Development of a "smart" tracer: The resazurin-resorufin assay of biological activity

A smart tracer is a tracer that provides, directly or through measurement of its concentration or in combination with another compound, at least 1 bit more information than a conservative tracer. In other words, the tracer provides information about conditions in the hydrologic system in addition to arrival time such as chemical conditions or biological activity. We have developed a smart tracer for quantifying biological activity along a flow path. We identified and characterized a suitable compound, resazurin (Raz), along with its reduction product resorufin (Rru). Resazurin is a weakly fluorescent redox-sensitive phenoxazin dye that undergoes an irreversible reduction to strongly fluorescent resorufin under mildly reducing conditions - most commonly in the presence of living bacteria. The Raz-Rru system has been widely used to test for the presence and viability of bacteria and other living cells. We characterized the decay, reaction, sorption and transport behavior of Raz and Rru in various waters and sediments. The compounds are stable (decay and reaction timescales of 100s-1000s of h) in natural water, except in the presence of strong light. Raz rapidly reacts to Rru in sediment (timescale of 1 h). The compounds are affected by significant sorption, with a Kd of 6.63 mL/g in sediment with 2.19% organic carbon. A set of mathematical models of the tracer concentrations were developed. Lastly, we conducted a successful field test of the tracer in the Riera de Santa Fe at Montseny, Catalonia, Spain. Results of these tests will be reported.