The bioaccumulation of nonyphenol and its adverse effect on the liver of rainbow trout (Onchorynchus mykiss)

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Abstract

Alkylphenol polyethoxylates (APEs) are widely used as nonionic surfactants. Nonylphenol (NP), one of the derivatives of APEs, has been found in the aquatic environment in ranges from nanograms per liter to milligrams per liter. In this study, juvenile rainbow trout were exposed to 0 (control), 66, 220, or 660 μg NP/L for up to 28 days. Fish remained healthy under NP exposures of 0, 66, and 220 μg/L for the length of the experiment. All fish died after 4 days of exposure to 660 μg NP/L. Time-dependent NP bioaccumulation was detected in the tissues of fish exposed to 220 μg NP/L (P<0.05) and histopathological changes were observed in the livers of fish exposed to 220 μg NP/L. Furthermore, an increase in the activity of glutathione-S-transferase (GST) was found in the liver of fish exposed to 220 μg NP/L for 1 week (P<0.05). There was an increase in GST activity in the liver of fish exposed to 66 μg NP/L but it did not occur before 2 weeks of exposure to NP. The GST activity then decreased in a time-dependent manner in treatment groups, and this decrease was lower in the livers of fish treated with 66 and 220 μg NP/L than in control fish after 3 weeks of exposure (P<0.05). These results indicated that sublethal doses of NP were accumulating in the bodies of the fish and causing histopathological and biochemical changes in the livers of rainbow trout.

Introduction

Some man-made chemicals have been shown to exert estrogenic activity (Jobling and Sumpter, 1993; Jobling et al., 1996). These chemicals are hormone-like compounds and are often referred to as environmental endocrine disrupters. A well known group of environmental endocrine disrupters is the alkylphenol polyethoxylates (APEs), which are widely used as nonionic surfactants and antioxidants in detergents, pesticides, herbicides, paints, cosmetics, and plasticware (reviewed in Nimrod and Benson, 1996). The annual worldwide production of APEs in 1997 exceeded 500,000 metric tons (Renner, 1997), with an estimated 60% of this production ending up in the bodies of water around the world.

Alkylphenol polyethoxylates undergo a biodegradation process to give derivatives such as octylphenols (OP), butylphenols (BP), and nonylphenols (NP). It has been shown that these derivatives have estrogenic (Jobling et al., 1996), toxic (Burkhardt-Holm et al., 2000; Hughes et al., 2000), and carcinogenic effects (Blom et al., 1998). For example, NP has been shown to stimulate the synthesis of female-specific egg-yolk protein, vitellogenin, in the livers of male fish, including rainbow trout (Jobling et al., 1996), eelpot (Christiansen et al., 1998), and carp (Gimeno et al., 1998). Furthermore, NP has been shown to inhibit spermatogenesis in adult rainbow trout (Jobling et al., 1996), induce sex reversal in Japanese medaka (Gray and Metcalfe, 1997), cause histopathological changes in the germ and Sertoli cells of the male eelpot (Christiansen et al., 1998), bring about proliferation in human breast cancer (Blom et al., 1998; Legler et al., 1999), and cause testicular and ovarian cancer in mice (Skakkebaek et al., 1998). Burkhardt-Holm et al. (2000) demonstrated the toxic effects of NP with an exposure of 10 μg NP/L, causing damage in the granulation pattern of epidermal mucous cells in rainbow trout. Schwaiger et al. (2000) also showed that estrogen, but not 15 μg NP/L, induced histopathological lesions in liver, kidney, and spleen of carp.

Alkylphenol polyethoxylate pollution has been reported in many countries. One of the derivatives of alkylphenol, NP, has been found in the rivers and lakes of many countries. For example, the concentrations of NP were between 2 and 336 μg/L in British rivers (Blackburn and Waldock, 1995), 0.3 and 45 μg/L in Swiss rivers (Ahel et al (1994a), Ahel et al (1994b)), and 3 and 300 μg/L in Canadian rivers (O’Halloran et al., 1999). Hale et al. (2000) reported that NP released into sewage effluent reached concentrations of up to 12 mg/L in the USA.

In the present study, the relationship between the environmentally observed concentrations of hazardous NP and its rate of bioaccumulation was investigated in one of the most widely consumed fish, rainbow trout (Onchorynchus mykiss).

It has been reported that NP exerts its biochemical effects by downregulating the activity of microsomal cytochrome P4501A (CYP1A) while stimulating an increase in the level and activity of the CYP3A protein in the livers of mammals (Lee et al (1996a), Lee et al (1996b)) and fish (Arukwe et al., 1997). It also has been reported that alkylphenols are metabolized in the liver and excreted through bile and urine. Therefore, a histological study was conducted to determine histopathological changes in the livers of rainbow trout. In the liver, glutathione-S-transferase (GST) is an important part of the cellular detoxification system and has evolved to protect cells against reactive metabolites (Landi, 2000). Since there has been no report concerning the activity of GSTs in response to alkylphenol exposure, we conducted a biochemical study in the liver of rainbow trout to determine the changes in GST activity.

Section snippets

Instruments and chemicals

The Shimadzu HPLC pump (Model LC-9A), spectrophotometer (Model UV-160A), and integrator (Model CR-6A) were employed in the study. We used HPLC-grade methanol obtained from Merck (Dormstad, Germany), a Luna C18 HPLC column (250×4.6 mm, 5 μL) and a C18 BOND ELUT octadecyl silica cartridge (100 mg/mL) obtained from Phenomenex (Aschaffenburg, Germany), and 4-nonylphenol purchased from Aldrich (Southampton, UK).

Fish

Six-month-old juvenile rainbow trout were supplied by the ER-SU fish farm (Kesikköprü,

Results

The HPLC assay to detect the NP in the fish was found to be linear, in the range of 5.5–220 μg/mL (Table 1). The minimum peak area obtained (Y) from HPLC increased linearly (P<0.01) in response to increasing concentrations of NP (X). The equation for the relationship of minimum peak area and NP concentration was Y=−370.63+53238.305X, r2=0.99.

Discussion

To date, the amount of alkylphenol pollution detected in aquatic environments ranges from nanograms per liter to milligrams per liter. For example, the amount of alkylphenol detected in water ranged from 0.11 μg/L in drinking water in the USA (Naylor et al., 1992) to 336 μg/L in the rivers of England (Blackburn and Waldock, 1995). Hale et al. (2000) reported that the amount of NP in the effluent reached 12,000 μg/L, whereas NP in sediment reached 70,000 μg/kg in the rivers of the USA.

In the present

Acknowledgements

The present study was a continuation of a field study conducted to determine the alkylphenol pollution in Turkish rivers, that was supported by the Directorate of Agricultural Research, Ministry of Agriculture and Rural Affairs. The authors thank the members of the Trabzon Fisheries Central Research Institute, namely, Yildiz Eroglu, Ilyas Tabak, Mustafa Zengin, and Yilmaz Yazar, Head of the Fisheries Department, for their help in providing the aquarium. The authors also thank Atilla Ertürk, the

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