Influence of sample volume on nitrate N and O isotope ratio analyses with the denitrifier method

Rationale Analyses of the isotope ratios of nitrogen (N-15/N-14) and oxygen (O-18/O-16) in nitrate (NO3-) with the denitrifier method require relatively high sample volumes at low concentrations (<= 1 mu M) to afford sufficient analyte for mass spectrometry, resulting in isotopic offsets compared to more concentrated samples of the same isotopic composition. Methods To uncover the origins of isotopic offsets, we analyzed the N and O isotope ratios of NO3- reference materials spanning concentrations of 0.5-20 mu M. We substantiated the incidence of volume-dependent isotopic offsets, then investigated whether they resulted from (a) incomplete sample recovery during N2O sparging, (b) blanks - bacterial, atmospheric, or in reference material solutions - and (c) oxygen atom exchange with water during the bacterial conversion of NO3- to N2O. Results Larger sample volumes resulted in modest offsets in delta N-15, but substantial offsets in delta O-18. N2O recovery from sparging was less complete at higher volumes, resulting in decreases in delta N-15 and delta O-18 due to associated isotope fractionation. Blanks increased detectably with volume, whereas oxygen atom exchange with water remained constant within batch analyses, being sensitive to neither sample volume nor salinity. The sizeable offsets in delta O-18 with volume are only partially explained by the factors considered in our analysis. Conclusions Our observations argue for bracketing of NO3- samples with reference materials that emulate sample volumes (concentrations) to achieve improved measurement accuracy and foster inter-comparability.