they formed (10 f 3 nmol/core), which appeared earlier and persisted until day 12. Due to the high quantity of sulfide present in the sediments, truly oxic conditions could not be maintained in these cores despite our efforts to supply
-
EX 0
- 100 0
2 -50
Y
2
0
0
4
8
12
16
Days Figure 4. Formation of gaseous products during incubation of sattmarsh slurries wlth 14C-labeiedmethyl iodide. (A) I4CH4(open symbols) and CH4 (closed symbols) In live samples (triangles) and BES-inhibited samples (squares). (B) I4CO2(open symbols) and CO2 (closed symbols) In llve samples (triang1es)and BES-inhlbltedsamples (squares). Symbols represent the mean of three experimental samples, and bars indicate flSD. The absence of bars indicates that the error Is smaller than the symbols.
them with 02 (they had a strong sulfide odor throughout the incubation). Effect of MeBr on Growth of Methanogens. The addition of MeBr to cultures of GS-16 strongly inhibited (-99.2 % ) methanogenesis. Controls with DMS as substrate, which lacked MeBr, produced 10.6 pmol of CH4 after 15-day incubation, while the corresponding MeBr tube formed only 0.8 pmol. A control which contained MeBr but was lacking in DMS formed only 0.01 pmol of CH4. No methane was formed (
