The use of in vitro bioassays to quantify endocrine disrupting chemicals in municipal wastewater treatment plant effluents

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Abstract

In vitro bioassays are widely used to detect and quantify endocrine disrupting chemicals (EDCs) in the influents and effluents of municipal wastewater treatment plants (WWTP). These assays have sometimes led to false positive or negative results, partly due to the low EDC concentrations in the samples. The objectives of the present study were: (a) to compare the estrogen screen (E-Screen) and the yeast estrogen screen (YES) bioassays using the 17β-estradiol (E2) or its equivalence and (b) to investigate if a combination of the E-Screen and YES assays can be used to improve the accuracy of EDC detection and quantification. The E-Screen bioassay was conducted with the MCF-7 (BOS) human breast cancer cell line while the YES bioassay employed two different types of recombinant yeast. The influent and effluent samples collected from the five WWTPs operated by the Greater Vancouver Regional District (GVRD) were analyzed by both the E-Screen and the YES bioassays. Since the results of the E-Screen and YES bioassays varied by up to 4-fold on the same split sample of a nominal E2 concentration, the mean value of the E-screen and YES bioassays was used to represent the EDC activity of a given WWTP sample. Results of these studies showed that the E2 equivalent concentration in each WWTP sample was consistently higher than 1 ng/L, a concentration that may potentially cause endocrine disruption in different aquatic species. The composition of selected EDCs in a subset of effluent samples was examined using a gas chromatograph–high resolution mass spectrometer (GC–HRMS). EDC composition in 10 WWTP samples correlated with the mean endocrine disrupting activities of the E-Screen and YES bioassays. Results also indicated that secondary treatment plants are comparable to the primary treatment plants in removing EDCs from the final effluents.

Introduction

In the aquatic environment, exposure of organisms to endocrine disrupting chemicals (EDCs) has been linked to endocrine effects in male fish such as vitellogenin induction and feminized reproductive organs (Tyler et al., 1998, Purdom et al., 1994, Aherne and Briggs, 1989, Routeledge et al., 1998). It is suggested that industrial and municipal effluents as well as urban and agricultural runoff are the major sources of EDC discharged into the aquatic environment (Desbrow et al., 1998, Snyder et al., 1999, Boyd et al., 2003). Therefore, when rainbow trout (Oncorhynchus mykiss) were kept in cages close to the discharges of wastewater treatment plant (WWTP) effluents, vitellogenin synthesis was induced in the male fish (Harries et al., 1997). Elevated levels of vitellogenin and decreased serum testosterone were also reported in male carp (Cyprinus carpio) caught near WWTP discharges (Folmar et al., 1996). Vitellogenin elevation and gonadal intersex also were observed in roach (Rutulis rutulis) and flounder (Platichthys flesus) caught near WWTP discharge sites (Jobling et al., 1998, Allen et al., 1999). Among the fish sampled in watersheds receiving WWTP discharges, about 70% of the fish were female (Hansen et al., 1998). These observations are consistent with the hypothesis of chemically induced feminization of fish at sites near WWTP discharges.

In response to the potential hazard of EDCs in the aquatic environment, several screening programs have been implemented using a variety of chemical analyses, in vitro and in vivo bioassays. Analytical methodologies based on gas chromatography–mass spectrometry or gas chromatography–tandem mass spectrometry have been developed and used for the ultra-trace determination of target EDCs in the aquatic environment (Desbrow et al., 1998, Johnson et al., 2000). Analytical techniques based on liquid chromatography–tandem mass spectrometry have also been used successfully for the determination of estrogens in different matrices (Draisci et al., 1998). Although chemical analysis can reveal the presence of EDCs in the aquatic environment, most chemical analysis is focused towards the determination of target substances in the matrices of interest. Considering the large number of EDC substances that can be present in complex environmental matrices, target chemical analyses could be limited in providing a complete account of all EDCs present in a specific environmental matrix. Moreover, mixture interaction is not taken into consideration and the biological effects of the chemical mixture cannot be determined. In contrast, in vitro bioassays which are based on the interaction between the EDCs and the estrogenic receptors can determine the total estrogenic activity of EDCs in a mixture (Legler et al., 1999, Routledge and Sumpter, 1996).

The Greater Vancouver Regional District (GVRD) is committed to a receiving environment monitoring (REM) approach of managing its liquid waste discharges in its Liquid Waste Management Plan (GVRD, 2001). This monitoring approach for the receiving environment of all five of GVRD's Wastewater Treatment Plants includes the characterization of WWTP effluent to define the nature of the effluent and potential effects. Within the GVRD there are two primary WWTPs that discharge into the marine environment, and three secondary WWTPs that discharge into the Fraser River (Fig. 1). Primary treatment is a mainly mechanical process that removes between 30 and 40% of biological oxygen demand (BOD) and 50% of the total suspended solids (TSS). Secondary treatment includes a biological process that removes up to 90% of BOD and the TSS. Neither primary nor secondary treatment processes are specifically designed to remove EDCs of which are at or below the detection limits (Folmar et al., 2002, Desbrow et al., 1998). However, there is a potential for even these low concentrations of EDCs to affect the endocrine system in fish. Therefore, it is important to assess the potential additive or synergistic effects of EDCs at these low concentrations because it may provide more information on the potential for effects in the receiving environment.

Hitherto, there is no consensus among scientists on the best screening methods for determining EDC activities in the aquatic environment. When in vitro bioassays such as the yeast estrogen screen (YES) (Gaido et al., 1997) and MCF-7 breast tumor cell proliferation (E-Screen) (Soto et al., 1995) are used in isolation, they may yield false negative or positive results (Folmar et al., 2002). The objectives of the present study were: (a) to compare the estrogenic potencies of 17β-estradiol (E2) using the E-Screen and the YES bioassays, (b) to determine the estrogenic potencies of the influents and effluents in the five municipal WWTPs operated by the GVRD using a combination of E-Screen and YES bioassays, (c) to identify and quantify selected EDCs in a subset of WWTP samples using gas chromatograph–high resolution mass spectrometry (GC–HRMS), and (d) compare calculated endocrine activity from GC–HRMS with measured activity from E-screen and YES bioassays.

Section snippets

Sample collection and preparation

The larger of the two primary plants (WWTP 2) provides primary treatment to wastewater from approximately 600,000 Vancouver residents before discharging it through a 7.5 km, deep-sea outfall into the Strait of Georgia. In 2003, the average annual flow was 597 million liters per day (MLD) with average total suspended solids (TSS) and biological oxygen demand (BOD) of 48 mg/L and 76 mg/L, respectively (GVRD, 2004).

The other primary plant (WWTP 5) provides treatment to wastewater from

EDC bioassays

E-Screen bioassay was performed according to the procedure of Soto et al. (2004) with modification. YES bioassay was conducted using two different recombinant yeast strains. The procedure of Lorenzen et al. (2004) was used to conduct the YES bioassay of the S. cerevisiae strain BJ3505. The procedure of Routledge and Sumpter (1996) was used to conduct the YES bioassay with S. cerevisiae culture. Permission to use cell lines for the E-Screen (Soto and Sonnenschein, 1995) and YES (Gaido et al.,

Results

The EC50 values of E2 were: 53.2 ± 7.2 pM (14.5 ± 2.0 ng/L) for E-Screen; 242 ± 28 pM (65.9 ± 7.6 ng/L) for Gaido's YES; 203 ± 67 pM (55.3 ± 18.2 ng/L) for Sumpter's YES. The EC50 values were calculated from 44 data points in each of the above bioassays. The EC50 values of E2 for the two YES bioassays were very similar. But the EC50 of E-screen was about 4-times less than those of the YES bioassays.

Complexicity of WWTP

Results of the present study show that the environmental fate of EDC in the WWTP is complex and no clear patterns associated with the treatment process could be identified. Among the five WWTPs operated by the GVRD, there are two primary treatment WWTPs and three secondary treatment WWTPs (GVRD, 2004) that discharge, respectively into the marine environment and the Fraser River (Fig. 1). The primary treatment process is essentially a mechanical process that removes 30–40% of biological oxygen

Conclusion

The environmental fates of estrogens in WWTP are very complex and there is no universally accepted bioassay or chemical technique to quantify EDCs in the aquatic environment. Chemical analysis of EDCs is sensitive and specific but limited in that only target substances are analyzed. In vitro bioassays which are based on the interaction between EDCs and estrogenic receptors can be very useful in determining the total estrogenic activity of EDCs in a mixture. Results of our studies indicate that

Acknowledgements

We would like to thank the following individuals: Dr. Ana Soto and Dr. Carlos Sonnenschein laboratory at Tufts University of Medicine (Boston, MA, USA) for supplying human breast cancer (MCF7-BOS) cells; and Drs. Ed Topp and Angela Lorenzen (Agriculture Canada, London, ON, CANADA) for supplying the recombinant yeast (Saccharomyces cerevisiae) strain BJ3505 in the Gaido YES bioassay. The recombinant yeast (S. cerevisiae) culture used in the Sumpter YES bioassay was provided by M. Fernandez of

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