Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

Science of the Total Environment 408 (2010) 4451–4460

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Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples Caroline Vanparys a,⁎, Sophie Depiereux b, Stéphanie Nadzialek b, Johan Robbens a, Ronny Blust a, Patrick Kestemont b, Wim De Coen a,c a b c

Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp, Belgium Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur, Belgium European Chemicals Agency (ECHA), Helsinki, Finland

a r t i c l e

i n f o

Article history: Received 10 November 2009 Received in revised form 17 June 2010 Accepted 19 June 2010 Available online 14 July 2010 Keywords: Estrogenicity E-screen Flow cytometry Sewage treatment Compound classification

a b s t r a c t In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC50 value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R² = 0.98), the estrogen receptor (ER) binding (R² = 0.84) and the ER transcription activation assay (R² = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies, supports the use of MCF-7 cell proliferation as estrogenicity screen for pure compounds and complex samples. © 2010 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: AS, activated sludge; BOD5, five-day biochemical oxygen demand; CASRN, chemical abstracts service registry number; COD, chemical oxygen demand; CV, coefficient of variation; DF, dilution factor; DMEM, Dulbecco's modified eagle medium; DMSO, dimethyl sulfoxide; E1, estrone; E2, 17β-estradiol; EDKB, Endocrine Disruptor Knowledge Database; EE2, ethinylestradiol; EEQs, estradiol equivalents; EF, effluent; ER, estrogen receptor; FBS, foetal bovine serum; ICCVAM, Interagency Coordinating Committee on the Validation of Alternative Methods; IF, influent; LOD, limit of detection; LOQ, limit of quantitation; N, nitrogen; P, phosphorus; PBS, phosphate buffered saline; PE, population equivalent; PI, propidium iodide; PNEC, predicted no effect concentration; RPE(max), (maximal) relative proliferative effect; RPP, relative proliferative potency; SD(s), standard deviation(s); SS, suspended solids; STP(s), sewage treatment plant(s); TA, transcription activation; YES, yeast estrogen screen. ⁎ Corresponding author. Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium. Tel.: + 32 3 265 3350; fax: + 32 3 265 3497. E-mail address: [email protected] (C. Vanparys). 0048-9697/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2010.06.049

The widespread occurrence and quantities of endocrine disrupting compounds in the environment are believed to pose an increasing threat on normal reproduction and developmental processes in humans and wildlife (Colborn et al., 1993; Tyler et al., 1998; Guillette and Gunderson, 2001; McLachlan et al., 2006). The estrogen-mimicking potential of a vast amount of waste stream chemicals is one of the most important factors associated with the majority of so far documented endocrine disruptive effects (Sharpe and Skakkebæk, 1993; Sumpter, 1995). It has frequently been stated that the various estrogenic effects observed in aquatic wildlife, such as intersex in fish, might be rescaled to a more defined point source problem, largely associated with discharge sites of sewage treatment plants (STPs) (Jobling et al., 1998, 2006; Vethaak et al., 2005). A range of industrial chemicals (bisphenol A, p-alkylphenols), as well as natural (estrone (E1), 17β-estradiol (E2)) and synthetic estrogens (17α-ethinylestradiol (EE2)) has been

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measured in the ng/L to μg/L range in municipal wastewaters and remain in substantial amounts (ng/L) in the effluents of STPs, sufficient to evoke hormonal disruption in aquatic organisms of the receiving water systems (Routledge et al., 1998; Larsson et al., 1999; Brion et al., 2004; Nash et al., 2004; Brian et al., 2005; Johnson et al., 2005). Several in vitro assays have been developed to screen for estrogenic activity of compounds or environmental samples (Zacharewski, 1997; Hilsherova et al., 2000). They allow the rapid screening of the estrogen-mimicking potential of fairly unknown compounds or samples, based on estrogen receptor interaction processes (Soto et al., 1995; Routledge and Sumpter, 1996; Legler et al., 1999). By comparison to the natural estrogen, 17β-estradiol, compounds or samples can be ranked according to their potency. Since the in vivo effects of estradiol are very well documented, this compound serves as a reference framework for compound prioritization or hazard identification of environmental matrices. In particular for complex samples, such as sewage, in vitro estrogenicity screens give an indication of the joint estrogenic activity of the total mixture, whereas chemical analysis only allows examination of known and suspected chemicals. This is a significant added value, since practice learns that it is very difficult to predict the estrogenic potential of the mixture based on the estrogenic activities of the individual compounds (Fent et al., 2006). Furthermore, in vivo studies showed that highly potent estrogenic compounds can be biologically active at only trace concentrations and have the potential to provoke additive estrogenic mixture effects at low doses, even at no observed effect levels (NOELs) (Thorpe et al., 2003; Brian et al., 2005; Fent et al., 2006). Although in vitro estrogenicity screens are far from complete to predict the risk for adverse effects in humans and wildlife, they are well suited to give a first rapid ranking of the estrogenic potency of compounds or to indicate the overall estrogenic load in environmental matrices and therefore add important information to identify estrogenic pollution. In the present study, we evaluated the performance of the flow cytometric MCF-7 proliferation assay to detect and rank pure compounds as well as more complex samples, such as influent and effluents of STPs. In this adapted E-screen assay, the re-induction of cell growth after synchronisation in the G0/G1 phase is flow cytometrically quantified as the percentage of cells in the S (ynthesis)-phase of the cell cycle (Vanparys et al., 2006). In a first step, the estrogenic-mimicking potency of a range of compounds was measured and qualitatively and quantitatively compared with other in vitro and in vivo estrogenicity screens, such as the ER binding, ER transcription activation (TA) and the rodent uterotrophic assay. Data from the literature (Fang et al., 2000), public databases such as the Endocrine Disruptor Knowledge Database (EDKB, 2009) and the ICCVAM reference list for validation of ER binding and ER TA tests (ICCVAM, 2003) were applied to allow a solid evaluation of the flow cytometric E-screen performance. Secondly, this assay was applied to compare the estrogenic activities of the influent and effluent samples of 10 predominantly municipal sewage treatment plants (STPs) in the Walloon region of Belgium. The classical E-screen has been used more often for the evaluation of estrogenicity of influent and/or effluents of STPs and has proven its value in comparing overall the estrogenic activity of different complex samples (Körner et al., 1999; Leusch et al., 2006b; Shappell, 2006). This study focuses particularly on the use of this assay to compare and rank the estrogenic removal efficacy of different sewage treatment processes in the field. 2. Materials and methods 2.1. Compound selection The selection of pure compounds for this study was based on the revised Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) list of 78 reference substances for

validation of in vitro ER and androgen receptor (AR) binding and transcription activation (TA) assays (ICCVAM, 2003). The initiative of this list was to ensure an adequate evaluation of new test systems to a broad range of chemical classes and responses. In total, 17 chemicals were selected, including 8 original ICCVAM reference substances (17β-estradiol, nonylphenol, genistein, kepone, testosterone, diethylhexylphthalate (DEHP), methoxychlor, and atrazine); 3 ICCVAM list chemicals indicated as substances considered but not included for validation (p,p′-DDT, lindane, and 2,3,7,8 tetrachloro-dibenzo-pdioxin (TCDD)) and 6 additional chemicals (α-endosulfan, toxaphene, tetrabromobisphenol A (TBBPA), dicyclohexylphthalate (DCHP), 7,12dimethyl-1,2-benz(a)anthracene (DMB), and benzo(a)pyrene (BaP)) including compounds of groups recognized as underrepresented in the ICCVAM list such as phthalates and polycyclic aromatic hydrocarbons. Chemical specifications are provided in Table 2. 2.2. Influent and effluent sampling and sample treatment Influent and effluent samples were collected from 10 large-scale STPs, selected on the basis of similar capacity and with at least 6 years of activity. These 10 STPs represent different purification procedures, including the three main secondary treatments such as lagoon, filter bed and activated sludge and different tertiary treatments such as elimination of nitrogen (N) and/or phosphorus (P) and UV disinfection. Details of the stations, their capacity expressed as population equivalent (PE; organic biodegradable load having a five-day biochemical oxygen demand (BOD5) of 60 g of oxygen per day; Directive 91/271/EEC, 1991), treatment type, influent flow (m³/d) and the overall purification performances (% removal of BOD5, chemical oxygen demand (COD), suspended solids (SS), nitrogen (Nkj, NNH4), and phosphorus (PPO4)) are indicated in Table 1. Influent and effluents were sampled in May 2008. Samples (2 L) were collected in dark glass bottles, transported refrigerated (4 °C) to the laboratory, acidified at pH 2 with H2SO4 and stored at 4 °C for no longer than 2 weeks until extraction. Influent and effluent samples were centrifuged at 4500 rpm (4 °C) for 30 min and filtered with Millipore glass fibre filters (AP2004700, pore size 0.2 μm) to remove particulate matter. The extraction procedure was performed according to Leusch et al. (2006a). Solid Phase Extraction (SPE) cartridges (Oasis HLB, Waters Corporation Milford, MASS U.S.) were preconditioned with methanol and ultrapure, deionized water (MilliQ, Millipore) and 1 L sample was extracted per disk. After elution of the cartridges with methanol, the samples were evaporated under gentle nitrogen stream and reconstituted in 250 μl methanol. According to Tan et al. (2007), this procedure allows a recovery of 80–120% for steroid hormones and 63– 117% for industrial chemicals such as bisphenol A, several phthalates and alkylphenols. To include the estrogenic activity inherent to the extraction procedure in the analysis, 1 L MilliQ water was treated in similar way as the STP samples and was used as filter blank control. Extracts were stored at 4 °C until analysis. 2.3. MCF-7 cell proliferation assay Cell treatment and flow cytometric cell cycle analysis were performed as previously described (Vanparys et al., 2006). In brief, human Caucasian MCF-7 breast adenocarcinoma cells (ECACC No. 86012803) were maintained as a monolayer culture in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1 mM sodium pyruvate, 4 mM L-glutamine, 1% non-essential amino acids, 50 IU/mL penicillin, 50 mg/mL streptomycin and 5% heat inactivated foetal bovine serum (FBS). Cells were grown under mycoplasma free conditions in a 37 °C incubator under a 5% CO2 atmosphere. At 80– 90% confluence, cells were split 1/10, with a maximum of 30 passages. All cell culture reagents were obtained from Gibco-BRL (Invitrogen LT, Merelbeke, Belgium).

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Table 1 Technical data of the selected STP stations. Station

PE

Rochefort Bastogne Rhin Trivières

23 700 17 500 19 000

Ciney

16 000

La Gueule Aval (Plombières)

24 750

Marche-en-Famenne

24 400

Malmedy Bertrix Morlanwelz

20 000 8 500 18 000

La Roche

11 500

Treatment

II: AS II: AS II: AS III: P removal (PC) II: AS III: P removal (PC + B) II: AS III: P removal (PC) III: N removal (B) II: AS III: P removal (PC) III: N removal (B) II: Lagoon II: Lagoon II: Bacterial/filter bed III: P removal (PC) III: N removal (B) II: AS III: P removal (B) III: N removal (B) III: UV disinfection

Influent flow (m3/d)

Purification performance (% removal) BOD5

COD

SS

Nkj

NNH4

PPO4

6316 4967 7490

96.2 91.6 96

88 91.3 80.3

91 95.8 87.8

85.7 86.2 94

88.5 83.4 /

43.5 62.7 80.4

5335

96

90.4

95

86

79.8

62.7

5616

97

95

98

96

98

90

9408

95

93

93

87

89

62

6327 3894 7872

93.5 92.4 94

89.5 88.9 76.7

97 96.2 85.7

48.8 69.2 73

17.2 -25 /

52.1 62.8 53

3987

91.8

90.5

92.6

82.5

90

78.6

PE: population equivalent; II: secondary treatment; III: tertiary treatment; AS: activated sludge; P: phosphorus; N: nitrogen; PC: physical-chemical; B: biological; BOD5: five-day biochemical oxygen demand; COD: chemical oxygen demand; SS: suspended solids; Nkj: nitrogen Kjedahl.

For cell proliferation analysis, MCF-7 cells were seeded in 12-well plates in standard growth medium at a density of 50,000 cells per well. Cells were allowed to attach overnight (24 h) after which growth medium was replaced by exposure medium containing phenol red-free DMEM with supplements but with 5% charcoal dextran treated foetal bovine serum (CDFBS) as a replacement of FBS (Payne et al., 2000). The conditioning of the cells in low-steroid medium minimizes the confounding estrogenic activity from serum and simultaneously synchronizes the cells in the G0/G1-phase of the cell cycle thereby maximizing the estrogen-sensitivity of the MCF-7 cells. After 72 h incubation, exposure medium was refreshed and pure chemicals or extracts of the STP samples were added. After 24 h exposure, cells were harvested, washed with PBS (with Ca²+ and Mg²+) and stained with 1 mL freshly made propidium iodide (PI, Molecular Probes, Invitrogen, Merelbeke, Belgium) staining solution (0.1% Triton X-100, 0.1% sodium citrate, 50 μg/mL PI and 10 μg/mL RNase A in PBS (with Ca²+ and Mg²+)). Cells were stored for 3 h in the dark at 4 °C prior to flow cytometric analysis. Flow cytometric measurements were performed using a LSR II flow cytometer (BD Biosciences, Erembodegem, Belgium), equipped with a 15 mW solid state argon-ion laser emitting at 488 nm. For each measurement, PI fluorescence signals of 10,000 single cell events were collected with a 575/25 nm band pass filter (orange-red fluorescence (FL2)) after linear amplification. Cell cycle histograms were analysed using ModFit LTTM 3.0 software packages (Verity Software House Inc., Topsham, ME, USA). Based on this programme, only cell cycles with low variation coefficient of the G0/G1 peak (CV b 5) and low Reduced Chi-Square (RCS)-values (RCS b 3) were used for further statistical analysis. The proliferative effect of a chemical in this assay is indicated by the percentage of cells in S(ynthesis)-phase of the cell cycle. The experimental set-up was in agreement with the ICCVAM standards for ER transcription activation (TA) assays (ICCVAM, 2003, 2006). For each compound or extract sample, a dilution series of respectively 8 or 6 concentrations was conducted in triplicate. Noncytotoxic concentrations were used, based on the AlamarBlue viability assay (Nociari et al., 1998). Chemical specifications and test concentration ranges are indicated in Table 2. Solvent concentration (ethanol or DMSO for the pure compounds, methanol for the extracts) in the culture medium did not exceed 0.1% and did not affect proliferation with the positive control, 17β-estradiol. Finally, each

compound or sample extract at a highly active concentration was coexposed with an anti-estrogen (100 nM ICI 182,780) to evaluate the estrogen receptor mediated mechanism of cell proliferation. By blocking the estrogen receptor, the proliferative response of the compound/extract is lost if its activity is solely estrogen receptor mediated.

2.4. Calculations of estrogenic activity and statistical analysis As a measure of estrogenic activity, cell proliferative responses are expressed relative to the positive control, 17β-estradiol, in terms of the relative proliferative effect (RPE) and the relative proliferative potency (RPP) for pure compounds or Estradiol Equivalents (EEQs) for sample extracts. For the RPE, the proliferative effect is calculated as the ratio between the cell yield of the compound or sample extract, versus the cell yield of the solvent control. The RPE (%) is then calculated as 100 × (proliferative effect − 1)extract or compound / (proliferative effect − 1)17β-estradiol. Since the filter blank control of the extraction procedure was not significantly different from the solvent control, solvent control values were used for all calculations. RPEmax can be understood as the maximal proliferative effect expressed as a percentage of the maximum effect achieved by 17β-estradiol, with 100% indicating a full agonist. For calculations of the RPP and the EEQs, logistic 4 parameter regression curves were conducted for all compounds and extract samples. Dose response curves were only considered for further analysis if R²N 0.97. The concentration evoking the half-maximum activity of the positive control, 17β-estradiol, was calculated based on the regression curve equation (SigmaPlot 11.0, Systat Software 2008) and referred to as the PC50 value. For the pure compounds, the RPP is considered as the ratio of the PC50 of 17β-estradiol to the PC50 of the compound, multiplied by 100, and is often base 10 transformed (expressed as LogRPP). The RPP of 17β-estradiol is therefore 100, the LogRPP is 2. For the extracted samples, this PC50 value is expressed as a dilution factor (DF) and the product of the PC50 value of the positive control (ng/L) and the DF of the extract gives the EEQ value expressed in ng/L (Körner et al., 1999). The % removal is calculated as (EEQIF − EEQEF) / EEQIF ⁎ 100 with IF and EF representing influent and effluent, respectively (Pothitou and Voutsa, 2008).

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Table 2 Chemical specifications and flow cytometric E-screen results. Chemical

Chemical name

CASRN

Supplier

Test concentration range (M)

RPEmax (%)

PC50 (M)

LogRPP

17β-Estradiol Genistein Nonylphenol Testosterone Kepone BaP p,p′-DDT Methoxychlor Toxaphene Lindane DCHP Endosulfan DMB TBBPA DEHP Atrazine TCDD

17β-estradiol

50-28-2 446-72-0 104-40-5 58-22-0 143-50-0 50-32-8 50-29-3 72-43-5 8001-35-2 58-89-9 84-61-7 959-98-8 57-97-6 121839-52-9 117-81-7 1912-24-9 1746-01-6

Sigma Sigma Sigma Fluka LGC Promochem Sigma Supelco LGC Promochem LGC Promochem Aldrich LGC Promochem LGC Promochem Sigma LGC Promochem LGC Promochem Supelco CN Smidt bv

1 × 10−16–1 × 10−9 1 × 10−12–1 × 10−5 1 × 10−8–5 × 10−5 1 × 10−10–1 × 10−6 4 × 10−8–1 × 10−5 1 × 10−9–1 × 10−5 1 × 10−7–5 × 10−5 6 × 10−8–5 × 10−5 8 × 10−8–2 × 10−5 2 × 10−7–1 × 10−4 1 × 10−8–3 × 10−5 6 × 10−7–8 × 10−5 4 × 10−8–3 × 10−4 1 × 10−6–1 × 10−4 6 × 10−7–2 × 10−5 2 × 10−7–1 × 10−4 1 × 10−10–1 × 10−6

100 78.8 77.4 80.8 88.2 76.5 77.4 59.0 97.5 63.7 117 84.2 59.7 63.0 10.7 16.4 4.33

2.01 × 10−12 1.40 × 10−8 1.09 × 10−7 2.08 × 10−7 5.44 × 10−7 8.83 × 10−7 1.01 × 10−6 2.65 × 10−6 3.31 × 10−6 3.34 × 10−6 5.57 × 10−6 5.84 × 10−6 8.78 × 10−6 6.74 × 10−5 / / /

2.00 −1.84 −2.73 −3.01 −3.43 −3.64 −3.70 −4.12 −4.22 −4.22 −4.44 −4.46 −4.64 −5.53 / / /

4-nonylphenol Chlordecone Benzo(a)pyrene 4,4′-DDT p,p′-methoxychlor Camphechlor Gamma-HCH (1,2,3,4,5,6-HCH) Dicyclohexylphthalate α-Endosulfan 7,12-dimethyl-1,2-benz(a)anthracene Tetrabromobisphenol A di-ethylhexylphthalate 2,3,7,8 tetrachloro-dibenzo-p-dioxin

PC50: concentration of a compound with 50% activity of the positive control, 17β-estradiol; RPEmax: maximal relative proliferative effect i.e. maximal induction of a compound relative to the maximal induction of 17β-estradiol; RPP: relative proliferative potency, relative to 17β-estradiol.

Comparison of the LogRPP values of the pure compounds with LogRPP values of other estrogenic assays and comparison of the estrogenic removal efficiency with the purification parameters of the STPs were performed with correlation and linear regression analysis (Statistica 6.0, StatSoft Inc.). 3. Results 3.1. Evaluation of the flow cytometric E-screen The reproducibility of the flow cytometric E-screen was evaluated using the half-maximal response of the reference estrogen, 17βestradiol, referred to as the half-maximal effective concentration or EC50 value. Nine independent experiments, spread over a 2 year period, indicated a mean EC50-value of 1.96 ± 0.44 pM with a coefficient of variation (CV) of 22% (Fig. 1A). This evaluation included different passage levels (passage 12–30 from the ECACC cell stock), different medium and serum batches, nitrogen-preserved MCF-7 cell stocks and lab technicians. The maximal responsiveness was reached at 100 pM and ranged between 5 and 10 times fold induction and was thus always greater than four-fold induction, one of OECD criteria for validation of ER TA estrogenicity assay (OECD, 2008). The limit of detection (LOD) or the lowest amount of estrogenicity that can be detected in a sample is defined as the mean blank value + 3 SDs (99% confidence interval). This LOD only represents the random variation in the blank values and can be considered as a rather qualitative measure (Armbruster et al., 1994). Therefore also a limit of quantitation (LOQ) value was determined, representing the lowest amount of estrogenicity which can be quantitatively determined with suitable precision and accuracy. This LOQ value is measured as the mean blank value + 10 SDs. The LOD and LOQ of the flow cytometric E-screen assay are 1 fM (0.3 pg/L) and 0.18 pM (0.05 ng/L) 17βestradiol, respectively. These concentrations evoked respectively 5.8% and 20% of the maximal inducible effect with 17β-estradiol. According to the OECD Draft Test Guideline for an ER TA assay, a compound/ sample is considered positive if the RPEmax N blank value + 2 SDs in at least 2 of 2 or 2 of 3 runs (OECD, 2008). However, the ICCVAM report on the test method nomination of the MCF-7 cell proliferation assay of estrogenic activity (ICCVAM, 2006) defined a compound as estrogenic if RPE N 15% and a clear dose response curve could be fitted. In this paper, an RPEmax N 20% (LOQ value) and a clear dose response curve were considered as criteria for defining positive estrogens. To allow comparison of compounds, equipotent concentrations need to be calculated. To avoid confusion among terms, a rather new term, the PC50 value was used in this paper, indicating the

Fig. 1. Performance of the flow cytometric E-screen assay and description of the PC50 term. A. Dose response curve of the proliferative effect of the positive control, 17βestradiol, expressed as % relative proliferative effect (RPE). An RPE of 100% indicates the maximal RPE of 17β-estradiol. Each dot represents mean of triplicate measures. The regression line indicates the mean dose response curve of 9 independent experiments. B. Description of the PC50 value as the concentration of compound x with 50% activity of the positive control (17β-estradiol, E2) and its relation to the more common used EC50 value.

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concentration evoking 50% activity of the positive control, 17βestradiol. Since not all compounds reach a RPEmax of 100% due to cytotoxicity or lack of solubility at high concentrations, the halfmaximal concentration or EC50 value of the logistic 4 parameter regression equation is not equal to the concentration evoking 50% activity of 17β-estradiol and therefore not suited for comparison of different compounds or samples (Hilsherova et al., 2000). The concept of this PC50 value is adopted from the OECD draft test guideline for ER TA assay (OECD, 2008) and is illustrated in Fig. 1B. MCF-7 cell proliferation results of the range of pure compounds, expressed as maximal relative proliferative effect (RPEmax), PC50 or LogRPP values, are indicated in Table 2. Chemicals with RPEmax values N 75% are considered full agonists (DCHP, genistein, nonylphenol, testosterone, kepone, p,p′-DDT, BaP, toxaphene, and endosulfan), with 20% b RPEmax b 75% partial agonists (methoxychlor, lindane, TBBPA, and DMB) and with RPEmax b 20%, representing the LOQ value in this assay, as not significant estrogenic (atrazine, DEHP and TCDD). For DEHP and TCDD, no significant response was measured. For atrazine, only the highest concentration tested (100 μM) evoked a significant response (16.4%), but no significant dose response curve was obtained due to solubility problems at higher concentrations. For all other chemicals, PC50 values could be calculated. The experiment with the anti-estrogen ICI 182,780 indicated that proliferative responses were predominantly estrogen receptor mediated for all compounds tested. Except for TBBPA, where a small, but significantly higher amount of cells remained in S-phase (8.29 ± 0.78%) despite blocking the ER-receptor, compared to the control situation (4.89 ± 1.39%). However, the maximal inducible effect of solely TBBPA (18.2 ± 0.66% cells in S-phase) is far higher than the remaining activity with the ER blocker, indicating that the majority of the cell proliferative effect of TBBPA can be explained as estrogenic. Comparison of the LogRPP values with E-screen data from the literature (Fang et al., 2000) showed high degree of similarity (Linear regression y = 1.09 × −0.08; R² = 0.98; n = 8; p b 0.001, Fig. 2). Also, for the yeast estrogen screen (YES; expressed as LogRPP) and the ER binding assay (expressed as the Relative Binding Affinity or LogRBA), significant relations were found (y = 0.94 × −0.37; R² = 0.87; n = 6; p b 0.001 and y = 1.13 × −1.34; R² = 0.84; n = 7; p b 0.001, respectively) (united data of Fang et al., 2000). Overall, a good qualitative agreement was found among flow cytometric E-screen results and ICCVAM reference compounds (ICCVAM, 2003) or uterotrophic assay data (EDKB, 2009) (Table 3). Compounds that were regarded unanimous positive (n = 6) or

Fig. 2. Linear regression analysis of the classical E-screen assay versus the flow cytometric E-screen assay. Dots indicate LogRPP values, the straight line represents the regression curve and dashed lines indicate 95% confidence intervals. The regression equation and R² value are indicated in the graph, p b 0.001.

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Table 3 Ranking of flow cytometric E-screen data, according to the ICCVAM reference list for ER binding and ER transcription activation (TA) assays and the EDKB Uterotrophic assay. Compound

17β-Estradiol Genistein Nonylphenol Testosterone Kepone Methoxychlor p,p′-DDT Lindane DEHP Atrazine TCDD

Flow cytometric E-screena

ICCVAM ER bindingb

ER TAa

Uterotrophic assayc

+++ ++ ++ ++ + + + + − − −

+++ ++ ++ +/−d ++ + + +/−d − + −

+++ + ++ +/−d + + + + − − ++

+ + + − + + +

EDKB

a +++ Indicates a substance with strong estrogenic activity (PC50 b 0.001 μM), ++ indicates a substance with moderate estrogenic activity (0.001 μM b PC50 b 0.1 μM), + indicates weak estrogenic activity (PC50 N 0.1 μM) and − indicates no estrogenic activity measured, adapted from the ICCVAM classification of ER TA data (ICCVAM, 2003). The PC50 value is the concentration with 50% activity of the positive control, 17β-estradiol. b +++ Indicates a substance with strong estrogen receptor binding activity (RBA value N 1), ++ indicates substance with moderate estrogen receptor binding activity (0.01 b RBA value b 1), + indicates substance with weak estrogen receptor binding activity (RBA value b 0.01), − indicates that no RBA value could be calculated, according to the ICCVAM classification (ICCVAM, 2003). c Based on mouse or rat uterotrophic assay results, derived from EDKB (2009). d Indicates an equivocal response (i.e. different results were found in different studies).

negative (n = 1) in the ICCVAM reference list for validation of ER binding and ER TA assays were also positive or negative respectively in the flow cytometric E-screen. Positive compounds in the uterotrophic assay were also positive in the flow cytometric E-screen (n = 6), however the negative compound, testosterone, showed clear ER mediated cell proliferation. 3.2. Removal of estrogenic activity in different sewage treatment procedures Ten different STPs, representing the three main types of secondary treatment (activated sludge (AS), lagoon and filter bed), with or without tertiary treatment (P/N removal) were included in this assay to compare estrogenic removal efficiencies. Overall, there is not a large difference in amounts of waste receiving among the different plants (less than 10-fold difference in PE and influent flow) (Table 1). The stations were intentionally selected this way, since these parameters are important factors influencing the performance of STPs that could hamper comparison of the different treatment procedures. Plombières (AS + P/N) has the highest PE, but also the highest purification performance for BOD5, COD, SS, N and P (Table 1). The lagoon plants, Bertrix and Malmedy, have very low N and P removal while the filter bed plant, Morlanwelz, has the lowest COD and SS removal. All extracts showed clear dose dependent cell proliferation, as indicated for Morlanwelz (filter bed) and Plombières (AS + P/N) in Fig. 3. Comparison of influent and effluent dose response curves already indicates that the influent samples are consistently much stronger estrogenic than the effluent samples, regardless of the treatment process. For nearly all influent and effluent samples, the estrogen receptor blocker experiment indicated that the proliferative responses are solely estrogen receptor mediated and therefore can be considered as estrogenic effects (Fig. 4). For the effluent of Bastogne (AS) however, half of the proliferative response remained present after blocking the ER-receptor, which might indicate some general toxic events such as cell cycle arrest. The maximal relative proliferative effect (RPEmax) of the influent sample was the highest for Bertrix (85.6%, lagoon) and the lowest in Trivières (71.1%, AS + P)

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Fig. 4. Evaluation of the estrogen receptor mediated effect of influent and effluent extracts of STPs. The % of cells in S-phase (mean ± SD, n = 3) of influent (IF) and effluent (EF) extracts of STPs, without (dark gray bars) and with (light gray bars) addition of the estrogen receptor blocker, ICI 182,780 are shown. For all STPs a 4 times dilution of the original concentration was selected as an active dose, except for the effluents of La Roche, Bastogne and Marche a 0.25 times dilution was chosen. The white line crossing the bars indicates the solvent control.

Fig. 3. Examples of dose response curves of influent and effluent samples of STPs as obtained with the flow cytometric E-screen assay. Two STPs are shown, Morlanwelz (filterbed) and Plombières (AS + P/N). The relative proliferative effect (RPE) values (mean ± error term; n = 3) are presented in function of the dilution factor of the influent (black dots) and effluent (white dots) extracts. A dilution factor of 1 represents the concentration of the original sample.

(Table 4). For the effluent sample, this was the highest in Bastogne (109%, AS) and the lowest in Ciney (45.1%, AS + P). In only 3 stations (Trivières, Ciney and Plombières), all with AS + P or P/N removal treatment, the RPEmax lowered in the effluent sample compared to the influent sample, with the highest difference in Ciney (45.6%). Comparison of the EEQs (Estradiol Equivalents), based on the PC50 values, showed that for the influent samples, the EEQ in Marche (AS + P/ N) was the highest (62.1 ng/L), while the EEQ in La Roche (AS + P/N) was the lowest (4.75 ng/L) (Table 4). In the effluent sample, the EEQs were overall 5–75 times lower than in the influent sample, with the highest EEQ value in Bertrix (8.53 ng/L, lagoon) and the lowest value in Plombières (0.52 ng/L, AS+ P/N). Based on these EEQ values, removal efficiencies were calculated (Table 4). The mean estrogenic removal efficiency was 83.7 ± 12.0%, with the lowest efficiency in Morlanwelz (62.6%, filter bed), followed by Trivières (72.1%, AS + P) and the highest efficiency in Marche (99.0%, AS + P/N). In Fig. 5, the dilution factor (DF) of the influent sample evoking 50% activity of 17β-estradiol (representing the PC50 value) is plotted against the equipotent DF of the effluent sample and a 90% removal trend line is indicated. This representation visually indicates the estrogenic load in the effluent in function of the initial load in the influent. For Ciney, the DF evoking only 40% activity was considered for influent and effluent, since the proliferative response of

the effluent of Ciney did not reach 50% activity of 17β-estradiol. Activated sludge treatment followed by tertiary treatment with removal of phosphorus and nitrogen, as for Marche, Plombières and La Roche, showed the lowest estrogenic load in the effluent samples. Considering the fact that the initial estrogenicity in the influent sample is highly different between La Roche, Marche and Plombières, it appears that the amount of estrogenic load remaining in the effluent is independent of the initial estrogenic load (influent sample). However, this implies that the efficiency of this purification procedure spans a rather wide range (83–99%). It might indicate that the efficiency of purification reaches a plateau level at low estrogenic loads. However, this is only a hypothesis and needs to be evaluated in more detail with larger sample groups. Morlanwelz (filter bed) and both Bertrix and Malmedy (lagoon) appeared the least efficient (b80%). To evaluate the relation between the physical purification performance of the STPs and the estrogen removal efficiency, correlation analysis of the different purification parameters and PE (Table 1) with the % removal of estrogenic activity was performed. A significant positive correlation was found between % estrogenic removal and % COD removal (Linear regression, R² = 0.79, p b 0.001)

Table 4 Estrogenicity of influent and effluents samples of 10 sewage treatment plants (STPs). STP

Rochefort Bastogne Trivières Ciney Plombières Marche La Roche Morlanwelz Malmedy Bertrix

Type

AS AS AS + P AS + P AS + P/N AS + P/N AS+ P/N + UV Filter bed Lagoon Lagoon

RPEmax (%)

EEQs (ng/L)

IF

EF

IF

EF

% Removal

80.7 ± 2.51 84.5 ± 1.96 71.1 ± 3.69 82.9 ± 1.68 79.2 ± 2.05 76.7 ± 3.19 75.3 ± 1.71 76.4 ± 2.12 76.4 ± 2.00 85.6 ± 1.85

82.1 ± 1.28 109 ± 3.89 57.6 ± 2.49 45.1 ± 3.17 58.2 ± 2.52 107 ± 2.88 90.9 ± 6.31 77.5 ± 3.66 78.3 ± 1.82 89.6 ± 1.81

23.9 33.3 9.67 12.1° 12.7 62.1 4.75 6.54 36.5 38.8

5.41 1.22 2.70 0.74° 0.52 0.82 0.80 2.44 7.04 8.53

77.3 96.3 72.1 93.8° 95.9 98.7 83.2 62.6 80.7 78.0

AS: activated sludge; P/N: phosphorus and nitrogen removal; RPEmax: maximal relative proliferative effect; EEQs: Estradiol equivalents; IF: influent; EF; effluent. °For Ciney, PC40 values were used, since a PC50 activity of 17β-estradiol was not reached.

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or % SS removal (Linear regression, R² = 0.51, p = 0.02) removal (Fig. 6). On the other hand, % BOD5 removal was negatively correlated to the maximal inducible effect of the sample (RPEmax) (Correlation analysis, r = −0.64, p = 0.047). 4. Discussion

Fig. 5. Visual representation of the estrogenic load of the effluent in function of the initial influent estrogenic load. Estrogenic loads are presented by the dilution factor (DF) evoking 50% response of 17β-estradiol. STPs with different secondary treatments are indicated with different symbols (AS, filter bed, and lagoon), the different tertiary treatments of AS are indicated with different colours (black, dark gray, light gray, and white). A 90% removal trend line is shown.

The E-screen assay or MCF-7 cell proliferation assay is frequently used for the evaluation of estrogenicity of pure compounds or complex samples, such as influents and effluents of STPs (Soto et al., 1995; Körner et al., 1999; Leusch et al., 2006b; Shappell, 2006; Lee et al., 2008). It is currently one of the most sensitive estrogenicity in vitro screens, comparable to the mammalian ER transcription activation assay (Soto et al., 1995; Andersen et al., 1999; Silva et al., 2007). Moreover, since its endpoint involves the interaction of the entire cell physiology, it might give a more complete picture of the estrogenic potential than solely receptor interaction. However, the fact that cell proliferation can be mediated through other pathways than those involving transcriptional activation of estrogen responsive genes and could also include anti-estrogenic effects, might complicate interpretation of the results, specially for complex samples (Murk et al., 2002; ICCVAM, 2003). Moreover, MCF-7 proliferation as an endpoint is not a direct proof of a substance being estrogenic senso stricto. So far, reasonable agreement among results of cell proliferation assays and the classical ER binding and TA assays has been documented (Fang et al., 2000) and it can be hypothesized that increased complexity of a test system might not always hamper in vivo predictiveness. Recently ICCVAM reported a test method nomination evaluating the MCF-7 cell proliferation assay for screening estrogenic activity (ICCVAM, 2006), indicating that further research on cell proliferation as an estrogenic endpoint is highly supported. In our study, an adapted format of the E-screen assay was performed, in which induction of cell proliferation was not quantified as the total cell count after 5– 6 days exposure (Soto et al., 1995), but as the percentage of cells with re-induced cell cycle progression after only 24 h exposure. Since cell growth in MCF-7 cells is highly estrogen-dependent, low-steroid conditions synchronize approximately 95% of the MCF-7 cells in the G0/G1 phase of the cell cycle. The percentage of cells re-entering the S-phase of the cell cycle after exposure to compounds or samples, can be consequently used as an indication of estrogenicity and is easily measured with flow cytometry (Vanparys et al., 2006). 4.1. Evaluation of the flow cytometric E-screen assay

Fig. 6. Linear regression of the % estrogenic removal in function of two physical purification parameters: % COD removal and % SS removal. The straight line represents the regression curve; dashed lines indicate 95% confidence intervals. Regression equations, R² values and significance levels are indicated in the respective graphs.

A series of 9 independent experiments with 17β-estradiol was performed in a 2 year period, covering different laboratory conditions (passage number, MCF-7 cell stocks, technicians), to assess the sensitivity and reproducibility of the assay system. Considering an EC50 value of 1.96 ± 0.44 pM and CV of 22%, the variation of this assay is acceptable compared to the variation observed in ER activity assays (12–25%) such as the Yeast Estrogen Screen (EC50 = 0.73 ± 0.13 nM, CV = 18%; Bovee et al., 2009), the MELN TA assay (EC50 = 33 ± 7 pM, CV = 21%; Berckmans et al., 2007) and ER-CALUX (EC50 = 6–20 pM, CV = 5–25%; Legler et al., 1999; Murk et al., 2002; Sonneveld et al., 2005). For the ER binding assay, overall 1000 times higher EC50 values are measured, with CVs ranging from 15 to 25% (Blair et al., 2000; Murk et al., 2002). The detection and quantification limit for the flow cytometric assay are 1 fM and 0.18 pM respectively and by far the lowest reported in literature. Oh et al. (2000) also stated an LOD value for the classical E-screen assay of 1 fM, however, exact data were not shown. Reported LOD values for ER-CALUX, YES and ER binding assays are 0.5–0.8 pM, 10 pM and 1 μM, respectively (Legler et al., 1999; Murk et al., 2002; Sonneveld et al., 2005). Considering the low EC50

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value and acceptable CV, the flow cytometric MCF-7 proliferation assay is currently one of the most sensitive assays for detecting estrogenicity. This is very reassuring, since the estrogenic response measured is far more complex and therefore expected to be somewhat more variable than the more straightforward ER binding or ER transactivation-reporter systems. The flow cytometric E-screen was further tested with 17 selected compounds, including 11 compounds of the ICCVAM reference list for validation of in vitro ER binding and ER TA assays (ICCVAM, 2003). Overall, good agreement was found with other in vitro and in vivo estrogenicity screens based on the qualitative positive/negative assignment of the ICCVAM reference compounds and in vivo uterotrophic assay data (ICCVAM, 2003; EDKB, 2009). Moreover, very strong linear relationships were found with the classical E-screen (R² = 0.98) but also with the YES test (R² = 0.87) and the ER binding assay (R² = 0.84) (Fang et al., 2000). In total, only 3 compounds were assigned negative (DEHP, atrazine and TCDD), while the remaining 14 compounds were all positive. Atrazine and DEHP are more commonly considered as non-estrogenic compounds in vitro (Legler et al., 2002). TCDD has been indicated as clearly non-estrogenic in cell proliferation, the ER binding and ER TA assay (Legler et al., 1999) however, is classified as a strong estrogenic compound in the ER TA assay of the ICCVAM reference list (ICCVAM, 2003). Due to its interaction with the Ah-receptor, it is however believed to evoke predominantly non-estrogenic effects (Krishnan and Safe, 1993). The estrogenicity of BaP is rather controversial, since no ER binding activity has been measured for the mother compound, although its metabolites exhibit affinity for the estrogen receptor (Charles et al., 2000; Fertuck et al., 2001). It has been hypothesized that the cell proliferative effect of BaP might be a combination of ER activated cell proliferation and S-phase arrest due to genotoxicity (Plíšková et al., 2005). This assay, however, confirms the cell proliferative activity of BaP on MCF-7 cells measured in a previous study (Vanparys et al., 2006) and moreover defines its activity almost certainly as solely ER mediated with the ER blocking experiment (data not shown). The positive compounds DCHP, toxaphene and endosulfan are not included in the ICCVAM reference list. For DCHP, a strong ERα transactivation activity, but also a strong anti-estrogenic ERβ inhibition activity have been documented (Takeuchi et al., 2005). Both toxaphene and endosulfan exhibit a weak estrogenic activity in vitro (ER binding, ER TA and E-screen) (Soto et al., 1994; Ramamoorthy et al., 1997), although also non-estrogenic activity and for toxaphene also antagonistic effects have been documented (Shelby et al., 1996; Jørgensen et al., 1997). Even for compounds mentioned in the ICCVAM list, papers indicating opposite results were often found, perhaps due to the wide diversity of estrogenic screening assays currently developed, particularly for the ER binding assay (mouse versus rat versus human ER) and the ER TA assay (yeast versus human cells, different reporter systems). 4.2. Removal of estrogenic activity in different sewage treatment procedures To date, the quality of an STP is mainly evaluated in terms of removal of biochemical (BOD) and chemical oxygen demand (COD), suspended solids (SS), phosphorus (P) and nitrogen (N) (Directive 91/271/EEC, 1991). Estrogenicity of the effluent is not a quality criterion for the evaluation of the functionality of an STP. However, in view of the considerable load of estrogenic compounds that remain in the effluent and associated alterations in the reproductive physiology of fish living near STP discharge points (Jobling et al., 2006), it seems warranted to evaluate estrogenic removal efficacies of the different sewage treatment plants in the field, and moreover, to assess the remaining estrogenic load of these effluents at discharge. An increasing amount of studies are currently published evaluating

estrogenic removal efficiencies of sewage treatment plants, by either chemical analysis or by biological activity screens (Vethaak et al., 2005; Leusch et al., 2006b; Hashimoto et al., 2007). However, most studies only evaluated two or less different treatment types operational in the field. Only in controlled laboratory conditions, a wider range of different treatment processes have been compared (Gunnarsson et al., 2009). In this study, the flow cytometric E-screen assay was applied to compare and rank 5 different field-operational sewage treatment processes according to their estrogen removal efficacy. In total, 10 full-scale STPs in the Walloon region of Belgium were selected, covering the major processes of secondary treatment used in this region, such as activated sludge (AS), filter bed and lagoon, and with or without tertiary treatment (removal of P and N). In Belgium, the whole territory of surface water is considered as a sensitive area for eutrophication (SPGE, 2007). For sensitive areas, the Urban Waste Water Treatment Directive (91/271/EEC, 1991) declares that all agglomerations with a PE N 10,000 should have a tertiary treatment including removal of N and P. Although the implementation deadline in the Directive was set at 31/12/2008, several agglomerations are not yet conform this Directive (SPGE, 2007). Of the stations selected in this study, Bastogne Rhin and Malmedy shall be adapted to tertiary treatment by 08/2011 and 12/2011, respectively (SPGE, 2007). All the influent and effluent samples in this study showed clear dose dependent cell proliferation responses, that were solely estrogen receptor mediated as indicated with the estrogen antagonist ICI 182,780, except for the effluent of Bastogne. Overall, EEQs found in influent and effluent samples of this study are in the same order of magnitude than EEQs found in literature (Körner et al., 1999; Leusch et al., 2006b). As expected, the estrogenic potencies of the effluent samples were consistently lower than that of the corresponding influent samples, regardless of the treatment procedure, as in agreement with chemical analysis and bioassay studies from literature (Leusch et al., 2006b; Lee et al., 2008). Estrogenic removal efficiencies of the different procedures can be ranked in following order: AS + P/N removal N AS N lagoon N filter bed. Moreover, as indicated in Fig. 5, AS followed by removal of P and N led to very low estrogenic loads in the effluent, apparently independent of the initially estrogenic load in the influent sample. If the 90% removal criteria is considered, both lagoons (Bertrix and Malmedy) and the filter bed (Morlanwelz) did not reach this criteria, while efficiencies of AS + P/N removal in 2 of 3 stations (Marche and Plombières) were far better than 90%. The third station with AS + P/N removal (La Roche) had slightly lower efficiencies, probably due to the very low initial estrogenic load in the influent sample. Activated sludge treatment has been previously shown to be very effective in removal of estrogen hormones. Based on chemical analysis, the average removal rates for AS processes are slightly higher for E2 (91%) than for E1 (80%) and EE2 (76%), however E1 is the most difficult estrogen to predict with reported removal rates between 45% and 99% (Nakada et al., 2006; Johnson et al., 2007). Industrial chemicals such as nonylphenol and octylphenol are less efficiently removed, with removal rates of 61–75% and 32–65%, respectively (Nakada et al., 2006). In the study of Leusch et al. (2006b), AS treatment removed N90% of the estrogenic activity in raw sewage, measured with the ER binding assay. In this study, most chemicals measured (E1, E2, octylphenol, and bisphenol A) in effluents were close to or below the detection limit, only nonylphenol was still measured in considerable amounts (μg/L) (Leusch et al., 2006b). The filter bed is generally considered to have less estrogenic removal capacities than the activated sludge treatment (Auriol et al., 2006). Although we only included one STP with filter bed treatment (Morlanwelz), it clearly confirmed this statement with only 62% removal of estrogenic activity and thus the lowest removal efficiency measured in this study. Comparison of the purification parameters of

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the different STPs (Table 1) also indicated Morlanwelz as the STP with lowest COD and lowest SS removal. Although the estrogen removal efficiencies of the STPs with lagoon treatment were higher (±80%) than the filter bed, effluents of lagoon treatment displayed highest remaining estrogenic loads, while also N and P removal was least efficient. An important difference between filter bed and lagoon is the time of contact between bacteria and water. In the filter bed, this time is very short; in the lagoon this time is very long. Due to the longer sludge ages and the sensitivity of natural steroid estrogens for photodegradation, lagoon treatments overall present a better removal of natural steroid estrogens than filter bed treatment (Leusch et al., 2006b; Johnson et al., 2007). Correlation of the different purification parameters defining the general efficiency of an STP with the estrogenicity endpoints derived in this study revealed that the estrogen removal efficiency was positively correlated with the removal efficiency of COD and SS, while the maximal inducible effect (RPEmax) was negatively correlated with the BOD5. This might indicate that the overall performance of an STP to remove estrogenicity is linked to the performance of an STP to remove waste. Although it should be stated that the purification data used for the correlation analysis are an indication of the overall purification performance and are not related to the influent and effluent samples analysed for estrogenic removal efficiency. Therefore, caution is needed before any conclusions can be drawn. Based on the EEQ values (0.52–8.53 ng/L) found in the extracts of the STP effluents, it remains uncertain if the remaining estrogenic activity would cause major adverse effects in wildlife when discharged. The derived Predicted No Effect Concentration (PNEC) for EE2 and E2 is 0.02 ng/L based on vitellogenin induction data in male rainbow trout with an assessment factor of 50 (chronic NOECs for two trophic levels) (Carlsson et al., 2006). For EE2, known to be much more potent in fish than E1 or E2, endocrine disruptive effects ranging from vitellogenin induction, intersex, reduced fecundity, embryo viability and impaired reproductive behaviour have been documented at concentrations from 0.1 to 15 ng/L (Nash et al., 2004). Moreover, EE2 is approximately 100× more potent in fish than measured in in vitro assays (Vethaak et al., 2005), therefore in vitro assays might underestimate the estrogenic hazard for fish substantially (Hugett et al., 2003). In previous studies in the Walloon region, no clear differences were detected in reproductive parameters (gonadosomatic index, histological testicular and ovarian stages, intersexuality, sex steroid levels and aromatase activity) of gudgeon (Gobio gobio) and stoneloach (Barbatula barbatula) living up- and downstream two STPs (Goffontaine and Wegnez) (Douxfils et al., 2007). Although recently, increased incidence of ovotestis were observed in gudgeon living in the Vesdre River downstream the Wegnez station (Nadzialek et al., 2010). The actual estrogenic load in a river, originating from STP sewage, depends in important matter on the size of the receiving water system, the flow dynamics and the season. For risk assessment purposes a default dilution factor of only 10 is considered for calculations of risks of sewage water entering surface water. However, local dilution factors might be varied and substantially variable in time (Carlsson et al., 2006) and small streams might be more susceptible to waste discharges, particularly in densely populated and industrialised areas (Körner et al., 2001; Triebskorn et al., 2001). Since the Estradiol Equivalents (EEQs) approach, comparable to the TEF approach for dioxin-like compounds, allows comparison of estrogenic activity in complex samples, this concept could be further evaluated as benchmark for activity criteria. In this way the minimal dilution of the effluent samples can be estimated to minimize risk. Considering a dilution factor of 10, estrogenic activity in this study remains largely above the reported PNEC value. To fully assess the endocrine disruptive risk for wildlife, however, extensive research of the estrogenicity of the STP effluents and the receiving aquatic systems are necessary over time and also other modes of action of

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endocrine disruption, such as aromatase inhibition and androgenic, anti-estrogenic and anti-androgenic effects should be assessed as they have been indicated as substantially important as well. An integrated picture can only be achieved if chemical analysis, endocrine activity screens and in vivo research in the field are combined (Nadzialek et al., 2010). In conclusion, this paper illustrates the flow cytometric E-screen and MCF-7 cell proliferation in general, as an appropriate assay for detection and ranking of estrogenic activity. Despite the higher complexity of the endpoint measured, this assay appears a highly responsive, sensitive and reproducible system that corresponds very well with other ER-based estrogenicity screens. Even for complex samples, such as influent and effluents of sewage treatment plants, this assay showed nearly solely ER mediated estrogenic responses. This indicates that the complexity of the endpoint measured does not imply increased interpretation difficulties but on the contrary includes a more physiological response than receptor interaction alone. This study, together with previous E-screen studies, therefore paves the way for a wider application of this assay in multiple domains, ranging from pure compound screening to hazard identification of complex samples and environmental monitoring. Acknowledgements Caroline Vanparys, Stéphanie Nadzialek and Sophie Depiereux are supported by the ‘Institute for the Promotion of Innovation by Science and Technology (IWT)’ in Flanders (Belgium), the Funds for Research in Industry and Agriculture (FRIA) and the Funds for Scientific Research (FRS-FNRS) in Belgium, respectively. The technical support by Femke De Croock is highly appreciated. The authors are grateful to the SPGE (Société Publique de Gestion de l'Eau, Walloon Region, Belgium) for providing access to influent and effluent samples of the different sewage treatment plants. References Andersen HR, Andersson A-M, Arnold SF, Autrup H, Barfoed M, Beresford NA, et al. Comparison of short-term estrogenicity tests for identification of hormonedisrupting chemicals. Environ Health Perspect 1999;107(Suppl. 1):89-108. Armbruster DA, Tillman MD, Hubbs LM. Limit of Detection (LOD)/Limit of Quantitation (LOQ): comparison of the empirical and the statistical methods exemplified with GC–MS assays of abused drugs. Clin Chem 1994;40(7):1233–8. Auriol M, Filali-Meknassi Y, Tyagi RD, Adams CD, Surampalli RY. Endocrine disrupting compounds removal from wastewater, a new challenge. Process Biochem 2006;41: 525–39. Berckmans P, Leppens H, Vangenechten C, Witters H. Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment. Toxicol Vitro 2007;21(7):1262–7. Blair RM, Fang H, Branham WS, Hass BS, Dial SL, Moland CL, et al. Estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. Toxicol Sci 2000;54:138–53. Bovee TFH, Bor G, Becue I, Daamen FEJ, van Duursen MBM, Lehmann S, et al. Interlaboratory comparison of a yeast bioassay for the determination of estrogenic activity in biological samples. Anal Chim Acta 2009;637(1–2):265–72. Brian JV, Harris CA, Scholze M, Backhaus T, Booy P, Lamoree M, et al. Accurate prediction of the response of freshwater fish to a mixture of estrogenic chemicals. Environ Health Perspect 2005;113:721–8. Brion F, Tyler CR, Palazzi X, Laillet B, Porcher JM, Garric J, et al. Impacts of 17β-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embyro-, larval-, juvenile- and adult-life stages in zebrafish (Danio rerio). Aquat Toxicol 2004;68:193–217. Carlsson C, Johansson A-K, Alvan G, Bergman K, Kühler T. Are pharmaceuticals potent environmental pollutants? Part I: environmental risk assessments of selected pharmaceutical ingredients. Sci Total Environ 2006;364:67–87. Charles GD, Bartels MJ, Zacharewski TR, Gollapudi BB, Freshour NL, Carney EW. Activity of benzo(a)pyrene and its hydroxylated metabolites in an estrogen receptor-α reporter gene assay. Toxicol Sci 2000;55:320–6. Colborn T, vom Saal FS, Soto AM. Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ Health Perspect 1993;101:378–84. Directive 91/271/EEC, European Urban Waste Water Treatment Directive of 21 may 1991. Douxfils J, Mandiki R, Silvestre F, Arnaud B, Leroy D, Thomé J-P, et al. Do sewage treatment plant discharges substantially impair fish reproduction in polluted rivers? Sci Total Environ 2007;372:497–514.

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