Seawater was collected on the research vessel Fay Slover using 5-liter (L) Niskin bottles along a gradient from the open ocean near the Chesapeake light tower into the Chesapeake Bay. Conductivity, temperature, and depth were measured using a Seabird SBE 32 CTD equipped with a Wetlabs fluorometer and a Wetlabs transmissometer (650 nanometers (nm)).

Field collection of ATP
Using biological oxygen demand (BOD) bottles, samples of 150 - 300 milliliters (mL) (depending on station) were taken from the Niskin bottles at five stations and six depths along the transect. Water was filtered through 25-millimeter (mm) GF/F filters using large capacity filter funnels at a vacuum of 200 millibars (mbar). Once filtered, and without keeping the filters running dry for more than a few seconds, the filters were quickly transferred into 1.5 mL cryovials filled with 1 mL of a solution of benzalkonium chloride (1% w/w fin. conc.) with phosphoric acid (5% w/w fin. conc.) in a 25 millimolar (mM) Tricine buffer (PBAC, Bochdansky et al. 2021). The filters were extracted in PBAC for 20 to 30 minutes before the cryovials (including the filters) were frozen and kept at -80 degrees Celsius (°C) before analysis. The location and methods were similar to those published in Bochdansky et al. 2024.

Laboratory analysis of ATP
Ten microliters (μL) of each sample (in triplicates) were transferred to 6 mL pony scintillation vials (Research Products International), and received 3 mL of ultrapure water, and 50 μL of CellTiter-Glo 2.0 (Promega Corporation). Internal standards were used by spiking a fourth vial with 50 μL of samples with 50 μL of 0.0164 micromolar (μM) ATP standard. Using internal standards instead of separate calibration curves corrects for matrix effects that change the luminescence signal caused by the presence of ions, acids, and organic material (Bochdansky et al., 2021). Luminescence was analyzed in a PerkinElmer liquid scintillation counter model Tricarb 3110 TR with a single photon counting protocol of 1 minute each. ATP was calculated using equation 2 in Bochdansky et al. (2021).

Particulate organic carbon and nitrogen
Between 250 and 500 mL of seawater (less in the inshore and more in the offshore stations) was filtered onto pre-combusted (450°C, 4 hours) GF/F filters. The filters were stored frozen (-20°C) and later dried at 50°C for approximately 24 hours, then acidified under concentrated HCl fumes in a desiccator, redried for another 24 hours at 50°C and then capped and stored in a desiccator. Filters were rolled in a tin wrap and pressed into pellets to be analyzed in a CHN analyzer (ODU Department of Chemistry Water Quality Laboratory).