Water Filtration and Nutrient Analysis

Upon return to the laboratory, all samples were gently rotated ~ 10 × to ensure water was well-mixed prior to processing. Then, using a 30 ml syringe, water from each replicate was filtered through a 25 mm, 0.7 μm pore size glass fiber filter (GF/F) (Whatman^®) into acid-washed (as described previously) 20 ml glass scintillation vials (~ 17 ml final volume) for DIN: nitrate + nitrite (NO₃⁻ + NO₂⁻; henceforth N + N, ammonia-N (NH₃ + NH₄⁺; henceforth AmN), dissolved inorganic phosphorus (DIP): orthophosphate (PO₄³⁻), total dissolved N (TDN), and phosphorus (TDP). Sample vials were then frozen (−20 °C) until analysis. Nutrient samples were analyzed within 1 h of thawing using a Hach^© Lachat QuikChem 8500 Flow Injection Analysis System (Strickland and Parsons 1984 ; Grasshoff et al. 1999 ). Protocol Minimum Detection Limit (MDLs) were as follows: AmN (0.05 μM; Lachat Applications Group 2017 ), N + N (0.014 μM; Egan 2008 ), PO₄³⁻ (0.0646 μM; Egan 2007 ), TDN and TDP (0.485 and 0.123 μM, respectively; Tucker et al. 2008a , 2008b ). MDLs were used when measured concentrations were below those values. DON and DOP concentrations were calculated per replicate as TDN–DIN and TDP–DIP, respectively. DOC samples were collected following standard procedures (Ducklow and Dickson 1994 ; JGOFS 1996 ), specifically by filtering water through a 30 ml syringe (previously soaked in 10% HCL for 24 h then rinsed 3 times with ddH₂O, equipped with a pre-combusted (2 h at 450 °C) GF/F) into a pre-combusted (4 h at 450 °C) 20 ml glass scintillation vial. Following addition of two to three drops of 10% HCl, vials containing samples were stored (4 °C) prior to analysis (in duplicates), and subsequently analyzed by the University of Wisconsin–Milwaukee. DOM was calculated as the sum of DON, DOP, and DOC.