ENVIRONMENTAL BIOTECHNOLOGY The effect of calcium ion on the biodegradation of octylphenol polyethoxylates, and the antiandrogenic activity of their biodegradates Atsushi Shibata & Yoichi Ishimoto & Yosuke Nishizaki & Akifumi Hosoda & Hiromichi Yoshikawa & Hiroto Tamura Received: 20 June 2007 / Revised: 23 July 2007 / Accepted: 29 July 2007 / Published online: 6 September 2007 # Springer-Verlag 2007 Abstract Because limes have been used as important fertilizers to neutralize acidified farmland in Japan, our interest in this study was focused on the effect of calcium ion on the biodegradation of octylphenol polyethoxylates (OPEOn) by a pure culture of Pseudomonas putida S5 isolated from a rice paddy field in Japan. In the presence of calcium ion, P. putida S5 accelerated the formation of octylphenol oligoethoxy carboxylates (OPECn) rather than that of octylphenol oligoethoxylates under an aerobic condition, indicating that more soluble biodegradates with terminal carboxyl group may liquate out easily to surface and ground water rather than more hydrophobic biodegra- dates with shorter ethylene oxide residues. Therefore, the androgen receptor (AR) activity of their degradation products was characterized using an in vitro reporter gene assay. As ethylene oxide chain length decreased, the biodegradates, OPEOn (n <3), increased their AR antago- nist activity. However, OPECn (n <3) were unable to determine their AR activity because of their cytotoxicity in our reporter gene assay system. Keywords Alkylphenol . Androgen receptor . Endocrine disruptor . MDA-kb2 . Pseudomonas putida . Reporter gene assay Introduction Because alkylphenol polyethoxylates (APEOn) are widely used surfactants in domestic and industrial products, alkylphenols (APs) are frequently found in an aquatic environment throughout the world, and are now well- known estrogen agonist, which are capable of interfering with nuclear receptor functions and causing reproductive and developmental disorders in mammals, animals inhabit- ing aquatic environments, and in birds (Soto et al. 1991; Jobling and Sumpter 1993; White et al. 1994; Renner 1997; Quiros et al. 2005). Recently, the Japanese government announced that octylphenol acts as endocrine disruptors, especially in fishes http://www.env.go.jp/press). Therefore, the growing public concern has led to efforts in identifying and characterizing the mechanisms of their environmental pollution in rivers and ground water caused by their excess use. Until recently, a number of research results have been accumulated on the fate and behavior of APs and APEOn in an aquatic environment (Ahel et al. 1994a, b, 1996; Field and Reed 1996; Ying et al. 2002; Yuan et al. 2004). Furthermore, because APEOn have also been employed in the formulation of pesticides as one of the most popular additives (Stevens and Bukovac 1987; Knoche and Bukovac 1993; Tamura et al. 2001), they have been suspected as one of the putative sources of AP contamination. However, their precise Appl Microbiol Biotechnol (2007) 77:195–201 DOI 10.1007/s00253-007-1138-y A. Shibata : Y. Ishimoto : Y. Nishizaki : A. Hosoda : H. Tamura (*) Department of Environmental Biology, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan e-mail: hiroto@ccmfs.meijo-u.ac.jp H. Yoshikawa Department of Life, Environmental and Material Science, Fukuoka Institute of Technology, Fukuoka 811-0295, Japan Present address: A. Shibata Department of Geotechnical and Environmental Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan