Abstract:
Mesocosms were established with a variety of marsh vegetation types to examine the impact to oil exposure and plant type on denitrification and nitrogen fixation. Replicated mesocosms were set up at Dauphin Island Sea Lab and contained buckets planted with combination of plants: the black mangrove, Avicennia germinans, black mangroves with either a monoculture or polyculture of the smooth cord grass, Spartina alterniflora, and monoculture and polyculture of the smooth cord grass. Buckets were oiled in October of 2015 with emulsified weathered oil. Sediments were collected from control and oil amended mesocosms 6 months (April, 2016) and 11 months (September 2016) from oiled (October 2015) and control treatments for rate determination. In April 2016 potential denitrification, nitrogen fixation as well as benthic fluxes of oxygen, nitrate, nitrite, phosphate, and ammonium were measured. In September 2016 potential denitrification, nitrogen fixation, and denitrification with the isotope pairing technique as well as total petroleum hydrocarbons were measured.
Data Parameters and Units:
Oiled was Macon surrogate obtained through GOMRI, weathered and emulsified (350 ml of weathered oil, 350 ml of seawater) and applied to the buckets (0.17 m2 surface area) For April 2016: Process: Processes measured were potential denitrification, potential N2 fixation, or benthic fluxes Date Sampled: Date the process was measured (MM/DD/YYYY) Mesocosm ID: Designation of the mesocosms (1 through 4) Mesocosm Treatment: Designates if the mesocosms were oiled or served as controls Bucket Number: Is the bucket designation Vegetation Type: Denotes the vegetation that were planted in each bucket (AV is the black mangrove (Avicennia germinans), SP Mono is Spartina alterniflora monoculture, SP Poly is Spartina alterniflora polyculture) Depth (cm): The depth interval of the sediment in cm that were collected with 2 being the top 2 cm, 5 being the top 5 cm and 7 being the 5 to 7 cm interval. Treatment: potential denitrification measured either with or without amendment of KNO3 (potassium nitrate 100 micromolar), or potential N2 fixation measured with (20 millimolar) and without molybdate Potential Rate (umol N m-2 hr-1): Denotes the rates of either potential denitrification or N2 fixation in units of micromols of N per m2 per hr SOD (umol O2 m-2 hr-1): Sediment Oxygen demand in units of micromoles of oxygen per m2 per hr Denitrification Capacity (umol N m-2 hr-1): Denotes total denitrification in units of micromoles of N per m2 per hr D15 (umol N m-2 hr-1): Denotes denitrification supported by the added nitrate in units of micromoles of N per m2 per hr D14 (umol N m-2 hr-1): Denotes denitrification supported by in situ nitrate in units of micromoles of N per m2 per hr NO2+3 Flux (umol N m-2 hr-1): Corresponds to the flux of nitrate plus nitrite in units of micromols N per m2 per hr PO4 Flux (umol P m-2 hr-1): Corresponds to the flux of phosphate in units of micromols P per m2 per hr NO2 Flux (umol N m-2 hr-1): Corresponds to the flux of nitrite in units of micromols N per m2 per hr NH4 Flux (umol N m-2 hr-1): Corresponds to the flux of ammonium in units of micromols N per m2 per hr NO3 Flux (umol N m-2 hr-1): Corresponds to the flux of nitrate in units of micromols N per m2 per hr chl a (ug g-1 wet sediment): Corresponds to the concentration of Chlorophyll a per gram wet sediment chl-a (mg m-2): Corresponds to the inventory of chlorophyll a per m2 TPH (mg kg-1): Corresponds to the concentration of total petroleum hydrocarbons in mg per kg of sediment n/a: Not samples or not applicable For September 2016: Process: Is the measured process, potential denitrification, potential N2 fixation, or TPH Date Sampled: Date the process was measured (MM/DD/YYYY) Mesocosm ID: Designation of the mesocosms (1 through 4) Mesocosm Treatment: Designates if the mesocosms were oiled or served as controls Bucket Number: Is the bucket designation Vegetation Type: Denotes the vegetation that were planted in each bucket (Bare is bare sediments with no vegetation; AV is the black mangrove (Avicennia germinans), SP Mono is Spartina alterniflora monoculture, SP Poly is Spartina alterniflora polyculture) Depth (cm): The depth interval of the sediment in cm that were collected with 2 indicating the top 2 cm, 5 indicating the top 5 cm, and 10 indicating the top 10 cm Potential Rate (umol N m-2 hr-1): Denotes the rates of either potential denitrification or N2 fixation in units of micromols of N per m2 per hr TPH (mg kg-1): Corresponds to the concentration of total petroleum hydrocarbons in mg per kg of sediment n/a: not sampled and or not applicable
Methods:
Sediment chlorophyll-α concentrations were determined from samples collected with a corer (1.4 cm I.D.) to a depth of 1 cm. Sediment wet weights were recorded prior to being stored at -80oC. Sediments were freeze-dried, dry weight were recorded, and were extracted in 10 mL of 90% acetone for 24 hours. Chl-α concentrations were determined fluorometrically according to Welschmeye (1994). Sediment total petroleum hydrocarbon (TPH) concentrations, determined by Fourier transform infrared spectroscopy (FTIR) (Standard Method 5520F, Standard Methods for the Examination of Water and Wastewater, 2012) at Envirochem, Inc. (Mobile, AL, USA). Potential denitrification rates were measured following the acetylene inhibition technique (Sørensen 1978). To triplicate serum vials, approximately 20 g of sediments and 0.7 ml filtered site water were added at various treatments (at ambient nitrate concentrations or amended with nitrate to a final concentration of 100 micromolar). Samples were sealed with a butyl rubber stopper, capped and flushed with N2 gas for 10 min. After the addition of C2H2 (10 % v/v) and a 1-h incubation, headspace gas samples were injected into evacuated 12 mL Exetainer vials and N2O production was quantified with a Shimadzu GC-2014 with an electron capture detector (GC-ECD) within 24 h. Potential N2 fixation rates were measured as ethylene (C2H4) production from acetylene (C2H2) reduction (Welsh et al. 1996) in triplicate from slurry assays containing 20 g of homogenized sediment and filtered (0.7 lm) site water. Rates of N2 fixation by sulfate reducing bacteria (SRB) were determined after the addition of sodium molybdate as a specific inhibitor of the sulfate reduction process (Capone 1993; Hardy et al. 1973). After C2H4 analysis on a Shimadzu gas chromatograph (GC-2014) with flame ionization detection (GC-FID), production rates of C2H4 were converted to potential N2 fixation rates using a C2H2:N2 reduction ratio of 3:1 (Capone 1993). Oiled and control cores were collected from the buckets for the determination of benthic fluxes. Cores were collected and placed underwater in an environmental chamber set to the average water temperature and allowed to equilibrate overnight with aerobic overlying water to ensure that no bubbles would be formed when the cores were capped. Incubations were carried out in the dark to prevent interference by photosynthetic algae and bubble formation. Cores were capped underwater ensuring no air bubbles were formed and each core was equipped with a magnetic stir bar to mix the overlying water. GF/F filtered site water, enriched to a final concentration of 50 µM of isotopically labeled sodium nitrate, was then flowed through each core at a rate of ~2.5 mL min-1. The positive displacement of the overlying water exited the core through an outflow tube (“outflow”) and was collected in a reservoir. After 24 hours of flow through, water samples were collected from the outflow and inflow from each core for determination of oxygen, nutrients and nitrogen gas isotope concentrations (described below). Following the sample collections, cores were gently uncapped and the top 5 cm from each was sectioned and homogenized for THP analysis. Sediment oxygen demand was determined from inflow and outflow water based on O2 concentrations measured with a Unisense® multimeter equipped with a calibrated O2 (Ox-500) sensor. Water samples were collected in triplicate from the inflow and outflow lines, filtered through GF/F and immediately frozen until analysis for inorganic nutrient concentrations as previously described. Inflow and outflow samples from each core were collected in triplicate in 12 ml Exetainers® by overflowing the vial’s volume twice for the analysis of isotopes of nitrogen gas with a membrane inlet mass spectrometer (MIMS), equipped with an in-line quartz column packed with copper turnings and heated to 600 °C (Eyre et al. 2002), to remove oxygen upstream of the mass spectrometer. Samples were then preserved with 250μL of 50% w/v ZnCl2, capped and stored underwater until analysis. Denitrification was determined according to the isotope pairing technique (IPT) according to Nielson (1992). In this approach, denitrification is explicitly calculated from the 29N2 and 30N2 fluxes calculated from dissolved 29N2:28N2 and 30N2:28N2 measured with the MIMS. Benthic fluxes were calculated according to F = (Ce - Ci)*V/A, where Ce is the outflow concentration, Ci is the inflow concentration, V is the flow rate, and A is the surface area of the sediment. References: Capone DG (1993) Determination of nitrogenase activity in aquatic samples using the acetylene reduction procedure. In: Kemp PF, Sherr BF, Sherr EB, Cole JJ (eds) Handbook of Methods in Aquatic Microbiology. Lewis, Boca Raton, pp 621-631 Eyre B, Rysgaard S, Dalsgaard T, Christensen PB (2002) Comparison of isotope pairing and N2:Ar methods for measuring sediment denitrification, assumption, modifi-cations, and implications. Estuaries 25(6):1077-1087. Hardy RWF, Burns RC, Holsten RD (1973) Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biology & Biochemistry 5:47-81. Nielsen LP (1992) Denitrification in sediment determined from nitrogen isotope pairing. FEMS Microbiology Ecology 86(4): 357-362. Sorensen, J. (1978). Dentirification rates measured in a marine sediment as measured by acetlyene inhibition technique. Applied and Environmental Microbiology, 36, 139-143. StandardMethodsforthe Examination of Water and Wastewater, 22nd ed.; Rice, E. W., Baird, R. B., Eaton, A. D., Clesceri, L. S., Eds.; Washington, DC, 2012. Welsh DT, Bourgues S, de Wit R, Herbert RA (1996) Seasonal variations in nitrogen-fixation (acetylene reduction) and sulphate-reduction rates in the rihizosphere of Zostera noltii: nitrogen fixation by sulphate-reducing bacteria. Marine Biology 125:619-628 Welschmeyer, N. A. (1994). Fluorometric analysis of chlorophyll a in the presence of chlorophyll b and pheopigments. Limnology and Oceanography, 39(8), 1985-1992.
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