Samples for physical, chemical, biological, and organic matter (dissolved and particulate organic matter, DOM and POM respectively) analyses were collected as part of the Neuse River Monitoring and Modeling Program (ModMon; http://paerllab.web.unc.edu/projects/modmon/) conducted by the University of North Carolina - Chapel Hill, Institute of Marine Sciences (UNC-CH IMS) (Paerl et al. 2018). Samples were collected from July 20, 2015 to July 28, 2016; bi-weekly from March through October and monthly from November through February. For each sampling date (n = 22), samples were collected at 11 stations in the NRE spanning the upstream-most location of salinity intrusion (Station 0) to the mouth of the estuary (Station 180). In situ measurements (water temperature, salinity, turbidity, percent dissolved oxygen) were collected at discrete depths on the sunlit side of the research vessel using a Yellow Springs Instruments (YSI Incorporated, Ohio) multiparameter sonde (Model 6600 or 6600 EDS-S Extended Deployment System) equipped with a YSI conductivity/temperature probe (Model 6560), a YSI pulsed dissolved oxygen probe (Model 6562), and a self-cleaning YSI turbidity probe (Model 6026 or 6136). The YSI sonde was coupled to a either a YSI 610 DM datalogger or a YSI 650 MDS Multi-parameter Display System datalogger. In situ measurements were performed at the surface (approximately 0.2 meters) and at the bottom of the water column (approximately 0.5 meters from the sediment layer).
For each sampling date, a surface (0.2 m below surface) and bottom (0.5 m above bottom) water sample were collected for chemical, biological, and organic matter analyses at each of the 11 stations using a peristaltic pump. Samples were maintained in the dark at ambient temperature and returned to UNC-CH Institute of Marine Sciences within ~6 hours of collection. Samples were filtered through pre-combusted (450 degrees C, 4 h) GF/F glass fiber filters (0.7 _m nominal pore size). The filtrate was collected and stored frozen at -20_C in the dark until dissolved nutrient and DOM quantitative and qualitative analysis. Filters were collected and stored frozen at -20 degrees C in the dark until chlorophyll-a analysis, conducted within one month of collection, and POM quantitative and qualitative analysis, as described below.
The freshwater flushing time for each station and date was calculated using the date-specific fraction of freshwater method (Alber and Sheldon, 1999) as described in Peierls et al. 2012. Briefly, the date-specific average discharge is an iterative calculation that averages the riverine discharge over the flushing time period.
Organic matter analysis
DOC concentration ([DOC]) was measured via high-temperature catalytic oxidation on a Shimadzu TOC-5000 analyzer (Peierls et al. 2003): Water samples were vacuum filtered (less than 25 kilopascal) using pre-combusted Whatman glass microfiber filters (GF/F). The filtrate was stored in pre-combusted glass scintillation vials with Teflon closures and frozen at -20 degrees Celsius until analysis. The Shimadzu TOC-5000A Analyzer uses high temperature catalytic oxidation followed by non-dispersive infrared analysis of the carbon dioxide produced. Samples were acidified to a pH less than 2 and sparged with air before they were analyzed for non-volatile organic carbon. Total dissolved nitrogen (TDN), nitrate + nitrite, and ammonium were determined colorimetrically using a Lachat QuickChem autoanalyzer (Peierls et al. 2003). Dissolved organic nitrogen ([DON]) was determined by subtracting the dissolved inorganic nitrogen species (DIN, as nitrate + nitrite and ammonium) from TDN. The molar ratio of dissolved organic carbon (DOC) to dissolved organic nitrogen (DON) or DOC:DON was calculated.
Particulate organic carbon concentration ([POC]) and particulate nitrogen ([PN]) were determined on one set of collected filters via high temperature combustion on a Costech ECS 4010 analyzer, after vapor acidification (HCl) to remove carbonates (Paerl et al., 2018). After drying at 60 degrees C, the filters were rolled in tin disks and injected into a Costech Analytical Technologies, Inc. Elemental Combustion System CHNS-O ECS 4010 for elemental analysis. Atropine standards were used to develop a calibration curve (C 70.56%, N 4.84%, and carbon response ratio of 0.025 +/-0.003). NIST Buffalo River Sediment Reference Material 8704 (C 3.351% +/-0.017, N 0.20% +/-0.04) and/or Acetanilide Bypass (C 71.09%, N 10.36%, carbon response ratio of 0.055 +/- 0.003) were used for calibration or as a check standard.
Samples for fluorescent base-extracted particulate organic matter (BEPOM) were extracted following Osburn et al. 2012. Briefly, seston on collected filters was extracted using 10 mL of 0.1 M NaOH and stored in the dark at 4 degrees C for 24 hours. Samples were then neutralized with concentrated HCl (~ 100 _L) to measured neutral pH (~ 7.0) and filtered through 0.2 _m porosity, PES filters. Filtered extracts were immediately analyzed for absorbance and fluorescence as described below. For absorbance and fluorescence, DOM and neutralized BEPOM samples were filtered through 0.2 _m mesh size, polyethersulfone (PES) filters immediately prior to analysis to ensure optical consistency.
Absorbance spectra for filtered DOM and extracted BEPOM samples were measured on a Shimadzu UV-1700 Pharma-Spec spectrophotometer. Absorbance spectra were corrected using a Nanopure water blank measured at the beginning of each day of analysis. All samples with > 0.4 raw absorbance units at 240 nm were diluted, and final results were corrected for dilution (Osburn et al. 2012). Absorbance values at 254 nm were converted to Napierian absorbance coefficients (a_, 1/m) (Spencer et al. 2013). Specific UV absorbance (SUVA254) (L/mg C/m) was calculated as decadal a254/[OC] (as [DOC] or [POC], respectively) for each sample (Weishaar et al. 2003).
Fluorescence spectra (i.e., excitation-emission matrices, EEMs) were measured on a Varian Cary Eclipse spectrofluorometer. Excitation wavelengths were scanned from 240 to 450 nm at 5 nm increments, and emission wavelengths were scanned from 300 to 600 nm at 2 nm increments. Instrument excitation and emission corrections were applied to each sample in addition to corrections for inner-filtering effects, calibrated against the Raman signal of Nanopure water, and standardized to quinine sulfate equivalents (Q.S.E.) (Murphy et al. 2013).
The humification index (HIX) and biological index (BIX) were calculated from measured fluorescence spectra and used as indicators of the relative quality of OM in estuaries from more terrestrial, humic-like OM to more biological, autochthonously produced OM (Huguet et al. 2009). HIX is the ratio of the H (435-480 nm) and L (300-345 nm) regions of fluorescence measured at an excitation wavelength of 254 nm. HIX is indicative of the degree of humification and aromaticity of the fluorescent organic matter (OM) in a sample and generally decreases down estuary. BIX is calculated as the ratio between the _ (380 nm) (Peak M) and _ (430 nm) (Peak C) regions of fluorescence measured at an excitation wavelength of 310 nm. BIX is an indicator of autochthonous, recently produced fluorescent OM and generally increases down estuaries (Huguet et al. 2009). In addition to fluorescent indicators such as HIX and BIX, peak-picking methods were used to identify previously selected and characterized EEM fluorescent peaks from the literature (Coble 2007; Fellman et al. 2010).
Chlorophyll-a (Chl a) analysis
Chl a concentration was measured using the modified in vitro fluorescence technique in EPA Method 445.0 (Welshmeyer 1994, Arar et al. 1997): Fifty milliliters of each water sample were vacuum filtered (less than 25 kilopascals) through duplicate filters at low ambient light conditions using 25 mm Whatman glass microfibre filters (GF/F). The filters were blotted dry, wrapped in foil and frozen immediately at -20 degrees C until analysis. Chl a was extracted from the filter using a tissue grinder and 10 mL of 90 percent reagent grade aqueous acetone. The samples remained in the acetone overnight at -20 degrees C. The extracts were filter-clarified using a centrifuge and analyzed on a Turner Designs TD-700 fluorometer that was configured for the non-acidification method of Welschmeyer (1994). The value reported is the average Chl a concentration measured from the two filters. The fluorometer was calibrated with a known concentration of pure Chl a that was determined using a TurnerDesigns Trilogy fluorometer. The calibration was checked daily against a solid secondary standard.
References
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