Biodegradation of Synthetic Base Fluid Surrogates in Gulf of Mexico

Sediment samples from hundreds of meters deep in the Gulf of Mexico were incubated at low temperatures (4 °C) and high hydrostatic pressure in steel ...
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Environ. Sci. Technol. 2006, 40, 5737-5742

Biodegradation of Synthetic Base Fluid Surrogates in Gulf of Mexico Sediments under Simulated Deep-Sea Conditions ALAN H. NGUYEN, DAVID HERMAN, AND DEBORAH J. ROBERTS* Civil and Environmental Engineering Department, University of Houston, Houston, Texas

In this study, an anaerobic marine biodegradability test was adapted to study the fate of synthetic base fluid (SBF) surrogates, ethyl oleate and tetradecene, by deep-sea microorganisms. Sediment samples from hundreds of meters deep in the Gulf of Mexico were incubated at low temperatures (4 °C) and high hydrostatic pressure in steel vessels. Stimulation of indigenous microbial communities to SBF biodegradation was evident in the fact that the rate of removal of ethyl oleate was greater in sediments that had some previous exposure to SBF (first-order decay coefficient k of -0.22 ( 0.02 week-1) compared to unexposed control sediments (first-order decay coefficient k of -0.11 ( 0.02 week-1). When sulfate-linked tetradecene degradation occurred within the test period, the activity could also be modeled as a first-order decay following an initial lag phase, with an average decay coefficient of k ) -0.05 ( 0.01 week-1. This study also revealed that the degradation of SBF surrogates by microorganisms collected from deep-sea sediments was not significantly effected by the hydrostatic incubation pressure.

Introduction Synthetic-based drilling muds (SBM) are made using a synthetic base fluid (SBF) as the mobile phase and are important in difficult deep water drilling operations. The most commonly used SBF are internal olefins (IO) and linear alpha olefins (LAO) (1, 2). SBM combine the technical advantages of oil-based muds and the low toxicity of water-based muds. When drilling mud and the cuttings it carries are returned to the surface, they are passed through separation processes, the drilling mud is pumped back to the mud tank and reused, and the cuttings (with adherent SBF) must be disposed of. The most economical disposal method is direct ocean discharge in compliance with USEPA permit regulations (3). The cuttings and adherent SBF that settle to the sea floor cause an organic rich environment that stimulates sediment microbial activity eventually turning the sediment anaerobic. Therefore, anaerobic biodegradation is expected to be the most significant mechanism for deep-sea SBF attenuation and thus environmental recovery. Concern about the environmental impact of the discharge of drill cuttings by the EPA has resulted in the enactment of rules requiring that all SBF be certified as biodegradable by microorganisms indi* Corresponding author phone: (250)807-8722; fax: (250)807-9850; e-mail: [email protected]. Corresponding author address: School of Engineering, 3333 University Way, University of British Columbia Okanagan, Kelowna, BC, Canada, V1V 1V7. 10.1021/es060873e CCC: $33.50 Published on Web 08/15/2006

 2006 American Chemical Society

genous to the marine sediment before their use in off-shore operations. The objective of this study is to determine SBF biodegradation rates in deep-sea sediments and to establish a priori information for use in a fate model. This is next to impossible to do in situ, so samples must be collected from the deep environment and returned to the lab for study. Deming (4) states that the maintenance of in situ (cold) temperature is more important than maintaining pressure to the study of organisms or the activity of organisms from deep environments. Deming also states that barophilic organisms from cold environments can survive exposure to atmospheric pressure, but the study of their activity should be done at pressure. Deming also points out the important difference between the hydrostatic (water) pressure and hyperbaric (gas) pressure (4). Microcosm incubations of sediments collected from beneath inactive drilling sites in the Gulf of Mexico and spiked with SBF surrogates ethyl oleate (an ester surrogate) and tetradecene (an IO surrogate) or left unamended (as controls) were performed to determine the decay rates of SBF in deepsea sediments. Incubations were performed at 4 °C at atmospheric pressure and at a higher pressure (97, 790, or 1700 psi) depending on the original depth of the sample. The depletion of sulfate and surrogate SBF were monitored.

Experimental Section Sediment Collection. The sediment samples were collected as part of the Gulf of Mexico Comprehensive Synthetic Based Muds Monitoring Program (5) using a box core. The sediments chosen for this study were from three sites: MP299 (65 m water depth; equivalent hydrostatic pressure of 97 psi), GC112 (535 m water depth; 790 psi), and VK916 (1135 m water depth; 1700 psi). Near field sediments were collected from within a 0-100 m radius beneath an inactive drilling platform and were believed to have been exposed to SBF prior to sampling given their proximity to the drilling platform. Far field sediments (3000-6000 meter radius away from an inactive drilling platform) were collected as controls and were not expected to have been exposed to SBF. The sediments were shipped immediately on ice (arriving still cold), stored, and worked with at 4 °C at all times. An initial screening analysis revealed only two near field sediments contained detectable residual SBF (Table 1). Table 1 also presents a brief history of SBF cuttings disposal in the sampled locations. Microcosm Development. The microcosms were developed similarly to the EPA approved closed-bottle test (CBT) (3, 6). In the typical EPA closed-bottle test, sediment is spiked with substrate mixed with seawater and poured into glass serum bottles. The headspace is then flushed of air with N2, and the bottles are sealed with a butyl rubber stopper. The production of gas (CO2 and CH4) are measured as an indication of degradation of the substrate. In this study, the incubation of the samples at the hydraulic pressure representative of the sampling depth was desired (4). The creation of elevated hydrostatic pressures in serum bottles was not deemed safe nor practical, so flexible-walled heat seal bags (Kapak pouches - SealPAK) were used to allow the transfer of hydrostatic pressure from a pressurized incubation chamber into the microcosms. Atmospheric incubations were performed in the typical serum bottle microcosms with crimp cap seals as described by Herman and Roberts (6). Three pressure chambers were constructed for the pressurized incubation of the test and control microcosms, one for each of the sediments tested in this study. Each chamber was made from 1 in. thick stainless steel tubes that were VOL. 40, NO. 18, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. History of SBM Discharges in Sediments sediment

location

depth (m)

pressure at depth (psi)

presence of SBFa (mg kg-1 dry sediment)

MP299

NF FF NF FF NF FF

66 65 535 454 1135 1114

97 96 787 667 1668 1638

not detected not detected 1068 ( 300 not detected 11717 ( 636 not detected

GC112 VK916 a

historyb 966 bbl of IO/LAO cuttings from 1962-2000 5470 bbl IO cuttings last discharge 1997 2510 bbl IO cuttings from Nov to Dec 2001

The SBF in the sediments were extracted and then analyzed with GC/FID. b The history data were taken from Continental Shelf Associates (5).

FIGURE 1. Pressurized incubation chamber. The top and body are not drawn to the same scale. 10 in. tall and had 10 in. internal diameters (Figure 1). Plates made of 1 in. stainless steel fit over the top and bottom of the tubes. The end plates had an extended inner core, which held two O-rings used to form an airtight seal with the inner wall of the stainless steel tube. The top plate had an inlet and an outlet valve for pressurization. Each pressure chamber could hold approximately 110 microcosm bags. The pressure was created by pumping 4 °C water into the chamber until all of the air was removed, closing the outlet valve, and then continuing to pump the water until the pressure gauge read 97, 790, or 1700 psi depending on the original depth of sediment collection. Sediments from NF and FF for each drilling site were prepared for use in the microcosms as follows. (1) Dry weight determination: three 5-g samples of sediment were removed from a sediment container, weighed, and dried overnight at 104 °C. The dry weight was used to determine the wet:dry ratio of the sediment. (2) Batch sediment preparation: the wet:dry ratio was used to calculate the total amount of wet sediment (approximately 2.5 kg) needed, which was then used to make one large batch of sediment. This was mixed with the equivalent of 2000 mg ethyl oleate kg-1 dry sediment or 2000 mg tetradecene kg-1 dry sediment or was not spiked (sediment control). Synthetic seawater (Crystal Sea Forty Fathoms Marine mix, Marine Enterprises International, Baltimore, MD) was added to achieve a final overall wet:dry ratio of 3:1 as described by Herman and Roberts (6). (3) Individual microcosm inoculation: 60 g of wet slurry was then transferred into replicate serum bottles and/or heatsealed plastic bags. The headspace of the serum bottles was flushed with nitrogen gas before sealing with a butyl rubber stopper. Any air bubbles in the bag microcosms were removed before the bags were heat sealed and transferred into pressure chambers for incubation. A total of 30-36 bottles/bags were prepared for each condition. All sample preparation and incubations were performed at 4 °C to prevent any loss of cold adapted organisms. Although the sediments were mixed in the ambient air, the NF sediments contained excess sulfide, which acts as an oxygen scavenger to remove any trace O2 introduced during mixing. The FF sediments were aerobic already, thus mixing in the ambient air would not change the O2 in the sediments greatly. 5738

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Two experiments were performed for quality assurance purposes. The first study determined if SBF removal could be attributed to abiotic factors and ensured that the heatsealed bags would not leak during high-pressure incubation. Shoreline sediment from Galveston Bay that had been sterilized by autoclaving was spiked with ethyl oleate or tetradecene and was divided into two sets. One set was incubated at atmospheric pressure in sealed serum bottles, and the other was incubated at 790 psi in heat-sealed bags. The concentrations of ethyl oleate and tetradecene were monitored over 6 months. A second experiment determined if sediment microcosms made using heat-sealed bags or serum bottles would produce similar results. Tetradecene-spiked shoreline sediment was incubated in sealed serum bottles and in heat-sealed bags. The bags were incubated in a water-filled pressure chamber that was open to the atmosphere in order to exclude pressure as a variable in this study. The control (no substrate amendment), tetradecene, and ethyl oleate amended microcosms used to acquire the data to determine the biodegradation kinetic parameters were established as described above and incubated at 4 °C at atmospheric pressure and/or at the pressure representative of the sample collection depth for 25-70 weeks, depending on the substrate removal progression. Incubations with VK916 NF sediment were repeated once. Sampling and Analysis. Triplicate samples of each treatment were collected immediately after microcosm setup and were analyzed to determine the initial concentration of surrogate and sulfate in the sediments (C0). Thereafter, triplicate microcosms (bottles or bags) were sacrificed each 4-6 weeks for analysis of the concentration of surrogate compound and sulfate (C(t)). At each sampling period the pressure chambers were brought to atmospheric pressure, triplicate microcosms for each condition were removed, and the chambers were pressurized again. Samples for sulfate analysis were centrifuged at 1000 rpm (∼275 × g) for 15 min in 5-mL conical plastic tubes. The supernatant (representing the sediment pore water) was collected, diluted 1:10 or 1:100 with distilled water, and filtered through a 0.2 µm syringe filter (Corning, Acton MA) into Dionex autosampler vials. Sulfate was analyzed using a Dionex DX-100 ion chromatograph with a carbonate/ bicarbonate eluent (Na2CO3 (2.7 mM) and NaHCO3 (0.3 mM)), at a flow rate of 1 mL min-1 through IonPac AG12A and AS12A columns. An ASRS-ULTRA 4 mm suppressor set at 100 mA current was in line before the conductivity detector. A five point external standard curve was run for each set of samples. Blanks and QA check samples were run every ten samples. Analyses for the surrogate SBF were performed by removing 20 g of sediment from each triplicate bottle or bag, spiking this with heneicosane (C21) and hexamethylbezene (HMB) (as internal standards) before sonication-enhanced extraction with dichloromethane (3 × 300 mL) as described by Herman and Roberts (6). The extracted dichloromethane layer was collected, dried over sodium sulfate, and concen-

TABLE 2. Kinetic Data for Substrate Degradationa microcosm

substrate

Co (mg kg-1 dry sediment)

k (week-1)

correlation (r2)

tlag (week)

GC 112 NF 790 psi GC 112 NF atm VK 916 NF average MP299 NF 97 psi MP299 NF atm. MP299 FF GC 112 FF VK 916 FF average GC 112 NF 790 psi GC 112 NF atm. VK 916 NF MP299 NF 97 psi MP299 NF atm. average MP299 FF GC 112 FF VK 916 FF

ethyl oleate ethyl oleate ethyl oleate

2341 2432 7289

1 0.995 0.929

ethyl oleate ethyl oleate ethyl oleate ethyl oleate ethyl oleate

3117 1960 2807 3015 2905

tetradecene tetradecene tetradecene tetradecene tetradecene

2338 2078 2507 2250 2124

2 2 4 2.7 ( 1 2 7 5 3 4 4.2 ( 2 4 4 >30 26 28

tetradecene tetradecene tetradecene

1776 1750 3059

-0.23 -0.2 -0.22 -0.22 ( 0.02 -0.1 -0.12 -0.13 -0.07 -0.13 -0.11 ( 0.02 -0.07 -0.05 -0.04 -0.04 -0.05 ( 0.01 -0.23b -0.05b -0.12b

a Data within a group and averaged together are not significantly different (p ) 0.05). and were not used to determine the k for sulfate reduction.

trated to 2 mL by rotary evaporation. SBF surrogate concentrations were determined using an HP 6890 gas chromatograph equipped with a 30 m HP-5 capillary column. Helium was used as a carrier gas, the injector port temperature was 275 °C, and the oven temperature was initially 100 °C and then was increased to 225 °C at a rate of 2 °C min-1. The column flow was 23.3 mL min-1 helium, and the FID temperature was 275 °C. New 5-point internal standard calibration curves were prepared and analyzed preceding each batch of samples. The detected surrogate concentrations (mg L-1) were converted to the reported surrogate concentrations (mg kg-1) in dry sediments using the calculated wet: dry ratios of the sediment slurry samples. Data Interpretation. Due to the presence of a lag-phase at the beginning of the degradation process, the determination of the decay coefficients from the raw data required preinterpretation. The duration of the lag-phase was first approximated directly from plots of the data. Then, the data points in the decay region of the plots were selected for linearization. The first-order decay equation of each selected point was expressed in its linear form:

ln (C(t))experiment ) -kt + ln (C0)

(1)

The decay coefficient (k) was estimated by finding the linear least-squares solution using the singular value decomposition method. The lag-times (tlag) and decay coefficients (k) were then used as initial parameters for the determination of the lag-times and decay coefficients in the entire data set. In this process, the integrated form of the first-order decay equation (with time delay) and the approximate values of tlag and k were used to generate the theoretical data pairs (C(t), t)theoretical, where

C(t)theoretical )

{

: 0 < t < tlag C0.e-k.(t-tlag) : t > tlag

C0

(2)

The sum of squared errors between the theoretical values and the experimental values were minimized to find the optimum decay coefficient k and the optimum lag-time tlag. The curves in all the figures were generated using eq 2 and the parameters from Table 2. Statistical analyses were performed using Sigmastat software using a students t-test or ANOVA. When an ANOVA analysis showed significant

b

0.982 0.998 0.986 0.991 0.965 0.983 0.991 0.829 0.958 0.971 0.984 0.768

14 10 12

These k values are most likely due to aerobic degradation

differences existed (p ) 0.05), a Holm-Sidak all-pairwise comparison of means was used to determine which means were significantly different.

Results and Discussion The experiment using sterile sediment spiked with ethyl oleate or tetradecene incubated in bags and pressurized in a pressure chamber showed that the SBF surrogates did not leak from the heat seal bags and that the SBF surrogates were not removed by sterile sediments due to abiotic processes (Figure 2a). A statistical comparison of the rate of sulfate removal in bags or bottles (Figure 2b) showed that there was no significant difference (p ) 0.05) in the microbial activity due to the incubation method. The removal of sulfate in microcosms spiked with tetradecene but not in the substrate free controls indicated that sulfate-reducing activity was linked to tetradecene removal in both bags and bottles (Figure 2b). The objectives of the microcosm studies were to determine whether deep-sea microorganisms can remove the SBF surrogate compounds and to determine the kinetics of degradation for use in the development of a fate model. The data could be described with a high degree of fit using typical first-order decay parameters after a lag period. As examples of typical data from the 24 test and control microcosm incubations, the results from incubations of MP299 NF sediment incubated at atmospheric pressure and 97 psi are presented in Figure 3, and those of GC112 NF incubated at atmospheric pressure and 790 psi are shown in Figure 4. The lines in the figure were generated using eq 2, the coefficients (k), and the lag times determined from the data and presented in Table 2. The table also presents the correlation coefficients (r2) for the fit of the theoretical curves to the raw data. The correlation coefficients are mostly above 0.9 suggesting that the data fit very well to typical first-order kinetics with an initial delay or lag. Pressure Effect. There was no effect of hydrostatic pressure on the removal of tetradecene, ethyl oleate, or sulfate from microcosms inoculated with sediments from MP299 (65 m, 97 psi) and GC112 (535 m, 790 psi). The curves for removal of the substrates under pressurized or atmospheric conditions were nearly overlapping (Figures 3 and 4). The first-order decay coefficients were not significantly different (P ) 0.05) when comparing pressurized incubation against VOL. 40, NO. 18, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Concentrations of (a) ethyl oleate and tetradecene in sterile sediments incubated in heat seal bags at 790 psi and serum bottles at ambient pressure and (b) concentrations of sulfate in near shore sediment microcosms spiked with tetradecene and incubated in bags or bottles. The average and standard deviation of triplicate microcosms are presented. incubation at atmospheric pressure. The lack of an effect of pressure on the removal of these compounds could be explained because all of the pressure sensitive organisms were lost when the sediment was depressurized during collection, but this seems unlikely since the degradation of ethyl oleate was immediate and rapid, suggesting the degradative community had not been devastated by depressurization. The suggestion is that the activities observed were not dependent on enzymes that were sensitive to these pressures. As Deming (4) suggests, the maintenance of in situ temperature is more important than in situ pressure. Ethyl Oleate Degradation. Ethyl oleate was degraded more rapidly and with a shorter lag phase than tetradecene in all sediments tested. Ethyl oleate was completely removed from the MP299 NF (Figure 3a) and GC112 NF sediments (Figure 4a) as well as the FF sediments from all sites (data not shown). It was only partially removed from the VK916 NF sediment (Figure 5a). Ethyl oleate (and other esters) are found as common constituents of microbial membranes and as such are familiar structures to degradative populations. All previous research using shoreline sediments has also shown that ethyl oleate was removed much faster than internal olefins (hexadecene) (6). A students t-test (p ) 0.05) showed that there were significant differences in ethyl oleate degradation rates between a group of the two NF samples that had measurable SBF still present (GC112 and VK916 NF) (k ) -0.22 wk-1) and a grouping composed of MP299 NF and the three presumably unexposed FF sediments (k ) 0.11 wk-1) (Table 2). This suggests that the microbial populations were more active and possibly in higher numbers in 5740

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FIGURE 3. Concentrations of (a) sulfate and ethyl oleate and (b) sulfate and tetradecene in MP299 NF sediment incubated at 97 psi or atmospheric pressure. The average and standard deviation of triplicate microcosms are presented. The lines were generated using the lag times and coefficients presented in Table 2 and eq 2. exposed sediments than in sediments that were not exposed to SBF (all FF sediments) or were exposed to low amounts of SBF and have recovered (MP299 NF). This must be due to an increase in the general population of organisms (of which many would possess the ability to degrade esters) since site history indicates that the sediments were exposed to IO SBF and not ester-based SBF. The linkage of sulfate removal to ethyl oleate degradation was strong in every case. Figure 3a shows the almost simultaneous removal of ethyl oleate and sulfate from the MP299 sediment. There was no change in sulfate levels in the control samples not spiked with SBF substrates in the MP299 sediment. Historically this sediment was exposed to 996 barrels of IO or LAO SBF between 1962 and 2000. The sample received by our laboratory showed no detectable SBF contamination, but samples processed by Continental Shelf Associates (5) showed an average of 4.5 mg SBF kg-1 dry sediment still unattenuated at this location. Figure 3 suggests that the removal of sulfate due to any residual SBF was insignificant in MP299. There were no significant differences (p ) 0.05) in the sulfate concentrations in substrate-unamended control sediments throughout the 60-week incubation period. The GC112 NF sediment was exposed to much larger amounts of SBF (5470 bbl) and still had detectable SBF in the sediment used for this study (1068 mg kg-1) (Table 1). The data from the atmospheric and pressurized incubations showed some sulfate removal in the unspiked control microcosms, presumably coupled to the removal of the residual SBF (Figure 4a). The data again suggest that ethyl oleate removal was coupled closely to sulfate removal and that sulfate removal was slightly faster than ethyl oleate removal,

FIGURE 4. Concentrations of (a) sulfate and ethyl oleate or (b) sulfate and tetradecene in GC112 NF sediment incubated at 790 psi or atmospheric pressure. The average and standard deviation of triplicate microcosms are presented. The lines were generated using the lag times and coefficients presented in Table 2 and eq 2. most likely due to sulfate consumption during the degradation of the residual SBF in the microcosms. This was also the case with VK916 NF sediment. In this sediment, the sulfate was depleted by the 11th week, and the removal of ethyl oleate immediately decreased at this time (Figure 5a). The model prediction line deviates from the observed data after 11 weeks of incubation, presumably due to the depletion of sulfate from the microcosm. The residual removal of ethyl oleate may be due to fermentation reactions or methanogenesis, although no gas production was detected. Tetradecene Degradation. Tetradecene was removed by GC112 and MP299 near field (Figures 3b and 4b) sediments at the same rate, but the lag times were quite different (Table 2). The lag time in the GC112 NF sediment was much shorter (4 weeks), again most likely due to the presence of a stimulated microbial population. The rate of tetradecene degradation in this pre-exposed sediment was not enhanced though, indicating the possibility of an abiotic limitation to the rate or due to the inherent difficulty in degrading this class of compounds. The bioavailability of the substrate could pose a rate limitation although neither ethyl oleate nor tetradecene were soluble in the test system (4 °C). The role of sulfate reduction in the anaerobic degradation of tetradecene was evident in both MP299 NF and GC112 NF sediments. During incubation of the GC112 NF sediment, sulfate in the pore water was depleted by about 37 weeks of incubation at which time the degradation of tetradecene in this sediment slowed and eventually stopped (Figure 4b, >37 weeks), indicating the sulfate was required for tetradecene removal. As discussed above, the depletion of sulfate was undoubtedly due to the presence of excess SBF in the preexposed sediment (Table 1).

FIGURE 5. Concentrations of sulfate and tetradecene in (a) VK916 NF and (b) VK 916 FF sediment microcosms incubated at 1700 psi. The average and standard deviation of triplicate microcosms are presented. The lines were generated using the lag times and coefficients presented in Table 2 and eq 2. Linking tetradecene reduction to sulfate removal was not as clear for the VK 916 NF and all three FF sediments. Tetradecene was not removed from the VK916 NF sediment microcosms in the 30-week incubation period (Figure 5a). Sulfate in these samples was not removed until after the 20th week of incubation, and when sulfate removal began, it was removed at a rapid rate, which was not correlated with tetradecene loss. The control samples showed low initial concentrations of sulfate, and sulfate removal occurred spontaneously in the absence of ethyl oleate or tetradecene, indicating sulfate was consumed by organisms in the sediment during degradation of the residual SBF in the sediment. The removal of tetradecene in the far field samples was more difficult to analyze. The rate of tetradecene removal was more rapid in the far field samples than in the near field samples but not conclusively linked to sulfate removal. An example of this is presented in the results of the VK916 FF incubations (Figure 5b). The tetradecene was removed by about 30 weeks of incubation, but the sulfate was never removed in these microcosms. This is most likely due to the oxygenated nature of the far field sediments. Site history suggests that these sediments had not been exposed to SBF. This is supported by the lack of detection of SBF in the samples (Table 1). The FF sediments also did not appear to be anaerobic in nature, i.e., they were not black and lacked the odor of sulfide that was prevalent in the NF sediments. The far field sediments were a brownish gray in color and did not possess a smell other than salty. The redox potential of the FF sediments was not measured in this study. Continental Shelf Associates (5) reported that the top 20-cm layer of the MP299 FF sediment had 79 nmol DO cm-2 and >100 mV redox potential; they also reported that the redox VOL. 40, NO. 18, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Stoichiometric Coefficients for Sulfate Usage (mg Sulfate L-1 Pore Water/mg Substrate kg-1 Dry Sediment) sediment test

ethyl oleate

tetradecene

predicted MP299 NF atm MP299 NF 97 psi MP299 FF GC112 NF atm GC112 NF 790 psi GC112 FF VK916 NF1700 psi VK916 FF 1700 psi mean (SD) pooled mean

4.4 1.38a 0.72a (LA)c 0.99 1.37 (LA)c 1.38 (LA)c 1.06 (LA)c 0.165e (LA)c 0.923 (LA)c 1.12 ( 0.26 1.2 ( 0.3

4.9 1.33 (LA)c 0.91 (LA)c NLb 1.75 1.38 NLb NAd NLb 1.34 ( 0.34

a These coefficients gave good matches to initial curves but not the tails of the curves. b NL - it was determined that for these sediments sulfate reduction was not linked to tetradecene removal. c LA - the lag times for substrate and sulfate degradation were adjusted to be equal for these analyses. d NA - not available. e Not used in average.

potential of the top 10-cm layer of the GC112 FF sediment was also greater than 100 mV. VK916 was not included in the Continental Shelf Associates report (5) but was also aerobic in appearance, similar to the other far field sediments. It is possible that the tetradecene was removed at the expense of low levels of dissolved oxygen in the sediment. Statistical analysis of the results for tetradecene degradation suggested that the main divisor for differences in the k values was whether sulfate use accompanied tetradecene degradation. The k values for NF sediments (regardless of whether pre-exposure to SBF was detected) were all very similar. The average k value for the removal of tetradecene was 0.05 ( 0.01 week-1. The lag times preceding the onset of tetradecene degradation were much more varied. In GC112 NF sediment, low levels of SBF were detected (Table 1), and the lag time was only 4 weeks. The exposure and probable acclimation of the organisms in this sediment undoubtedly played a role in the shortened lag times. The MPN299 sediment was exposed to low levels of SBF over a long period and had recovered from its exposure (Table 1) thus the organisms could have lost the ability to begin degrading tetradecene rapidly, thus causing a 30-week lag period. Sediment from the VK916 drilling site had a recent exposure to high levels of SBF (Table 1), and it appears that the specialty group of SRB capable of degrading tetradecene had not been stimulated yet. The link in sulfate degradation to the removal of ethyl oleate or tetradecene was examined further by determining the stoichiometric coefficient that could be used to predict the sulfate removal curve from the ethyl oleate or tetradecene removal curve. A direct comparison cannot easily be made since the sulfate measurements had units of mg L-1 pore water, and the substrate measurements were in terms of mg kg-1 dry sediment. Although the units can be related by determining the amount of pore water in the bulk sediments, a more useful term would relate the two directly. The data from this study were used to calculate a stoichiometric coefficient with the units of mg sulfate L-1 pore water/mg substrate kg-1 dry sediment. The coefficients were determined by using

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Microsoft Office Excel solver to minimize the squares of the errors between a line predicted by multiplying the change in substrate to generate the change in sulfate concentration and are presented in Table 3. Once the differences in the lag times were adjusted, the fits of the generated lines to the original data were very good in most cases. The calculated stoichiometric coefficients ranged from 0.72 to 1.38 for ethyl oleate with an average of 1.12. The range of stoichiometric coefficients for tetradecene was slightly higher (0.91-1.75) resulting in a higher average of 1.34. Tetradecene was either not degraded, or there was no link between tetradecene degradation and sulfate removal in 4 of the tests. In summary, Gulf of Mexico sediments contained microorganisms capable of the biodegradation of SBF under sulfate-reducing conditions under high barometric pressures associated with the depth of sampling or at atmospheric pressure. SBF degradation could be described using firstorder decay kinetics, taking into account the initial lag phase. The main factors shown to affect SBF removal from sediments were the form of SBF compound (ester-based versus olefin based), prior history of exposure to SBF, and the amount of residual SBF present in the sediment prior to the initiation of surrogate experiments. The incubation pressure had no determinable effect on the removal of SBF surrogate or sulfate.

Acknowledgments This research was conducted as part of a cooperative agreement (1435-01-01-CA-31179) between the University of Houston and the U.S. Department of Interior - Minerals Management Service. The authors are also grateful to Mr. Al Hart of Continental Shelf Associates for providing the deepsea samples and Mr. Martin Kowis for his expertise in building the pressure chambers.

Literature Cited (1) Darley, H. C. H.; Gray, G. G. Composition and properties of drilling and completion fluids, 5th ed.; Gulf Publishing Co.: Houston, TX, 1988. (2) Neff, J. M.; McKelvie, S.; Ayers, R. C. Environmental impacts of synthetic based drilling fluids; U.S. Department of the Interior Minerals Management Service Gulf of Mexico OCS Region: New Orleans, LA, 2000. (3) Final NPDES permit for new and existing sources and new dischargers in the offshore subcategory of the oil and gas extraction category for the western portion of the OCS of the Gulf of Mexico. Fed. Regist. 2004, 60150-60151. (4) Deming, J. W. Unusual or extreme high-pressure marine environments. In Manual of Environmental Microbiology, 2nd ed.; Hurst, C. J., Crawford, R. L., Knudsen, R. K. G., McInerny, M. J., Stetzenbach, L. D., Eds.; ASM Press: Washington, DC, 2002. (5) Continental Shelf Associates. Gulf of Mexico comprehensive synthetic based muds monitoring program final report; 2004. http://www.gomr.mms.gov/homepg/regulate/environ/synthetic_muds/Volume%20I%20-%20Technical.pdf (accessed Aug 8, 2006). (6) Herman, D.; Roberts, D. J. A marine anaerobic biodegradation test applied to the biodegradation of synthetic drilling mud base fluids. Soil Sediment Contam. 2005 14, 433-447.

Received for review April 11, 2006. Revised manuscript received July 17, 2006. Accepted July 19, 2006. ES060873E