In a previous work, a reproducible procedure to produce a new biosynthetic tracer was developed. This new tracer is an MS2 bacteriophage with enzymatic probes grafted Hi its surface, which can induce enzymatic activity of the tracer. In this paper, the biochemical and physicochemical characteristics of this new tracer are determined. A protocol was developed to determine the specific enzymatic activity kcat(TRACER) of the tracer, which was found to be 2.93 +/- 0.78 x 10(4) min(-1) on average. Physicochemical characterizations of this new tracer showed that it is representative of viruses and nay thus be used as a virus surrogate to assess the virus retention of membrane systems inline. Notable, the mean diameter and molecular weight of the tracer were found to be respectively 64.1 +/- 0.3 nm and 12140 +/- 3654 kDa, which are within the size and molecular weight ranges of pathogenic viruses carried by water. The tracer surface was also studied and revealed the considerable porosity of the grafted probe layer, with a mean porosity of 88%, which could explain why the zeta potential of the tracers (-14.34 +/- 1.66 mV) was nearly the same as that of the native MS2 phages. Finally, a comparison between filtration of the reference microorganism used for membrane performance assessment (the MS2 phage) and the tracer suspensions showed the same filtration behaviour. (C) 2011 Elsevier B.V. All rights reserved.