| Summary: | 碩士 === 國立成功大學 === 環境工程學系 === 102 === SUMMARY
There is a growing concern of organisms exposed to endocrine disrupting chemical (EDCs), which have potential impact on endocrine systems. To understand the occurrence of EDCs in Taiwnese rivers, yeast-based reporter gene assays and liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to analyze EDCs in samples collected from the aquatic environment. Bioassay results showed that (anti-)estrogenic, anti-androgenic, and anti-thyroid hormonal activities were detected in most samples. In addition, natural estrogens, including estrone, 17β-estradiol and estriol, and synthetic compounds, such as bisphenol A, nonylphenol, triclosan, and triclocarban were detected by LC-MS/MS. The results indicated that there was a potential ecological after calculating the risk quotients (RQ) of different compounds. According to the results of combined effects assessment, anti-androgenic and estrogenic compounds showed concentration additive effects in the yeast bioassays, and the the results of iceberg mixture indicated that there were still a lot of unknown EDCs not detected by LC-MS/MS. It is essential to continuously monitor the effects of EDCs on organisms in the aquatic environment
Key words: Endocrine disrupting chemicals (EDCs), Yeast-based reporter gene assays, River water, LC-MS/MS, Combined effects
INTRODUCTION
Emerging Contaminants (ECs) are chemicals that pose potential risk to human and animals associated with their presence, frequency of occurrence, or source may not be known. According to US Environmental Protection Agency, ECs contain pharmaceuticals and personal care products (PPCPs), endocrine disrupting chemicals (EDCs), persistent organic pollutants (POPs), etc. Among ECs, EDCs have been an important concerns for decades because they are widespread in the environment.
EDCs are chemicals which interfere with the endocrine systems of organisms, including synthesis, transport reproduction, etc. A large variety of EDCs have been reported to show estrogenic, androgenic or thyroid hormone disrupting effects. These compounds are introduced into the aquatic environment via industrial, municipal and livestock effluents, and their influence are difficult to evaluate using traditional water qualities, such as pH, DO, BOD5, etc. Many studies have investigated the occurrence of EDCs by using bioassays and chemical analysis. Bioassays, including in vivo and in vitro assays, are used to evaluate the effects on organisms by exposing to EDCs. Chemical analysis using intrument, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), is qualitative and quantitative measurement of known EDCs..
Organisms in environment are often exposed to many EDCs in the same time, therefore, it’s essential to consider the combined effects induced by EDCs.
In this study, both bioassays and chemical analysis were carried out to assess the amount of EDCs in Taiwanses rivers, including water phase and suspended solid (SS) phase samples. In addition, since organisms in environment are often exposed to many EDCs at the same time, the artificial mixtures were also analyzed by bioassays to evaluate the combined effects of EDCs.
MATERIALS AND METHODS
Water grab samples were collected from ten rivers in Taiwan. All water samples (0.5L) were separated into water phase and SS phase by filtration. Water phase samples were extracted by solid phase extraction (SPE) using Oasis HLB Plus cartridge (Waters, USA). Cartridges were activated with 3 mL of 50% methanol and 3 mL of water, and then water phase samples were forced through the cartridges using peristaltic pump. After extraction, the cartridges were washed with 3 mL of 5% methanol and eluted with 1 mL of methanol and 4 mL tert-butylmethylether. SPE extracts were then evaporated by centrifugal vacuum concentrator (EYELA, Japan) and then dissolved in 0.5 mL dimethyl sulfoxide (DMSO) for 1000-fold concentrated samples. SS samples were dried at 105 °C to remove water, and then extracted by Soxhlet extraction using approximately 200 mL of hexane and acetone mixture (1:1, v:v) for 24 h. The extracts were then concentrated to about 3-5 mL by rotary evaporation, and then nitrogen blowing concentrator was used to remove hexane after adding 0.5 mL DMSO for 1000-fold concentrated samples. In this study, yeast-based reporter gene assays used were able to detect estrogenic receptor (ER), androgenic receptor (AR) and thyroid hormone recptor (TR) disrupting activities.
Chemical analysis was carried out using LC-MS/MS to detect the known EDCs. The LC system was Agilent 1260 Infinity (Agilent, USA), and the column was Xbridge BEH C18, 75×2.1 mm i.d., 2.5 μm (Waters, USA).and the mobile phases used in LC were water and methanol. The MS/MS system was Thermo TSQ Quantum Ultra (Thermo, USA) using electrospray ionization (ESI). The mode of detection was multiple reaction monitoring (MRM).
The samples used for combined effect assessment consisted of three parts. The first part was equipotent samples, which were normalized to their effect concentration in yeast-based reporter gene assays so that each EDC should have the same contribution to the mixture effects. The second part was threshold value mixtures, which were based on predicted no-effect concentrations (PNECs) and the values of augmentation of drinking water supplies of Australia. The third part was iceberg mixtures, which were based on the concentrations of EDCs detected in Taiwanese river samples.
RESULTS AND DISCUSSION
Bioassay results demonstrated that high estrogenic activity was detetect in water phase samples in Erren river (ND~346.6 E2-EQ ng/L) and Agodian river (ND~60.5 E2-EQ ng/L). Anti-estrogenic activity was often detected in SS phase samples, especially in Erren river (ND~7.7 OHT-EQ μg/L). Androgenic or thyroid hormonal activities were not detected in all samples. In contrast, high anti-androgenic activity was detected in water phase samples in Erren river (ND~7489.1 FLU-EQ μg/L) and Agodian river (ND~3329.3 FLU-EQ μg/L). Most of water phase samples showed anti-thyroid hormonal activity, especially samples collected from Erren river (ND~47.5%) and Agodian river (30.7~55.1%).
LC-MS/MS results demonstrated that among natural estrogens, estrone was detected most frequently, and the detection frequencies in water phase and SS phase samples were 90.6% and 41.7%, respectively. The highest concentrations of total natural estrogen in water phase samples were detected in Erren river (ND~455.8 ng/L), following by Agodian river (7.3~327.7 ng/L). The detection frequencies of bisphenol A (BPA) were 100% both in water phase and SS phase samples, and the highest concentrations in water phase samples were detected in Erren river (775.3~613258.3 ng/L). The detection frequencies of nonylphenol (NP) were 91.7% and 45.8% in water phase and SS phase samples, respectively, and the highest concentrations in water phase samples were detected in Agodian river (74.6~2086.7 ng/L). The detection frequencies of triclosan (TCS) were 68.8% and 18.8% in water phase and SS phase samples, respectively, and the highest concentrations in water phase samples were detected in Erren river (ND~509.7 ng/L). The detection frequencies of triclocarban (TCC) were 7.3% and 45.8% in water phase and SS phase samples, respectively, and the highest concentrations in SS phase samples were detected in Tsengwen river (ND~1192.2 ng/L).
To evaluate the potential effects of target EDCs measured by LC-MS/MS on organisms, risk quotient (RQ), which represents the ratio of measured environment concentrations of target compounds in water samples and their PNECs was calculated. The results showed that the concentrations of estrone, TCS and BPA had potential ecological risk to the aquatic environment of Taiwan.
In order to clarify the relation between yeast-based reporter gene assays and chemical analysis, the target EDCs were analyzed by using bioassays to determine their estrogenic or anti-androgenic activities relative to 17β-estradiol or flutamide, respectively, and then the EEQLC-MS/MS and FEQLC-MS/MS were obtain to compared with the EEQbioassay and FEQbioassay. The results showed that EEQLC-MS/MS and FEQLC-MS/MS of most samples were lower than EEQbioassay and FEQbioassay, indicating that there were still a large amount of EDCs not detected by LC-MS/MS analysis.
Bioassay results of equipotent samples showed that EDCs exhibited concentration additive estrogenic and anti-androgenic activities in yeast-based reporter gene assays. The results of threshold value mixtures showed that mixture samples may produce significant effects even all concentrations of EDCs were under PNECs via concentration addition. The iceberg mixtures were also analyzed by bioassays after LC-MS/MS analysis, and the results worked in concert with the relation between bioassay and chemical analysis, which indicated that there were some unknown EDCs in the environmental samples.
CONCLUSION
The yeast-based in reporter gene assay adopted in the present study were used to estimate the (anti-)estrogenic, (anti-)androgenic and (anti-)thyroid hormonal activities in Taiwanese rivers. The results showed that estrogenic and anti-androgenic activities were detected in water phase samples, while anti-estrogenic activities were present in SS phase samples. In addition, TR agonist and antagonist activities were also detected both in water phase and SS phase samples . Among these samples, Erren river and Agodian river showed higher endocrine disrupting activities. By using LC-MS/MS, natural estrogens, BPA, NP, TCS and TCC were detected in samples, and the results of RQ posed potential risk of EDCs to local aquatic organisms. The combined effects assessment conducted in this study indicated that EDCs showed concentration additive effects in yeast assay, which meant that even the chemical concentrations under PNEC might also induce significant effects. Therefore, the combined effects must be considered when evaluating the effects of EDCs on organisms.
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