Sediment nutrient fluxes under acidification

We collected sediment cores from Waquoit Bay MA, on two occasions: Metoxit Point in September 2021 and Sage Lot Pond in October 2021. At each site we collected 12 sediment cores using clear polyvinyl chloride (PVC) core tubes via a pole corer. The pole corer has a check valve system designed to keep the delicate sediment water interface intact (Gardner et al. 2009). Once collected, we capped the sediment cores and placed them in a cooler filled with water to maintain field temperatures during transport. We transported the sediment cores to an environmental chamber at Boston University set to 29 (±1) °C within 7 hours of collection. We uncapped the cores and gently bubbled the overlying water with ambient air using aquarium pumps (Cornwell et al. 2016). We kept the cores in the dark and let them bubble overnight (~12 hours) before starting incubation set-up the following morning. Cores from Sage Lot Pond sat in the chamber for an additional ~24 hrs before starting incubations to let them acclimate to the warmer temperatures. We selected six cores for the first incubation which were exposed to the extreme (pH 6.3) treatment while the other six cores were bubbled with ambient air in the dark. Of the six cores, we designated three for the acidified treatment and three for the control. In preparation for the incubation, we carefully removed the overlying water using a siphon and replaced it with a ~4 cm headspace of treated bottom water. Once filled, cores and the overlying water were sealed with gas-tight plungers fitted with PEEK tubing for inflow and outflow ports located ~ 3 cm above the sediment (overlying water volume ~314 mL).Water from the reservoir tanks was pumped through the inflow tubing at a rate of 1.5 (± 0.2) mL min-1 using a peristaltic pump (Masterflex L/S, Coleman-Palmer) and Viton (gas-impermeable) tubing (overlying water residence time ~209 min; McCarthy et al. 2013). Once capped we left the cores under continuous flow through conditions in the dark overnight (~12 h) until sampling began in the morning (Newell et al. 2016; Li et al. 2021). We collected samples at 0, 6, 12 and 24 hours via the inflow and outflow ports for temperature, dissolved O2 concentrations, and pH measurements (FireSting®-PRO using TSUB21, OXROBSC, PHROBSC-PK7 probes). We also collected 20 mL of water for nutrients (NOx, NH4, DIP), and sulfate (SO42-) and dissolved iron (Fe2+) analysis. These samples were filtered using a syringe and polyethersulfone (0.2 µm) filters. Four, two mL aliquots were placed into microcentrifuge tubes with screw on caps and immediately frozen for nutrient analyses. Two, three mL aliquots were collected into 5 mL centrifuge tubes with screw on caps, acidified with 1/100 v/v 1 N HCl and stored in the fridge (4 °C) for Fe2+and SO42- analysis (Anschutz and Charbonnier 2021). As soon as the last sample of the incubation was collected, the flow through system was turned off, sediments cores were uncapped, measured for post incubation O2 profiles, and sub-cored. Due to logistical constraints moderate and extreme treatments were not conducted at the same time, but began one day after the first incubation was finished. We repeated the process with the remaining six cores and exposed them to a moderate (pH 7.3) pH treatment. Nutrient samples were melted at room temperature, shaken vigorously, and analyzed for dissolved inorganic nitrogen (DIN: nitrite + nitrate (NOx), ammonium (NH+)), and dissolved inorganic phosphorus (DIP). Samples for SO42- and Fe2+ were taken out of the fridge and warmed up to room temperature. All analyses were conducted using micro-colorimetric assays based on a standard 96-well microplate format and read on an absorbance microplate reader (SpectraMax ABSPlus; San Jose, CA, USA). NH4+ and DIP were analyzed following the protocol outlined by Ringuet et al. (2010). NOx analysis was adapted from Doane and Horwáth (2003). SO42- and Fe2+ analyses were adapted from (Anschutz and Charbonnier 2021). Values below MDL were assigned to the concentration of the method detection limit. <strong>Sampling Stations</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 Fluxes:</strong> SOD = Sediment oxygen demand, µmol O2 m-2 hr-1 NH4_Flux = ammonium flux, µmol NH4+ µmol m-2 hr-1 NOx_Flux = nitrite + nitrate, µmol NOx µmol m-2 hr-1 DIN = ammonium + nitrite + nitrate flux, µmol N µmol m-2 hr-1 DIP = dissolved inorganic phosphorus flux, µmol DIP m-2 hr-1 DIN_DIP = nitrite + nitrate + ammonium to dissolved inorganic phosphorus ratio <strong>Water Characteristics:</strong> Inflow_NOx = nitrite + nitrate concentration from the inflow port, µmol L-1 Inflow_NH4 = ammonium concentration from the inflow port, µmol L-1 Inflow_DIN = nitrite + nitrate + ammonium concentration from the inflow port, µmol L-1 Inflow_DIP = dissolved inorganic phosphorus concentration at the start of incubations, µmol L-1 Inflow_O2 = oxygen concentration from the inflow port, µmol L-1 Inflow_Temp = temperature from the inflow port, °C Inflow_pH = pH from the inflow port Outflow_O2 = oxygen concentration from the outflow port, µmol L-1 Outflow _Temp = temperature from the outflow port, °C Outflow _pH = pH from the outflow port <strong>Aknowledgements</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, Emily Miao, Kwetzpallin Mexika, Melissa Ederington Hagy, Nia Bartolucci, Paulina Azzu, and Ryan Shipley for their help with sample prep, field work and experiments. We also thank Dr. Cédric Fichot and Dr. Nilotpal Ghosh for help with CN sample analysis and instrumentation use. <strong>References: </strong> Anschutz P, Charbonnier C (2021) Sampling pore water at a centimeter resolution in sandy permeable sediments of lakes, streams, and coastal zones. Limnol Oceanogr Methods 19:96–114. https://doi.org/10.1002/LOM3.10408 Cornwell JC, Owens MS, Boynton WR, Harris LA (2016) Sediment-Water Nitrogen Exchange along the Potomac River Estuarine Salinity Gradient. J Coast Res 32:776–787. https://doi.org/10.2112/JCOASTRES-D-15-00159.1 Doane TA, Horwáth WR (2003) Spectrophotometric determination of nitrate with a single reagent. Anal Lett 36:2713–2722. https://doi.org/10.1081/AL-120024647 Gardner WS, McCarthy MJ, Carini SA, et al (2009) Collection of intact sediment cores with overlying water to study nitrogen- and oxygen-dynamics in regions with seasonal hypoxia. Cont Shelf Res 29:2207–2213. https://doi.org/10.1016/J.CSR.2009.08.012 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 Ringuet S, Sassano L, Johnson ZI (2010) A suite of microplate reader-based colorimetric methods to quantify ammonium, nitrate, orthophosphate and silicate concentrations for aquatic nutrient monitoring. J Environ Monit 13:370–376. https://doi.org/10.1039/c0em00290a