A Critical Assessment of Relating Resazurin-Resorufin Experiments to Reach-Scale Metabolism in Lowland Streams

The reactive tracer resazurin is frequently used to investigate metabolic processes and hyporheic exchange in streams, because its transformation to resorufin is considered a function of oxygen turnover in the presence of living cells and thus of ecosystem respiration. This has been investigated and confirmed in a number of laboratory and batch studies but so far not unanimously been verified in the field. In this study we conducted 13 tracer tests with resazurin in different small, low-gradient streams typical of lowland regions. We determined tracer processing rates and ratios based on metrics commonly used to assess reactive-tracer tests with resazurin and compared them to flow characteristics and respiration rates determined from diel-oxygen recordings following a two-station approach. We found no statistical relationship between tracer processing and parameters of transport, indicating that resazurin-to-resorufin transformation cannot be explained by hydrological parameters alone. The statistical relationship between resazurin processing and respiration rates based on diel-oxygen profiles was strongest if resazurin-to-resorufin transformation ratios were used, and uptake lengths were also found to be a good proxy. However, the relationships were not linear, and weak for individual streams, contrary to what was expected based on previous laboratory and batch studies. We attribute this finding to a lack of process understanding of resazurin reactions under field conditions, and generally insignificant hyporheic exchange fluxes in lowland streams. Our findings highlight important limitations of the tracer technique under field conditions. Plain Language Summary Resazurin is a compound that can be used to track the flow of water and metabolic processes in rivers. This has been tested in laboratory experiments before, but until now the method has not been fully verified in the field. In this study we apply resazurin to 13 different small rivers and measure its transformation. We compare our results to other, independent methods that are often used to determine metabolic processes. We find good relationships between the two different methods for all rivers together, but not for individual rivers. Also, the relationships are not linear, which makes predicting metabolic rates from the transformation of resazurin difficult.