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Methodology from\u00a0Smyth, A. R., Piehler, M. F. and Grabowski, J. H. (2015), Habitat context influences nitrogen removal by restored oyster reefs. J Appl Ecol, 52: 716\u2013725. doi:10.1111/1365-2664.12435
\nWithin 4\u00a0h of collection, sediment cores were set up in a continuous flow core incubation system to measure steady-state nutrient and dissolved gas fluxes, described in Piehler & Smyth (2011). Briefly, cores were sealed with gas-tight lids, which had an inflow and outflow port. Water from a reservoir was pulled over the cores at a flow rate of 1\u00a0mL min\u22121. Triplicate dissolved gases and duplicate dissolved inorganic nitrogen samples were collected from the outflow and inflow periodically over the next 24\u00a0h. To examine how sediments from different habitat contexts responded to nitrate pulses, nitrate concentration in the reservoir water was elevated with NaNO3\u00a0(~800\u00a0\u03bcm) after 48\u00a0h of sampling. Dissolved gas and inorganic nitrogen samples were then collected for an additional 48\u00a0h. Incubations were conducted in the dark and at ambient temperature (30\u00a0\u00b0C).
\nWater samples from laboratory experiments were\u00a0analysed\u00a0immediately upon collection for dissolved gasses (N2,\u00a0O2\u00a0and Ar) with membrane inlet mass spectrometry (MIMS). Concentrations of dissolved N2\u00a0and O2\u00a0were determined using the ratio with Ar (Kana\u00a0et\u00a0al.\u00a01994). Coefficients of variation for N2/Ar were 0\u00b705% and 0\u00b704% for O2/Ar. Water samples from laboratory experiments for dissolved nutrient determination were filtered through Whatman GF/F glass\u00a0fibre\u00a0filters (25\u00a0mm diameter, 0\u00b77\u00a0\u03bcm nominal pore size) and frozen until analysis. Dissolved inorganic nutrients were analysed with a Lachat Quick-Chem 8000 automated ion analyser for\u00a0 [math formula] + [math formula] \u00a0(reported as NOx) and\u00a0 [math formula] \u00a0concentrations using standard protocols (Lachat Instruments, Milwaukee, WI, USA:\u00a0 [math formula] / [math formula] \u00a0method 31-107-04-1-A,\u00a0 [math formula] \u00a0method 31-107-06-1-A; detection limits: 0\u00b704\u00a0\u03bcm\u00a0NOx, 0\u00b718\u00a0\u03bcm\u00a0 [math formula] ; CV(%): 0\u00b79% NOx\u00a0and 2\u00b76%\u00a0 [math formula] ).
\nUpon completion of the incubations, the upper 2\u00a0cm of sediment in each core was sampled for organic matter content by mass difference from dried sediments before ignition (105\u00a0\u00b0C for 6\u00a0h) and after ignition (525\u00a0\u00b0C for 3\u00a0h).
\nWater Quality Data:
\n\n\u00a0
Nutrient flux data from several landscapes in coastal North Carolina.
Methodology from\u00a0Smyth, A. R., Piehler, M. F. and Grabowski, J. H. (2015), Habitat context influences nitrogen removal by restored oyster reefs. J Appl Ecol, 52: 716\u2013725. doi:10.1111/1365-2664.12435
\nFluxes across the sediment\u2013water interface were calculated as (Co\u2212Ci)\u00a0\u00d7\u00a0f/a, where\u00a0Co\u00a0is the outflow concentration (\u03bcmol L\u22121),\u00a0Ci\u00a0is the inflow concentration,\u00a0f\u00a0is the flow rate (0\u00b706\u00a0L h\u22121), and\u00a0a\u00a0is the sediment surface area (0\u00b70032\u00a0m2). Successive measurements from each core (triplicates for dissolved gas and duplicates for dissolved inorganic nutrients) were averaged to give core-specific values. This results in a net N2flux (gross denitrification \u2013 gross nitrogen fixation) and does not distinguish between the sources of N2. Consequently, denitrification refers to net N2\u00a0production. Oxygen fluxes were calculated using the concentrations of O2\u00a0obtained from the MIMS, presented as sediment oxygen demand (SOD), and serve as an indicator of organic matter quality, such that more labile organic matter is associated with higher SOD (Ferguson, Eyre & Gay\u00a02003). To determine the influence of oyster reefs on sediment N2\u00a0fluxes, the change in denitrification between the control and reef habitat pair in each zone was calculated (Kellogg\u00a0et\u00a0al.\u00a02014). Denitrification efficiency was computed as the percentage of the dissolved inorganic nitrogen efflux that was N2\u00a0(Piehler & Smyth\u00a02011).
\nStatistical analyses were performed using\u00a0r\u00a02.13.1 (R Foundation for Statistical Computing\u00a02011). Linear mixed-effects models (lme\u00a0in\u00a0R nlme\u00a0package), where habitat nested in sampling location was included as a random effect for the intercept, were used to investigate the effects of oyster reef presence, habitat context, nitrate concentration (ambient vs. elevated) and the interaction between these factors on response variables. Fluxes of N2, NOx\u00a0( [math formula] \u00a0+\u00a0 [math formula] )\u00a0 [math formula] , denitrification efficiency and SOD were analysed using all three fixed effects. For sediment organic matter, only habitat context and reef presence were included as fixed effects. The effects of ambient vs. elevated nitrate concentration and habitat context on oyster reef-mediated changes in denitrification were also analysed with a mixed-effects model (fixed effects: nitrate concentration\u00a0\u00d7\u00a0habitat context; random effects: habitat nested in location). Relationships between oyster density and habitat context were made using a mixed-effects model (fixed effects: habitat context; random effects: habitat nested in location). Comparisons were conducted using linear contrasts and judged against an alpha level of 0\u00b705. Interactions were assessed using Tukey's HSD (lsmeans\u00a0in\u00a0R lsmeans\u00a0package). Assumptions of homogeneity were tested using Levene's tests. Regression analyses were used to investigate the effect of oyster density on denitrification. Models with the lowest Akaike's information criterion corrected for small sample sizes (AICc) were chosen.
\nBCO-DMO Processing Notes:
\n- column names reformatted to comply with BCO-DMO naming standards.
\n- lat and lon\u00a0columns added to correspond with locations.