Despite the importance of behavioral ecotoxicological tests, few researches have been conducted to assess the quality of freshwater using behavior of test species, in part due to lack of standardized test method for quantifying the response. In this study, we developed a new test methods using non-biting midge larva’s burrowing behavior to assess field water quality and a new index, AUC (area under curve), to compare burrowing behavior quantitatively. Four kinds of field samples (residential, natural, agricultural and industrial areas) were tested using the method to validate our methodology and index. There were not significant differences in 96 h mortality and heavy metal concentration between samples, but AUC from the industry sample was significantly decreased.