An isotope dilution method to measure nitrification rates in the northern Gulf of Mexico and other eutrophic waters

An isotope dilution method is described to determine nitrification rates in the northern Gulf of Mexico (NGOMEX) and other eutrophic waters using high performance liquid chromatography (HPLC). A N15H4+ isotope tracer method was not successful for NGOMEX waters due to excessive levels of unlabeled nitrogen (N) captured on sample filters. Nitrification rates were estimated by measuring the dilution of added N15O3− by N14O2−+N14O3−(=14NOX) generated from in situ  N14H4+ during sample incubations. NOX in pre- and post-incubation samples was reduced to NH4+ with zinc (Zn) dust following sample acidification. Samples were analyzed by HPLC for NH4+ concentration and atom% 15N composition. Results were corrected for background natural abundance NH4+ concentrations measured in sub-samples collected before the reduction step. Nitrification rates were calculated using the difference between initial and final corrected NOX isotopic ratios and concentrations before and after the incubations. Laboratory experiments demonstrated that Zn reduction of NOX to NH4+ averaged 102% (±0.2–0.8%) within 30 min. Different levels of N14O3−were added to sub-samples of coastal sea water containing a pool of added N15O3− to simulate a range of nitrification rates. Analysis of the resulting solutions produced a linear relationship (R2=0.9895; p  <0.01) between added N14O3− concentration and isotope ratio and indicated a detection sensitivity of better than 2 atom%, making the method suitable for eutrophic waters with high nitrification rates. Nitrification rates estimated using both isotope dilution and tracer methods in coastal waters produced comparable results. Nitrification rates of up to 150 nmol N L−1 h−1 were measured in the NGOMEX hypoxic zone by isotope dilution. Advantages of the method include: (1) unaltered substrate (NH4+) concentrations, (2) small sample volume, (3) minimal sample handling, and (4) direct 24 h nitrification rate assessment.