Sediment nitrous oxide and methane fluxes under acidification

We collected twelve sediment cores from Metoxit Point (MP) in August 2021 and from Sage Lot Pond (SLP) in September of 2021. We collected the cores using a pull corer which maintains the vertical structure of the sediment cores and preserves the delicate sediment-water interface (Foster and Fulweiler 2019). We also collected filtered bottom water (0.2 µm) in acid-washed plastic carboys. We transported the cores back to Boston University within 7 hours of collection and placed them in an environmental chamber set to 29 (±1) °C. We conducted two separate incubations examining the effect on acidification on fluxes of nitrous oxide (N2O) and methane (CH4). We included experimental conditions representing control (pH 8.0), moderate (pH 7.3) and extreme (pH 6.3) pH conditions in Waquoit Bay (Baumann and Smith 2018). All treatments were conducted in triplicate. We divided the filtered site water into two 30-gallon acid-washed polycarbonate reservoir tanks. One reservoir tank was acidified to the appropriate pH treatment by bubbling it with 10% CO2 controlled using a Qubit pH/CO2 controller system (Qubit Systems Inc., Ontario, CA). The second tank was bubbled with compressed air to maintain a pH of 8.0. In preparation for each incubation, we carefully siphoned off the overlying water of six sediment cores and gently replaced it with a ~4 cm headspace of treated bottom water from the corresponding station and treatment (McCarthy et al. 2013). Cores and the overlying water were capped with gas-tight plungers fitted with gas-tight PEEK inflow and outflow tubing above the sediment-water interface (overlying water volume ~314 mL). The inflow tubing was connected to a peristaltic pump (Masterflex L/S, Coleman-Palmer) with Viton (gas-impermeable) pump tubing to deliver 1.5 (± 0.2) mL min-1 of treated bottom water (overlying water residence time ~209 min; McCarthy et al. 2013). Cores were incubated in the dark under continuous flow through conditions overnight (~12 h) until sampling began the next morning (Newell et al. 2016; Li et al. 2021). During incubation, we collected water samples from inflow and outflow ports at 0, 6, 12 and 24 hours for dissolved GHG samples in 12 mL Exetainers (Labco, UK) and preserved them with 25 µL of saturated ZnCl2. Samples were kept in the refrigerator at 4 °C and analyzed within one month of sampling. After completing the first set of incubations, we repeated the process with the remaining six cores and exposed them to a moderate pH treatment. Concentrations of dissolved N2O and CH4 were measured directly from the sample using a headspace equilibration technique and measured on a gas chromatograph (~4 mL; GC2014, Shimadzu, Japan; Foster and Fulweiler 2016). The GC was equipped with a flame ionization detector to measure CH4 and an electron capture detector to measure N2O. We estimated concentrations by comparing the area under the produced peak against a standard curve. Standard curve concentrations were determined using a linear regression of custom gas concentrations (CH4 5084 ppb; N2O: 495 ppb in N2) made by Airgas (Radnor Township, Pennsylvania, USA). Each standard curve had at least six time points and a R2 ≥ 0.995. Detection limits during sample analysis were 83.21 ppb for CH4 and 16.83 ppb for N2O. Email questions and comments to cmazur@bu.edu <strong>Sampling Sites:</strong> Metoxit Point = 41° 34' 8.04" N 70° 31' 17.76" W Sage Lot Pond = 41° 34' 8.04" N 70° 30' 30.20" W <strong>Experiment Information:</strong> Collection_Date = Date when sediment cores were collected Incubation_Date = Date when incubations of sediment cores began Experiment_Type = For each station we conducted two incubations. The first incubation conducted was the Extreme treatment (pH 6.3) which included 3 control cores and 3 cores acidified to a pH of 6.3. The second experiment conducted was the moderate treatment (pH 7.3). Treatment = The treatment each cores were exposed to: Control = pH 8.0 pH 6.3 = Extreme pH 7.3 = Moderate Core_ID = Number/Letter identifying the core Time_Point = The hour of the incubation when samples were collected <strong>Abbreviations and Units:</strong> Date = month - dd (day) - yy (year) <strong>Sediment-Water Greenhouse Gas Fluxes:</strong> N2O_Flux = nitrous oxide, nmol N2O m-2 hr-1 (nanomoles per meter squared per hour) CH4_Flux = methane, nmol CH4 m-2 hr-1 (nanomoles per meter squared per hour) Sediment-Water NOx Fluxes: NOx_Flux = nitrate + nitrite, µmol m-2 hr-1 (micromoles per meter squared per hour) <strong>Acknowledgements:</strong> We would like to thank the Waquoit Bay National Estuarine Research Reserve (WBNERR) for their support of our research. Sediment cores for this study were collected using WBNERR boats. We are particularly grateful for Dr. Megan Tyrrell and Tonna-Marie Rogers who were instrumental in helping us carry out fieldwork and for making sure we had all the resources necessary for a successful field day. We would also like to thank members of the Fulweiler Lab, Alia Al-Haj, Amanda Vieillard, Catherine Mahoney, Kwetzpallin Mexika, Melissa Ederington Hagy, Nia Bartolucci, Paulina Azzu, and Ryan Shipley for their help with sample prep, field work and experiments. <strong>References: </strong> Baumann H, Smith EM (2018) Quantifying Metabolically Driven pH and Oxygen Fluctuations in US Nearshore Habitats at Diel to Interannual Time Scales. Estuaries and Coasts 41:1102–1117. https://doi.org/10.1007/s12237-017-0321-3 Foster SQ, Fulweiler RW (2019) Estuarine Sediments Exhibit Dynamic and Variable Biogeochemical Responses to Hypoxia. J Geophys Res Biogeosciences 124:737–758. https://doi.org/10.1029/2018JG004663 Foster SQ, Fulweiler RW (2016) Sediment Nitrous Oxide Fluxes Are Dominated by Uptake in a Temperate Estuary. Front Mar Sci 3:1–13. https://doi.org/10.3389/fmars.2016.00040 Li S, Twilley RR, Hou A (2021) Heterotrophic nitrogen fixation in response to nitrate loading and sediment organic matter in an emerging coastal deltaic floodplain within the Mississippi River Delta plain. Limnol Oceanogr 66:1961–1978. https://doi.org/10.1002/lno.11737 McCarthy MJ, Carini SA, Liu Z, et al (2013) Oxygen consumption in the water column and sediments of the northern Gulf of Mexico hypoxic zone. Estuar Coast Shelf Sci 123:46–53. https://doi.org/10.1016/j.ecss.2013.02.019 Newell SE, McCarthy MJ, Gardner WS, Fulweiler RW (2016) Sediment Nitrogen Fixation: a Call for Re-evaluating Coastal N Budgets. Estuaries and Coasts 39:1626–1638. https://doi.org/10.1007/s12237-016-0116-y <strong>Funding</strong> This work was funded by MIT SeaGrant through grants to RWF and by the NOAA Margaret A. Davidson Graduate Fellowship awarded to CIM. CIM received additional support from the Mount Holyoke Alumnae Fellowship, the Warren-McLeod Graduate Fellowship from the Boston University Marine Program, and from Boston University’s Department of Earth &amp; Environment.

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