Xenoestrogen Status of Wuling Farm to Surrounding Water Bodies: An Application of Biochemical Parameters Using Onychostoma barbatulum

Abstract

The aim of this study was to apply biochemical parameters to Onychostoma barbatulum to understand whether Wuling Farm activities have caused organic pollution from xenoestrogens to flow into surrounding streams and thereby affected area aquatic ecosystems. Individuals of the Taiwan shovel-jaw carp (O. barbatulum) were collected by fyke netting in the Cijiawan, Kaoshan, and Yusheng Rivers in the protected area of Shei-Pa National Park and in the Ikawan River outside the protected areas from 4–5 June 2015 to 20–21 September 2015. The collected male individuals of O. barbatulum were divided into two groups. In the first group monooxygenase (Mon) activity, glutathione S-transferase (GST) activity, and vitellogenin (VTG) concentration in the liver were measured right after they were captured. Those in the second group were acclimated for 14 days and then exposed to 0 (control) or 10 ng/L 17β-estradiol for 10 days and then measured for changes in Mon, GST, and VTG in their livers. The value of VTG concentrations in O. barbatulum individuals collected from the Ikawan River was 2.12 ± 1.62 μg PO₄²⁺/g protein, which was the highest among all samples, followed by those collected from the Yusheng River (0.78 ± 1.00 μg PO₄²⁺/g protein). Individuals collected from the Yusheng River had the highest liver Mon activity (4.16 ± 1.08^△A650/30 min/g protein) and the highest GST activity (1.58 ± 1.13 ^△A340/20 min/g protein), followed by those from the Ikawan River (Mon: 3.63 ± 1.13 ^△A650/30 min/g protein; GST: 1.24 ± 0.73 ^△A340/20 min/g protein). Comprehensive analyses showed that Mon and GST activities and VTG concentrations in livers measured right after being collected were lower than individuals exposed to 10 ng 17β-estradiol/L in the laboratory. Induced VTG concentrations were 9.87 ± 0.89 μg PO₄²⁺/g protein, Mon activity 8.02 ± 1.74 ^△A650/30 min/g protein, and GST activity 3.24 ± 0.62 ^△A340/20 min/g protein, indicating that farming activities have not significantly affected these aquatic organisms. However, pollution sources are still releasing pollutants containing xenoestrogens into the Yusheng River and Ikawan River. The impact of xenoestrogens is worthy of continuous long-term follow-up monitoring.

1. Introduction

Wuling Farm is part of Shei-Pa National Park. It is located in the upper reaches of the Dajia River, which is the main habitat of Taiwan’s treasured national fish, the cherry salmon (Oncorhynchus masou formosanus) [1]. In its early days, Wuling Farm mainly cultivated deciduous fruit trees and vegetables. After the establishment of Shei-Pa National Park, the focus of Wuling Farm gradually changed from farming to tourism-based activities, though some farming activities (planting fruit trees and vegetables on sloping fields) still continue. Chemical substances such as pesticides and fertilizers used in farming can be carried into streams by rainfall and thus affect local water quality [2]. If pesticides function as endocrine-disrupting chemicals (EDCs), they will affect individual organisms, and even organism communities and the stream ecosystem [3].

In recent years, the environmental impact of EDCs has received great attention. Many of them are xenoestrogens that mainly interfere with the physiological functions of animals, such as metabolism, behavior, reproduction, and sexual differentiation [4]. The impact of EDCs is currently a popular topic of scientific research, and among EDCs, xenoestrogens are the most studied [4,5]. Exposure to xenoestrogens can induce vitellogenin (VTG) in male organisms, a protein that they do not normally express. Therefore, VTG in salmonids and cyprinids [6] is an important physiological indicator of the presence of xenoestrogens in water bodies.

Chemical substances can enter organisms through inhalation, ingestion, or skin contact, after which they are transported to different body parts through different physiological functions, blood circulation, or the lymphatic system [7,8]. The monooxygenase (Mon) process (also known as the cytochrome P450 system) is a phase I detoxification system, and is expressed mostly in hepatocytes [9,10]. When toxic substances undergo a series of oxidation, reduction, and hydrolysis reactions in the Mon system, the functional groups of toxic substances change to increase their hydrophilicity to facilitate their excretion. Glutathione S-transferases (GSTs) are indispensable and important enzymes of phase II detoxification that can convert toxic substances into less active and more hydrophilic materials that are easier to excrete. Therefore, the activity of metabolic enzymes such as Mon and GST in an organism can be measured to determine whether the organism is contaminated, thus making them sensitive and reliable biological indicators [11].

The Taiwan shovel-jaw carp (Onychostoma barbatulum) is a member of the Cyprinidae family. O. barbatulum has high water-quality requirements, surviving only in streams with water temperatures of 17–25 °C and having little pollution [12,13]. It is an omnivorous fish that feeds on algae attached to rocks as a staple food, as well as on organic detritus and aquatic insects such as stoneflies and caddisflies. There is a large population of O. barbatulum in the Cijiawan River and neighboring streams, so it is quite suitable as an indicator fish species in the Wuling area. In this study, we used O. barbatulum as the experimental organism to compare VTG concentrations and Mon and GST activities in male fish within and outside the protected areas of Shei-Pa National Park. Other male individuals of O. barbatulum were exposed to 0 or 10 ng/L 17β-estradiol (E2) for 10 days after being collected from sampling sites. The enzyme activities in O. barbatulum individuals exposed to 10 ng/L E2 were compared with those in O. barbatulum individuals exposed to 0 ng/L E2 in order to investigate whether human-produced pollutants in the Wuling area contained xenoestrogens and whether xenoestrogens have affected its water bodies.

2. Materials and Methods

To understand whether human activities have discharged organic pollutants containing xenoestrogens in the area, this study collected male O. barbatulum by fyke netting at four study sites in rivers around Wuling Farm from 4–5 June 2015 to 20–21 September 2015. Three of the study sites were in the protected area of the Shei-Pa National Park: Cijiawan River downstream, 24°22.825′ N, 121°18.577′ E; Kaoshan River downstream, 24°21.470′ N, 121°18.557′ E; and Yusheng River downstream, -24°20.841′ N, 121°18.620′ E. The fourth site was in the Ikawan River (24°19.423′ N, 121°17.000′ E), which is outside the park’s protected area (Figure 1). The collected O. barbatulum males were divided into two groups. The first group was measured for Mon activity, GST activity, and VTG concentration in their livers. The second group, having similar body sizes, were acclimated for 14 days and then exposed to 0 (control group) or 10 ng/L E2 for 10 days prior to analyses for Mon, GST, and VTG in their livers.

2.1. Exposure of O. barbatulum to Different Concentrations of Estrogen

This study used an exposure test to establish the dose-response relationship between VTG and estrogen. After being collected from the wild, O. barbatulum males were cultured in a fiberglass-reinforced polymer (FRP) barrel having a diameter of 100 cm, a height of 120 cm, and a volume of approximately 200 L. During the acclimation period, dissolved oxygen was kept above 5 mg/L, water temperature at 20 ± 2 °C (the same as the test temperature), and light duration at 12 h/day (light:dark = 12 h:12 h). Water quality was maintained by a circulating filter device. After 14 days of acclimation, individuals sized 3.5~4.5 cm were selected for the static exposure test. During this test, the water was changed every 48 h to control E2 concentration. All experiments were performed in a thermostatic chamber with a temperature of 20 ± 1 °C and a light cycle of 12 h. During the experiment, O. barbatulum individuals were exposed to 0 (control group, only acetone was added) or 10 ng/L E2 (lowest-observed-effect level in freshwater fish [14]), each treatment in independent tanks. After 10 days of exposure, Mon and GST activities and VTG concentrations in the fish liver were determined.

The male O. barbatulum were evaluated for levels of vitellogenin-like protein in the liver within 10 days. Estradiol equivalent concentration from fish (EEQ) values were calculated based on the E2-VTG curve from levels of vitellogenin-like protein. The E2-VTG curves, according to the concentration of vitellogenin-like protein and 17β-estradiol exposure, were estimated based on the regression equations calculated in the SigmaPlot 10.0 software (Palo Alto, CA, USA). The value of the estradiol concentration was the estradiol equivalent concentration in the study site.

2.2. Biochemical Examination

Each sample that included exposed test fish or those collected from study sites was anesthetized with MS-222 and their livers homogenized with a Teflon pestle (Kontes, Vineland, NJ, USA) in ice-cold 25 mM Tris-HCl containing EDTA (0.5 M, pH 8). The homogenate was then centrifuged at 10,000× g for 20 min at 4 °C, the supernatant collected and stored at −20 °C, and the analysis was completed within 48 h.

2.3. Determination of Vitellogenin (VTG)

The vitellogenin-like protein level in the liver supernatant was determined using the alkali-labile phosphate (ALP) measurement assay previously described by Huang et al. [15]. A mixture of supernatant (50 μL) and ice-cold 20% trichloroacetic acid (50 μL) was incubated at room temperature for 15 min, followed by centrifugation at 10,000× g for 10 min at 4 °C. The protein pellet was resuspended in 200 μL 1 M NaOH and then heat-treated at 75 °C for 60 min before measurement. The level of free phosphate was determined by the phosphomolybdenum method [16], and optical absorbance at 600 nm was recorded by a microplate reader (Power ware 340, BioTek, Hampton, VA, USA).

2.4. Determination of Monooxygenase (Mon) Activity

Monooxygenase activity was measured based on the modified method previously described by Brogden et al. [17] and Hemingway [18]. Briefly, the assay was started by adding 2 μL of supernatant into 200 μL of working reagent in a microplate. The working reagent was prepared by dissolving 10 mg 3, 3¹,5,5¹ -tetramethyl benzidine in 5 mL methanol and 15 mL of 0.25 M sodium acetate. Optical absorbance at 650 nm was recorded by a microplate reader (Power ware 340, BioTek, Hampton, VA, USA) following the further addition of 25 μL of 3% hydrogen peroxide onto the plate. MO activity was calculated as ΔA_650mm per hour [19,20].

2.5. Determination of Glutathione-S-Transferase (GST) Activity

Glutathione-S-transferase activity was assayed by the method provided by Brogden and Barber [19], which used 1-chloro-2, 4-dinitrobebzene (CDNB) and reduced glutathione (GSH) as substrates for the enzyme reaction. When measuring, 20 μL of liver supernatant was added to 200 μL of working reagent, which was made by adding 125 μL 63 mM CDNB solution into 2.5 mL 10 mM GSH solution. Optical absorbance at 340 nm was recorded by a microplate reader (Power ware 340, BioTek, Hampton, VA, USA), and GST activity calculated as ΔA_340mm per min.

2.6. Statistical Analysis

SPSS ver. 20.0 [21] was used for statistical analysis. Hepatic MO activity, GST activity, and level of VTG protein values from the different study sites (including the values for exposures to 0 and 10 ng 17β-estradiol/L for 10 days in the laboratory) were statistically compared by one-way ANOVA and post hoc Duncan’s analysis.

3. Results

To understand whether human activities in Shei-Pa National Park have discharged organic pollutants containing xenoestrogens, this investigation studied male O. barbatulum as the experimental organism to analyze for induced VTG concentration, Mon activity, and GST activity.

Laboratory exposure experiments showed that male O. barbatulum individuals exposed to 0 (control) and 10 ng/L E2 for 10 days had, respectively: induced VTG concentrations of 0.06 ± 0.02 μg PO₄²⁺/g protein and 9.87 ± 0.89 μg PO₄²⁺/g protein (Figure 2), Mon activity of 2.28 ± 0.78^△A650/30 min/g protein and 8.02 ± 1.74 ^△A650/30 min/g protein (Figure 3), and GST activity of 1.09 ± 0.40 ^△A340/20 min/g protein and 3.24 ± 0.62 ^△A340/20 min/g protein (Figure 4).

The analysis of VTG concentrations in the livers of wild male O. barbatulum individuals showed that those captured from the Ikawan River had the highest VTG concentrations (2.12 ± 0.66 μg PO₄²⁺/g protein), followed by those from Yusheng River (0.78 ± 0.25 μg PO₄²⁺/g protein) and Cijiawan River (0.59 ± 0.81 μg PO₄²⁺/g protein). Those from the Kaoshan River (0.33 ± 0.62 μg PO₄²⁺/g protein) had the lowest VTG concentrations. VTG concentrations in livers of O. barbatulum measured immediately after collection from the four study sites were lower than O. barbatulum individuals exposed to 10 ng/L E2 in the laboratory (p < 0.05, Figure 2). The livers of wild O. barbatulum collected from the Yusheng River had the highest Mon activity (4.16 ± 1.08 ^△A650/30 min/g protein) and the highest GST activity (1.58 ± 1.13 ^△A340/20 min/g protein), followed by those from the Ikawan River (Mon: 3.63 ± 1.13 ^△A650/30 min/g protein; GST: 1.24 ± 0.73 ^△A340/20 min/g protein). Samples from the Kaoshan River and Cijiawan River had the lowest values. The Mon and GST activities in Yusheng River O. barbatulum livers were significantly different than in O. barbatulum individuals collected from the other three sampling sites (p < 0.05, Figure 3 and Figure 4).

4. Discussion

Agricultural activities are the main source of pollution in the area surrounding Wuling Farm, among which the impacts of pesticides are the most important. MO and GST are important enzymes that play critical roles in the detoxification systems of organisms. In addition, the expressions and activities of enzymes have been reported to be affected by certain xenobiotics such as pesticides and aflatoxin in a dose- or time-dependent manner [15,17,22,23]. After exposure to organic chemicals such as aldicarb [24], carbamate insecticide, DDT [25], dioxin [26], PCB [27], and polycyclic aromatic hydrocarbons [28], an increase in hepatic MO activity in animals has been observed and studied [17,18]. Similarly, enhanced hepatic GST activity has been reported in rainbow trout (Oncorhynchus mykiss) exposed to pesticides [29]. Therefore, the elevated Mon and GST activities observed in these study sites are clues that O. barbatulum is exposed to organic chemicals.

A value of E2 higher than 5 ng/L in water starts to induce VTG in male crucian carp (Carassius auratus) and common carp (Cyprinus carpio) [30,31]. Concentrations of 16–20 ng E2/L initiates abnormal reproductive behavior in medaka (Oryzias latipes) and mosquito fish (Gambusia affinis), and reduces hatching rates in their offspring [32,33]. After reaching concentrations of 30–40 ng/L, E2 causes gonadal atrophy and alteration of the female-to-male ratio in the offspring of the guppy (Poecilia reticulata) and sheepshead minnow (Cyprinodon variegatus) [34,35]. Our experimental results indicate the presence of organic pollution in the Ikawan and Yusheng Rivers. A farm producing hardy vegetables in the lower reaches of the Yusheng River (which converges with the Cijiawan River) causes water pollution, since fertilizers applied to the soil enter the groundwater system with surface runoff or flow directly into the river. The Ikawan River is a confluence of several water bodies, which we speculate is one of the reasons for it having higher pollutant concentrations than the other three rivers. The estradiol equivalent concentration (EEQ) is used to assess the concentration of xenoestrogens in a water body and speculate about the possible harm that xenoestrogens present to the environment and its ecology. In this study, VTG concentrations in O. barbatulum induced by laboratory exposure to E2 (0, 10 ng/L) were used for reference. VTG concentrations in O. barbatulum individuals measured right after collection from the Cijiawan, Yusheng, Kaoshan, and Ikawan Rivers were used to estimate EEQ values at the four sampling sites, which were 0.54 (95% CI = 0.26–1.26), 0.73 (95% CI = 0.49–1.43), 0.28 (95% CI = 0.01–1.13), and 2.10 (95% CI = 0.32–2.87), respectively. We think that the concentration of xenoestrogens in the water at the four sampling points in this study is likely to be lower than the lowest concentration (5 ng/L) of E2 that would generally affect organisms in water bodies. Therefore, although farming activities have introduced organic pollutants containing xenoestrogens into the water bodies around Wuling Farm, they should not have had much impact on their aquatic organisms.

5. Conclusions

Shei-Pa National Park has been gradually recovering from agricultural usage since 1997. However, farming activities still introduce pollutants containing xenoestrogens into the Yusheng and Ikawan Rivers. Therefore, we recommend that pollutants containing xenoestrogens in the rivers in this area be tracked over long periods to keep the aquatic ecosystem in this area healthy.