Reference materials are important tools for maintaining high-quality assurance standards, including for biological materials. A significant number of environmental international standards, including biodegradability and toxicity, involve utilization of activated sludge (AS) inocula. The absence of inoculum standardization in these tests is a potential source of error influencing the results. In this study three defined microbial consortia, two commercial inocula and a designed bacterial inoculum, were evaluated as an AS alternative seed for the Zahn-Wellens test, using diethylene glycol as the reference chemical. The results showed that to achieve diethylene glycol biodegradation with these inocula, a number of 10(5) colony-forming units per milliliter of effective degrader microorganisms had to be present. Moreover, the addition of AS supernatant to the test mixtures improved inocula performance (the biodegradability curves), bringing them closer to those obtained with AS inocula. Among the three defined consortia, the designed inoculum replicates bested the AS behavior in the range of concentrations tested, with degradation completed in 12-14 days. Comparisons of the community substrate utilization profiles corroborated these results, showing that the designed inoculum profile was the most similar to the AS profile. The biodegradability and metabolism results provided support for the assertion that the designed inoculum can be used in the Zahn-Wellens test and as a base to develop reference inocula for other biodegradability and toxicity tests.