Data Parameters and Units:
% hydrocarbon loss -- Sample ID, Incubation time (hours), Mean % loss relative to hour 0 (%), Standard deviation of loss total relative to hour 0 (%), Total alkane (mg/g sediment), SD of total alkanes (mg/g sediment), Mean concentration of individual PAH (ug/g sediment, uM). time series data -- Incubation time (hour), total alkane (mean, mg/g sediment), SD of total alkanes (mg/g sediment), %loss of total alkanes relative to hour 0 (mean, %), SD of %loss total alkanes relative to hour 0, Short-chain alkanes (less than C17) (mean, mg/g sediment), SD of short-chain alkanes, %loss of short-chain alkanes (mean, %), SD of %loss short-chain alkanes relative to hour 0, Long-chain alkanes (greater than C17) (mean, mg/g sediment), SD of long-chain alkanes %loss of long-chain alkanes relative to hour 0, SD of %loss long-chain alkanes, Napthalen (mean, ug/g sediment), SD of Napthalen, %Loss of Napthalen relative to hour 0 (%), SD of %loss of Napthalen relative to hour 0 Fluorene (mean, ug/g sediment), SD of Fluorene, %Loss of Fluorene relative to hour 0 (%), SD of %loss of Fluorene relative to hour 0, Pyrene (mean, ug/g sediment), SD of Pyrene, %Loss of Pyrene relative to hour 0 (%) SD of %loss of Pyrene relative to hour 0, Acenaphthene (mean, ug/g sediment), SD of Acenaphthene, %Loss of Acenaphthene relative to hour 0 (%), SD of %Loss of Acenaphthene relative to hour 0, Fluoranthene (mean, ug/g sediment), SD of Fluoranthene, NO3-N+NO2-N dissolved (mean, uM), SD of NO3-N+NO2-N dissolved, Phosphate dissolved (mean, uM), SD of phosphate in overlying water Dissolved organic carbon (mean, uM), SD of dissolved organic carbon, Ratio of less than C17 greater than or equal to C17 alkanees, SD of ratio of less than C17: greater than or equal to C17 alkanees, low molecular weight PAHs (mean, ug/g sediment), SD of low molecular weight PAHs, high molecular weight PAHs (mean, ug/g sediment), SD of high molecular weight PAHs, low mecular weight /high molecular weight PAH ratio, SD of low mecular weight /high molecular weight PAH ratio. NOTE: mean and SD standard deviation for all replicate bottles.
Methods:
Sample Collection On March 17, 2011, seawater and sediment samples were collected just seaward of a salt marsh on the coastline SW of Bayou La Batre, Alabama (30.38°N, 88.30°W), which is located in the Gulf Coastal Plain and approximately 10 miles west of the Mobile Bay (Fig. 1). The sediments collected are typical of many areas along the Gulf Coast, consisting of inorganic clays, poorly-sorted sands, silty-sand, and sandy-clay (Rentschler, 2013). The seawater had a pH of 7.70 and total alkalinity of 83.07mgCaCO3/L (Rentschler, 2013). All containers and sampling equipment to be in direct contact with water samples were either combusted at 450°C for 5 hours (glass materials), or acid soaked (10% HCl or 10% HNO3) and thoroughly rinsed with Milli-Q water (plastic materials). Seawater was collected using polypropylene bottles which were rinsed with seawater, then filled and capped under the water surface. The upper 15 – 30 cm of sediment was collected into a five gallon plastic bucket. All samples were transported on ice to the lab, where they were refrigerated in the dark until the microcosm experiment was conducted. Microcosm Incubations Each microcosm consisted of 326.94 g wet sediment (mass equivalent to 200 g dry sediment) and 173.06 g seawater in a 500 mL glass jar with Teflon-lined lids. Samples were spiked with 10g Macondo oil (MC 252 oil that was collected from the wellhead by the British Petroleum), thoroughly mixed, and placed on a shaker table set to 100 rpm for 24 hours. The incubations then started and lasted 14 days in the dark. All jars were kept closed and consistently shaken to ensure a thorough mixture of sediments and water throughout the experiment. Two jars were sacrificed at 0, 6, 12, 24, 48, 168, and 336 hours. One control sample that was not treated with oil was sacrificed at hour 336. Over the course of the incubation, the concentrations and compositions of alkanes and PAHs in the sediments, as well as the concentrations of dissolved organic carbon (DOC) and nutrients in aqueous solution were determined. Chemical Analyses Microcosm sample collection. After the duplicate jars were sacrificed at their respective time points, the aqueous (seawater) and solid (sediment) phase samples within each glass jar were thoroughly mixed before they were sampled. Aqueous samples were collected by siphoning the upper liquid layer before and after centrifuging sediments at 750 rpm for 20 minutes. The aqueous samples were then filtered through 0.45 micron filters and stored frozen until analysis for DOC and nutrients (NO3-+NO2-, PO43-) concentrations. Hydrocarbon determination in sediments. Solid phase samples were homogenized and then stored frozen in glass jars prior to hydrocarbon analysis. Hydrocarbon extraction followed the method described in Risdon et al. (2008) with modifications. Approximately 6g the incubated sediments were mixed with Hydromatrix (Agilent Technologies) to remove water. Normal hexadecane-d34 (C16D34) and phenanthrene-d10 were added as surrogates to the samples prior to the extraction. Samples were then ultrasonically extracted with 4mL Acetone for 2 minutes at 20°C to ensure thorough mixing between the sediments and the surrogates. Twenty mL of acetone:hexane (1:1, vol:vol) was then added to the samples, ultrasonically extracted for 10 minutes at 20°C, and stored at 4°C overnight. Short copper ribbon was used to remove sulfur during the extraction process. Samples were extracted again ultrasonically for 20 minutes at 20 ºC before the liquid portion of the samples was collected with disposable pipettes and concentrated with an ultra high purity (UHP) nitrogen stream. Three mL of acetone, 5 mL of hexane, 4 mL of Milli-Q water and a spatula of sodium chloride were added to the extracts, which were then manually shaken for 30 seconds and allowed to settle for 20 minutes. The top hexane layer was siphoned via disposable glass pipette as the hydrocarbon fraction and the more polar compounds dissolved in the lower acetone and water layer were discarded. The hydrocarbon fraction was then separated by silica column chromatography into aliphatic and aromatic hydrocarbon fractions, which were eluted by 10 mL of hexane and 12 mL of dichloromethane respectively. Aliphatic fractions were quantified and identified using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), respectively. The initial temperature for the GC oven was 50°C, held for one minute, followed by a 6°C/min increase until 310°C was reached, then holding for 15 minutes. UHP helium was the carrier gas used at a flow rate of 1.48 mL/min. The quantification of objective compounds was done by comparing their peak areas to the peak areas of n-Hexadecane-d34. Different instrumental responses for various compounds were corrected by regularly running a standard containing a series of alkane compounds (C7 – C40 normal alkanes) and n-Hexadecane-d34. Aromatic components were identified using GC-MS by comparing their relative retention times and mass spectra to those of multiple known PAHs in a standard mixture. They were quantified by comparing their peak areas to Phananthrene-d10. The initial temperature of the GC oven was 50°C, held for one minute, followed by a 20°C/min increase until 140°C was reached, and then a 6°C/min increase until 310°C was reached, holding for 15 minutes for a total run time of 47 minutes. The carrier gas was UHP helium with a flow rate of 1.48 mL/min. DOC concentration. The DOC concentration of aqueous samples was determined using a Shimadzu TOC-VCPN Total Organic Carbon Analyzer, with a KHP calibration standard solution and a Consensus Reference Material deep seawater DOC check standard (Hansell Laboratory, http://yyy.rsmas.miami.edu/groups/biogeochem/CRM.html). Dissolved nutrients. Water samples were submitted to the Dauphin Island Sea Laboratory (AL, USA) for nutrient (nitrate + nitrite, phosphate) analyses using a Skalar San + continuous flow autoanalyser with wet chemistry colorimetric modules designed for the individual analytes (NO2- and NO3- consistent with EPA method 353.2, and PO43- consistent with EPA method 365.3). Sample absorbance was compared with regression statistics based on a five-point standard curve for each analyte, and the results were baseline- and drift- corrected throughout the sample run.
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