Abstract
Retinoic acid receptor (RAR) antagonists are potential toxic compounds that can cause teratogenesis in vertebrates. This study was conducted to evaluate the occurrence of RAR antagonist contamination in aquatic environments and identify its potential sources in detail. To accomplish this, the RAR antagonistic activities of surface waters of two rivers (the Yodo River and the Ina River) and influents and effluents of municipal wastewater treatment plants (WWTPs) in the Kinki region of Japan were investigated using a yeast two-hybrid assay. In the investigated rivers, remarkable RAR antagonistic activities were detected relatively consistently in specific regions, although the levels varied with time, and tended to increase downstream of municipal WWTPs. Investigations of WWTPs also revealed that RAR antagonists were present at remarkably high levels in municipal wastewater, and that RAR antagonist contamination remained in effluent after activated sludge treatments. Comparison of the concentration factors that reduced 50% of the RAR agonistic activity of 10(-7) M all-trans retinoic acid (IC50) for selected river water and WWTP effluent samples revealed that the contamination levels were greater in effluent (IC50: concentration factors of 92-313) than river water (IC50: concentration factors of 10.2-68.9). These results indicate that municipal WWTPs could be an important source of RAR antagonist contamination in the receiving rivers. Fractionations with high-performance liquid chromatography directed by the bioassay indicated that there were multiple RAR antagonists in municipal wastewater. Although a trial to identify the causative compounds in municipal wastewater was not completed, multiple bioactive peaks that should be studied further were isolated. This study clarified the occurrence of novel endocrine disrupting chemicals (i.e., RAR antagonists) in the aquatic environment at the watershed level and identified their possible source for the first time, which suggests the need of further studies to identify the causative compounds and to assess possible ecological risks associated with the contamination.