Excitation emission matrix (EEM) of fluorescence intensity has been often used to characterize dissolved organic matter in the aquatic environment. On EEM, there is a peak (Peak X) around excitation (emission) wavelengths of 490 (520) nm that is detected only in municipal wastewater-related samples. Peak X has been reported in very few papers. We investigated the characteristics of substances associated with Peak X from various perspectives. Based on that information, we hypothesized that Peak X was derived from fluorescein. This hypothesis was supported by the results of several estimates in this study, and it was revealed for the first time that Peak X on EEM was derived from fluorescein contained in colored bath salts widely used in Japan. We used Peak X to quantify how effluent from a wastewater treatment plant (WWTP) was dispersed in the outfall area of the plant and to evaluate its impact on the water quality to the area. By using Peak X, it was shown that the influence of the WWTP effluent on the discharged area extended several hundred meters. In other words, it was quantitatively demonstrated that the effluent significantly contributed to the increase in nutrient concentrations and primary production. These findings are expected to be highly useful for the control of nutrient concentrations in river water using the WWTP effluent, which has been the focus of much attention in recent years.