Aquatic Toxicology 67 (2004) 167–179
Effects of triclosan on the early life stages and reproduction of medaka Oryzias latipes and induction of hepatic vitellogenin Hiroshi Ishibashi a , Naomi Matsumura a , Masashi Hirano a , Munekazu Matsuoka a , Hideki Shiratsuchi a , Yasuhiro Ishibashi b , Yuji Takao c , Koji Arizono a,∗ a
Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502, Japan b Environmental Protection Center, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan c Faculty of Environmental Studies, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan Received 11 September 2003; received in revised form 5 December 2003; accepted 8 December 2003
Abstract Triclosan (2,4,4 -trichloro-2 -hydroxydiphenyl ether) is widely used as antibacterial agent in various industrial products, such as textile goods, soap, shampoo, liquid toothpaste and cosmetics, and often detected in wastewater effluent. In this study, the effects of TCS on the early life stages and reproduction of medaka (Oryzias latipes) were investigated. The 96-h median lethal concentration value of TCS for 24-h-old larvae was 602 g/l. The hatchability and time to hatching in fertilized eggs exposed to 313 g/l TCS for 14 days were significantly decreased and delayed, respectively. An assessment of the effects of a TCS 21-day exposure period on the reproduction of paired medaka showed no significant differences in the number of eggs produced and fertility among the control and 20, 100 and 200 g/l TCS treatment groups. However, concentrations of hepatic vitellogenin were increased significantly in males treated with TCS at 20 and 100 g/l. In the F1 generations, although the hatching of embryos in the 20 g/l treatment showed adverse effects, there was no dose–response relationship between hatchability and TCS treatment levels. These results suggest that TCS has high toxicity on the early life stages of medaka, and that the metabolite of TCS may be a weak estrogenic compound with the potential to induce vitellogenin in male medaka but with no adverse effect on reproductive success and offspring. © 2003 Elsevier B.V. All rights reserved. Keywords: Triclosan; Oryzias latipes; Early life stage; Reproduction; Vitellogenin
1. Introduction Recently, a number of studies have been performed worldwide that examine endocrine-disrupting chemicals (EDCs) and their interactions with the develop∗ Corresponding author. Tel.: +81-96-383-2929x486; fax: +81-96-383-6062. E-mail address:
[email protected] (K. Arizono).
ment and functioning of various systems in animals and humans (Colborn et al., 1993; Colborn et al., 1996; Jobling et al., 1998). Altered hormone status or gonad histology has been reported in fish inhabiting water released from wastewater treatment plants and the induction of plasma vitellogenin (VTG, egg yolk protein precursor) has been recorded in male rainbow trout (Oncorhynchus mykiss) caged in a polluted stream (Harries et al., 1997; Steven et al., 1997).
0166-445X/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2003.12.005
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These alterations in the physiology of fish may be linked to declines in fish populations; therefore, various screening and testing systems for EDCs have been established (Organization for Economic Cooperation and Development, 1999; U.S. Environmental Protection Agency, 1998). Some laboratory assays report a reduction in fecundity and/or fertility of fish exposed to estrogenic compounds, such as 17-estradiol (Kang et al., 2002), 4-nonylphenol (Giesy et al., 2000), and bisphenol A (Kang et al., 2002). Many of these chemicals have been detected in aquatic environments (Kolpin et al., 2002), and can adversely affect the reproductive health of freshwater and marine fish populations. Therefore, there is a need to evaluate the reproductive biology of fishes exposed to EDCs. Triclosan (TCS, 2,4,4 -trichloro-2 -hydroxydiphenyl ether) is widely used as an antibacterial agent in liquid toothpaste, soap, shampoo, and cosmetics (Black et al., 1975; Yuge, 1983), and is frequency found in wastewater effluent. Water samples collected near the outfall of a wastewater treatment plant in Rhode Island, USA, showed 10–20 g/l of TCS in the effluent and 80–100 g/g of TCS in the sediment (Lopez-Avila and Hites, 1980). TCS and its chlorinated derivatives are readily converted into various chlorinated dibenzo-p-dioxins by heat and ultraviolet irradiation (Kanetoshi et al., 1987, 1998a, 1998b). Miyazaki et al. (1984) have also detected methylated TCS in fishes and shellfishes from Tokyo Bay and the Tama River of Japan. The U.S. Geological Survey used five newly developed analytical methods to measure the concentrations of 95 chemical compounds (such as pharmaceuticals, hormones, and other organic wastewater contaminants) in water samples from a network of 139 streams across 30 states during 1999 and 2000 to provide the first nationwide reconnaissance of the occurrence of organic wastewater contaminants in water resources and reported that TCS was one of the most frequently detected compounds (maximum contaminant levels; 2.3 g/l) (Kolpin et al., 2002). Orvos et al. (2002) studied the aquatic toxicity of TCS in activated sludge microorganisms, algae, invertebrates, and fish and reported a median effective concentration for Daphnia magna of 390 g/l at 48 h and median lethal concentrations for Pimephales promelas and Lepomis macrochirus of 260 and 370 g/l, respectively, at 96 h. Additionally, the chemical structure of TCS closely resembles known non-steroidal
estrogens (e.g. diethylstilbestrol and bisphenol A). Recent studies have demonstrated changes in fin length and non-significant trends in the sex ratio of medaka exposed to TCS, and suggested that it was potentially a weakly androgenic contaminant (Foran et al., 2000). Hanioka et al. (1997) reported that 7-benzyloxyresorufin O-debenzylase (BROD) and 7-pentoxyresorufin O-depentylase (PROD) activities, which are associated with CYP2B1 activity, were remarkably induced by all doses of TCS in rats. These results suggested that TCS induces the P450 isoforms of the CYP2B subfamily in the rat liver, and that the induced P450 isozymes were closely related to the toxicity of TCS or its chlorinated derivatives. However, the potential for TCS to act as an EDC has not been studied in depth nor has it potential to disrupt reproductive function in fish. Medaka (Oryzias latipes) is widely used in ecotoxicology and has been proposed to be a suitable fish for evaluating EDCs. Medaka is also a suitable test organism for assessing reproductive effects because its large eggs with clear chorions can easily be observed. Early life stage toxicity tests using medaka are also considered to be sensitive biosensors. In this study, we used medaka as a test organism, and investigated the effect of TCS on the early life stage and reproduction of medaka. Furthermore, we evaluated the induction of hepatic VTG in male medaka, 7-ethoxyresorufin O-deethylase (EROD) and PROD activities in female liver microsomes exposed to TCS, and also measured the estrogenic activity of TCS using a yeast two-hybrid assay in vitro.
2. Materials and methods 2.1. Test chemical TCS (>98.0% purity) was obtained from Wako Pure Chemical Industries Ltd., Tokyo, Japan and dissolved in dimethyl sulfoxide (DMSO, Wako Pure Chemical Industries) to prepare test solutions. 2.2. Test organisms Sexually mature medaka were purchased from a local fish farm in Kumamoto, Japan, and were maintained in glass tanks in our laboratory. In the
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pre-exposure period, 40 mating pairs were selected and each pair was placed in a 1-l glass beaker under a 16:8 light:dark photoperiod at 25 ± 1 ◦ C. The fish were fed a diet of Artemia nauplii (
