Biological Activity of Wastewater Assessed Using in Vitro Cell-Based Assays

Bioanalytical tools, namely in vitro bioassays, can be employed in tandem with chemical analyses to assess the ecacy of wastewater treatment and the potential for adverse effects from the discharges of wastewater into receiving waters. In the present study, samples of untreated wastewater (i.e. inuent) and treated wastewater (i.e. euent) were collected from two wastewater treatment plants and a wastewater treatment lagoon serving municipalities in southern Ontario, Canada. In addition, grab samples of surface water were collected downstream of the lagoon discharge. After solid phase extraction (SPE) using ion-exchange columns for basic/neutral and acidic compounds, respectively, the extracts were analyzed for a suite of 16 indicator compounds. The two SPE extracts were combined for analysis of biological responses in four in vitro cell-based bioassays. The concentrations of several indicator compounds, including the estrogens, 17β-estradiol and 17α-ethinylestradiol, were below the limits of detection. However, androstenedione and estrone were detected in several inuent samples. The concentrations of these steroid hormones and some of the other indicator compounds declined during treatment but acesulfame K, carbamazepine, trimethoprim and DEET persisted in the euent. The MTS- CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) indicated that cell viability was not affected by exposure to the extracts. The Qiagen Nuclear Receptors 10-Pathway Reporter Array indicated that several cellular pathways were upregulated, with the greatest upregulation observed with the estrogen receptor (i.e. induction ratios 12 to 47) and the liver X receptor (i.e. induction ratios 10 to 45). The ERα CALUX assay indicated that estrogenic activity was lower in euents compared to inuents, with the greatest estrogenic activity observed for grab samples of inuent from the lagoon (i.e. 56-215 ng L -1 17β-estradiol equivalents). Finally, the results of the Nrf2 Luciferase Luminescence Assay indicated a lower oxidative stress in the euent samples. Overall, the present study demonstrates that chemical analyses are limited in their ability to predict or explain reductions in the toxicity of treated wastewater. There are thus advantages to using a combination of chemical analyses and in vitro bioassays to monitor the treatment eciency of wastewater treatment plants and to predict the potential impacts of wastewater discharges into receiving waters. The assay with the Nrf2 luciferase reporter MCF7 stable cell line, hereafter referred to as the Nrf2 assay was employed to determine whether exposure to sample extracts resulted in an oxidative stress response. Nrf2 cells were obtained from Signosis Inc. (Santa Clara, CA, USA) and were cultured in Dulbecco’s Modied Eagle medium (DMEM) supplemented with 10% FBS, 1× GlutaMAX, 0.8 mg mL − 1 geneticin (G418 sulfate), 100 units mL − 1 penicillin and 100 µg mL − 1 streptomycin. All media components were from Life Technologies. Once 75–95% conuent in T75 asks, cells were trypsinized with 0.25% trypsin-EDTA (Life Technologies) and plated onto white, opaque 96-well plates at a density of 5×10 4 cells mL − 1 (i.e. 100 µL of cells added per well) for luminescence analyses. Cells were allowed to grow for 24-h prior to the addition of sample extracts. Following 24-h exposure to the extracts, the Bright-Glo™ Luciferase Assay System (Promega) was employed to quantitate rey luciferase expression. Once more, luminescence measurements were performed on a Berthold Technologies Mitras LB 940 Multimode Microplate Reader. quantitation of the oxidative stress response, a dilution series of tert-butylhydroquinone (t-BHQ, Sigma-Aldrich) at concentrations ranging from 1×10 − 5 – 1.6×10 − 7 M was included on each opaque plate. Oxidative stress was expressed in terms t-BHQ equivalents. reagent Reduced 96-well (Greiner)

Due to advances in analytical methods, it is now possible to monitor thousands of chemical contaminants in water and wastewater (Richardson and Ternes 2018). Regulating wastewater discharges based on the analysis of a wide range of target compounds is an expensive and time-consuming approach (Jia et al. 2015). In addition, limiting contaminant monitoring to a number of known pollutants may underestimate the risks to the environment and possible hazards to human health (Smital et al. 2013) and do not take into account mixture effects (Snyder and Leusch 2018). A complementary approach is to use bioanalytical tools to screen for different modes of toxicity in samples of water and wastewater. These screening assays can be combined with subsequent analytical approaches to assess the effectiveness of wastewater treatment (Escher and Leusch 2012, Jia et al. 2015).
In the present study, extracts from samples of treated and untreated wastewater, and surface water were analyzed for the concentrations of 16 target analytes (i.e. indicator compounds), which included speci c compounds selected from the classes of estrogens, androgens, antibiotics and pharmaceuticals, pesticides, arti cial sweeteners and personal care products and results were analyzed using two approaches described in Neale et al. (2020): comparison between in uent and e uent for the evaluation of treatment process e ciency and comparison to effect-based trigger values for the evaluation of treated water quality. The extracts were also tested for toxicity using four mammalian cell-based in vitro assays. In the case of in vitro toxicity testing, extracts from water and wastewater were initially screened using the Qiagen Nuclear Receptors 10-Pathway Reporter Array to provide information regarding the regulation of multiple nuclear receptors. The estrogenic activity of the extracts was subsequently tested with the ERα CALUX assay. The capacity of extracts to induce oxidative stress was tested with a Nrf2 Luciferase Luminescence Assay. Finally, the MTS assay was employed to evaluate nonspeci c cytotoxicity in order to ensure that responses observed with the reporter gene-based assays were not in uenced by decreased cell viability. In tandem with chemical analyses, the in vitro toxicity data were used to evaluate whether toxic substances are removed effectively from wastewater and to evaluate the sensitivity of the assays to detect changes in the levels of speci c classes of chemical contaminants in water and wastewater. Samples of wastewater were collected from two WWTPs and a wastewater lagoon (WL) serving municipalities in the southern region in the province of Ontario, Canada, and from river water 2.0 km downstream of the lagoon discharge.

Indicator Compounds
The 16 indicator compounds analyzed in extracts of water and wastewater are listed in Table 1, along with information on the class of the contaminant, the supplier, and the stable-isotope labelled surrogate used as an internal standard for quanti cation. An external standard stock solution (1000 ppm) for these indicator compounds was prepared in methanol (Fisher Optima, LC/MS grade) and stored in the dark at 4 o C for preparation of fresh analytical standards.

Sample Collection
In 2014, samples of wastewater were collected at two WWTPs in the West Central Region of southern Ontario (WWTP 1 and WWTP 2). Both treatment plants employ conventional activated sludge wastewater treatment, but WWTP 2 also has a tertiary treatment train. At these WWTPs, 24-h composite samples of untreated wastewater (i.e. in uent) and treated wastewater (i.e. e uent) were collected once a month for 5 months from April to August. A wastewater lagoon located in the Central Region of southern Ontario (WL) was also sampled in the spring and fall of 2014 during periods of intermittent discharge. In this case, grab samples of in uent and e uent were collected in May, June and twice in September. Grab samples of surface water were also collected from a river approximately 2.0 km downstream of the lagoon during discharge. Information regarding the WWTPs and WL is summarized in Table S1 in Supplementary Information.
The Laboratory Services Branch of the Ontario Ministry of the Environment, Conservation and Parks (Etobicoke, ON, Canada) analyzed all samples of water and wastewater for a wide range of water quality parameters. Samples were analyzed for a range of cations and metals, chemical oxygen demand (COD), carbonaceous biochemical oxygen demand (cBOD), pH, nitrogen species, phosphorous species, total and dissolved organic carbon, suspended and dissolved solids, etc. Samples were analyzed according to standard protocols developed by the Ministry.

Sample Extraction
Subsamples of water and wastewater were extracted using two solid phase extraction (SPE) methods, including extraction with Oasis® MCX cartridges (6 mL, 150 mg) to concentrate base/neutral compounds and extraction with MAX cartridges (6 mL, 400 mg) to concentrate acidic compounds. The SPE cartridges were purchased from Waters (Milford, MA, USA). Prior to extraction, samples (110 mL) were ltered through 1 µm glass-ber lters (Fisher Scienti c, Ottawa, ON, Canada) and the pH was adjusted to pH = 2.5 for extraction using MCX cartridges and to pH = 8.0 for extraction using MAX cartridges. For subsamples prepared for analysis of contaminants (n = 3), the samples were spiked with the internal standard solution of labeled surrogates. Subsamples prepared for bioassays (n = 3) were not spiked. The methods for SPE extraction were previously described by Baalbaki et al. (2017). Following extraction with both types of cartridges, elution solvents were evaporated to near dryness and reconstituted in either 0.4 mL of a 1:1 methanol-water solution (Fisher Optima HPLC grade) for chemical analyses, or in 0.4 mL of DMSO (Sigma-Aldrich Bioreagent, molecular biology grade) for bioassays. The MCX and MAX extracts were analyzed separately for contaminants, as described below. Prior to conducting the in vitro bioassays, the MCX and MAX extracts were pooled for each sample. Extracts were placed into amber high performance liquid chromatography (HPLC) vials with polytetra uoroethylene (PTFE) tops and stored at -20 o C until analysis. The overall pre-concentration factor was 275× (i.e. 0.11 L of sample concentrated to 400 µL extract). Chemical analysis and bioassays were run on triplicate samples. To evaluate whether there was contamination from the extraction process and to allow for the calculation of SPE recoveries, procedural blanks were prepared by extracting 110 mL of Milli-Q water. Prior to extraction, the procedural blank was spiked with the internal standard solution of labeled surrogates and the pH was adjusted for extraction using MCX and MAX cartridges, as described for the water and wastewater samples.

Chemical Analysis
The concentrations of the 16 indicator compounds in procedural blanks and in extracts from water and wastewater were quanti ed by liquid chromatography with high resolution mass spectrometry (LC-HRMS) or by liquid chromatography with tandem mass spectrometry  Table S2 in Supplementary Information describes which of these three analytical methods was employed for each of the indicator compounds. For LC-HRMS, a 6-point calibration curve covering the range of anticipated analyte concentrations was used for external calibration. The concentrations were adjusted according to the recovery of the internal standards in order to compensate for the effects of the sample matrix on ionization and for variations in the recoveries during sample extraction.
The limits of detection (LODs) and limits of quanti cation (LOQs) were determined by analyzing serial dilutions of a 1:1 methanol-water standard solution containing all compounds. LOD and LOQ are de ned as the indicator compound concentration producing a peak with a signal-to-noise ratio of 3 and 10, respectively. The LODs and LOQs for all analytes are listed in Table S3 in Supplementary Information.
Two samples spiked with the indicator compounds at concentrations of 12 and 30 µg L − 1 were extracted and analyzed for quality control purposes. For these spiked samples, the relative error between expected and measured quality control concentrations was 20% for all indicator compounds.

Nuclear Receptors 10-Pathway Reporter Array
The Nuclear Receptors 10-Pathway Reporter Array purchased from Qiagen (Germantown, MD, USA), hereafter referred to as the 10-Pathway Reporter Array, was used as a screening assay to determine which cellular signalling pathways are affected or regulated by exposure to sample extracts. MCF7 breast cancer cells were selected to carry out this assay. This cell line expresses a majority of the transcription factors monitored by the Qiagen 10-Pathway Reporter Array. Recently, Kittler et al. (2013) systematically "mapped the genomic binding sites of all nuclear receptors expressed in MCF-7 breast cancer cells". In total, they "mapped the genomic binding sites of a total of 33 proteins whose corresponding genes are expressed at moderate to high levels in MCF-7 cells". Amongst these proteins, 9 of the 10 transcription factors were identi ed, whose activity can be monitored by the Qiagen reporter array. The only transcription factor that will likely not be encountered in the MCF7 cells is HNF4 (hepatocyte nuclear factor 4). However, in Escher et al. (2014), HNF4 (hepatocyte nuclear factor 4) that is not expressed by the MCF7 cell line is not a major player in the MOA in any of the toxicity pathways.
All cell lines employed in this study were cultured at 37 o C, 5% CO 2 and 95% humidity. All cell culture materials manufactured by Life Technologies (Rockville, MD, USA) were supplied by Thermo Fisher. The MCF7 cells were cultured in Dulbecco's Modi ed Eagle Medium (DMEM) media supplemented with 10% fetal bovine serum (FBS), 1x GlutaMAX supplement, 100 units mL − 1 penicillin and 100 µg mL − 1 streptomycin. To carry out this assay, cells were transfected with Qiagen Reporter Array DNA. This was achieved employing the Attractene Transfection Reagent (Qiagen) as described by the manufacturer. Prior to transfection, 75-95% con uent MCF7 cells growing in T75 plates were trypsinized with 0.25% trypsin-EDTA and plated onto white, opaque 96-well plates at a density of 4×10 4 cells mL − 1 (i.e. 100 µL per well) in Opti-MEM® I Reduced Serum Media (Invitrogen) supplemented with 5% FBS and 1% NEAAs. Cells were plated onto transparent polypropylene 96-well plates. Following 24-h growth, media was removed from the cells and the cells were rinsed with 100 µL of phosphate buffered saline (PBS) prior to the addition of 88 µL per well fresh antibiotic and Opti-MEM® I Reduced Serum Media (FBSfree). At this point, cells were exposed to the transfection materials.
Transfection materials were prepared in 15 mL polypropylene centrifuge tubes. The total volume of the transfection materials prepared was determined by the number of wells on the 96-well plate to be transfected. Brie y, into each centrifuge tube, 0.08 µg per well (i.e., 0.8 µL per well) of Reporter Array DNA was added to 12 µL per well of Opti-MEM® I Reduced Serum Media. Next, 0.4 µL per well of Attractene Transfection Reagent was added to the DNA and the mixture was vortexed. The DNA complexes were then incubated at room temperature for 15 minutes, before being added to the MCF7 cells in the 96-well plates. Cells were then incubated under normal growth conditions for 48-h. Following incubation with the transfection reagents, the Dual-Luciferase Reporter Assay System (Promega) was utilized to monitor luminescence. As described by the supplier for the Dual-Luciferase Reporter Assay System, re y luminescence and Renilla luminescence were quanti ed on the luminometer, which was a Berthold Technologies Mitras LB 940 Multimode Microplate Reader. Positive and negative control transfections were also prepared and included on each plate.
Responses for the various reporters in the array were quanti ed in terms of Induction Ratios (IR), which in luminescence-based assays is de ned as the ratio of the relative light units (RLU) measured for an experimental sample relative to a suitable control sample, as described in Eq. 1 (Jia et al. 2015). For the assay used, an IR value > 5 is recommended as evidence of a signi cant response. This value was later compared to Escher et al. (2014) who reported an IR of 1.5 as evidence of a positive biological response in other in vitro assays and further suggest that the ratio was su cient to ensure that a signi cant effect was observed.

ERα CALUX Assay
The ERα Chemically Activated LUciferase eXpression® assay, hereafter referred to as the ERα CALUX assay was employed to determine the estrogenic activity of sample extracts. This reporter gene assay is based on the ERα U2OS.Luc cell line, which can be stably transfected with ERα or ERβ and a luciferase reporter gene and was obtained under license from Biodetection Systems B.V. (Amsterdam, the Netherlands). The recombinant construct for this reporter-gene assay and the basis for the response in the assay has been previously described (Quaedackers et  The ERα U2OS.Luc cells were cultured using the methods recommended by the supplier, with minor modi cations. As stated previously, all cell lines employed in this study were cultured at 37 o C, 5% CO 2 and 95% humidity. All cell culture materials were from Life Technologies. Brie y, the cells were cultured in T75 asks containing 22 mL of DMEM/F12 media supplemented with 10% Fetal Bovine Serum (FBS), 100 units mL − 1 of penicillin and 100 µg mL − 1 of streptomycin, 0.8 mg mL − 1 geneticin and 1x non-essential amino acids (NEAAs). All assays were carried out in 96-well plates containing assay medium without phenol red and supplemented with 10% USDAapproved charcoal stripped FBS, 100 units mL − 1 penicillin and 100 µg mL − 1 streptomycin and 1x NEAAs (note: no geneticin was added to the assay media). Phenol red-free media was employed because of the ER-agonist activity of phenol red (Berthois et al. 1986). Furthermore, charcoal stripped FBS was employed to minimize exposure to any estrogens present in the culture media.

Statistical Analysis
For all data on the concentrations of indicator compounds and the responses in in vitro assays, the statistical analysis was done using the statistical package available on Excel.

Wastewater Quality Results
A wide range of wastewater quality parameters were monitored in the in uent and e uent of WWTP 1, WWTP 2 and WL. As illustrated with a selected number of these parameters, there was a marked improvement in water quality as a result of wastewater treatment (Fig. 1). WWTP2 was particularly effective at treating the wastewater, possibly because of the tertiary treatment system at this plant. The wastewater lagoon was also effective at improving wastewater quality before discharge into receiving waters (Fig. 1).

Concentrations of Indicator Compounds
The apparent recoveries for the 16 indicator compounds were rst evaluated using Milli-Q water. Of the 16 indicator compounds analyzed, 12 were found to have mean recoveries > 80%, including the estrogens, estrone (113 ± 47%), 17ß-estradiol (113 ± 48%) and 17αethinylestradiol (121 ± 47%). There were lower recoveries for DEET and sulfamethoxazole of 57 ± 30% and 57 ± 28%, respectively. A high apparent recovery of 604 ± 426% for acesulfame K indicates that there was signal enhancement for this compound; probably because a constituent of the sample matrix increased the ionization e ciency. The responses to the internal standards spiked into all samples of water and wastewater were used for quantitation of all indicator compounds.
The analytical data for wastewater samples collected at WWTPs 1 and 2 are summarized in Table 2 and the data for WL and receiving waters are summarized in Table 3. The estrogens, 17ß-estradiol and 17α-ethinylestradiol, the pharmaceutical, gem brozil, and the herbicides, atrazine, bentazon and MCPA were not detected in any of the samples. Acesulfame K and carbamazepine were detected in all in uent and e uent samples from WWTP 1 and were widely detected in samples collected from WWTP 2 ( Table 2) and WL (Table 3).
Carbamazepine is known to be poorly removed in WWTPs (Blair et al. 2013), and the arti cial sweetener, acesulfame K is also poorly removed by wastewater treatment (Subedi and Kannan 2014). The antibiotics, sulfamethoxazole and trimethoprim were widely detected in both in uent and e uent samples from WWTP 1 and WWTP 2 ( Table 2) but these compounds were detected less frequently in grab samples from WL ( Table 3). The non-prescription analgesic, ibuprofen and the antibacterial compound, triclosan were frequently detected in in uent samples collected in WWTP1, but not in e uent samples, which is consistent with the high removals usually reported for these compounds in WWTPs (Blair et al. 2013).
It is notable that androstenedione, which is an intermediate in the biosynthesis of testosterone, was detected at concentrations > 100 ng L − 1 in several in uent samples from the WWTPs (Table 2). There are only limited monitoring data for androstenedione in the literature.
However, Baalbaki et al. (2017) detected androstenedione in in uent samples but not in e uent samples collected from WWTPs, indicating that this compound is effectively removed by wastewater treatment. Estrone was detected in selected in uent samples collected in June and August from WWTP 1 ( Table 2) and from in uent samples collected in May and September in WL ( Table 3). The concentrations of estrone were lower in the e uent. This intermediate in the biosynthesis of 17ß-estradiol was present at concentrations < LOQ in selected in uent and e uent samples collected from WWTP 2 in June and August (Table 2).
For DEET, the active ingredient in some insect repellents, concentrations were highest in the months of June and July at WWTP 1 and WWTP 2 ( Table 2), presumably because of the higher numbers of biting insects in the summer. However, at WL, DEET levels peaked in May (Table 3). Overall, DEET appears to be partially removed by wastewater treatment, with generally lower levels detected in e uent samples. The herbicide, 2,4-D was detected in wastewater samples collected in June and July at WWTP 1 and occasionally detected in samples collected from WWTP 2 ( Table 2) and WL (Table 3). We assume that this herbicide made its way into domestic wastewater from storm water over ows. However, since 2,4-D has been banned since 2009 in Ontario for cosmetic weed control, it is di cult to speculate on the sources of this herbicide. Acesulfame K, DEET and ibuprofen were frequently detected in the surface water samples collected from a river 2.0 km downstream of the discharge from the wastewater lagoon (Table 3). The river sub-watershed is approximately 31% agricultural, 17% urban, 3% roads, 3% golf courses, and 3% industrial with the remainder being natural heritage features.
Caution should be used in interpreting the data on the relative concentrations of the target compounds in in uent and e uent samples as an indicator of the removals of contaminants by wastewater treatment. The hydraulic retention times for wastewater of 1-3 days in WWTPs and even longer in some wastewater lagoons means that in uent and e uent samples collected on the same day are not synchronized in terms of the composition of the wastewater (Ort et al. 2010). This is especially problematic when interpreting the analytical results from the grab samples of in uent and e uent collected at the wastewater lagoon. To overcome this problem, we recently used a modelling approach to estimate the removals of contaminants of emerging concern in WWTPs (Baalbaki et al. 2017). Table 2 Mean concentrations (ng L -1 ; ± %SD) of microcontaminants in wastewater sampled in 2014 from in uent and e uent of WWTP 1 and WWTP 2. ND = Not detected at concentrations > LOD; P = Present at concentrations < LOQ; NA = Not analyzed.

COMPOUND MEAN CONCENTRATIONS (± %SD)
(ng L − 1 ) In  Table 3 Mean concentrations (ng L -1 ; ± %SD) of microcontaminants in wastewater sampled in 2014 from in uent (INF) and e uent (EFFL) of WL and in a river surface water 2.0 km downstream of the lagoon discharge. ND = Not detected at concentrations > LOD; P = Present at concentrations < LOQ; NA = Not analyzed.

Results of MTS Assay
In working with human cell lines, the endocrine endpoint generally exhibits greater sensitivity than endpoints of toxicity (Kolkman et al. 2013). Nonetheless, it is important to evaluate whether a reduced response in vitro assays is caused by cytotoxicity (i.e., decreased cell viability). The MTS assay indicated that cell viability was not affected by exposure to the sample extracts at all dilutions examined (data not presented). Note that all cell lines employed (i.e., MCF7, Qiagen transfected MCF7, U2OS, Nrf2 cells) were tested for cell viability using the MTS assay. Thus, any change in response using these cell lines can be attributed to pathway-speci c impacts.

Results of 10-Pathway Reporter Array
The results of the 10-Pathway Reporter Array were used to select which cellular responses should be investigated more closely. Table 4 summarizes the mean IR values (n = 3) for the various receptors following exposure to selected wastewater extracts. All sample extracts induced upregulation of the estrogen receptor and liver X receptor ( Table 4). IRs of 12 to 47 were recorded for the estrogen receptor, while IRs ranged from 10 to 45 for the liver X receptor. There were no obvious reductions to upregulation of these receptors in treatments with e uent samples relative to treatments with in uent samples. There was also upregulation of the vitamin D and retinoid X receptors in some treatments, with IRs ranging from 2 to 16 and from 3 to 19, respectively (Table 4). For the progesterone receptor, the IR values were  (Table 4). In uent 30 ± 16 2 ± 1 7 ± 3 3 ± 1 11 ± 6 5 ± 2 8 ± 2 9 ± 5 17 ± 6 E uent 28 ± 8 1 ± 1 2 ± 1 1 ± 0.3 5 ± 4 1 ± 0.2 4 ± 1 3 ± 1 11 ± 4 Surface Water While there was some evidence that the in uent extracts were more potent than the e uent extracts in upregulating some of the receptors, it was not possible to determine if the assay is sensitive enough to reproducibly detect differences between treatments with in uent versus e uent extracts. There was no signi cant upregulation of the androgen receptor or the retinoic acid receptor, with maximum IR values of 3 and 4, respectively ( Table 4). The lack of a signi cant response for upregulation of the androgen receptor is worth noting, since androstenedione was detected in several in uent samples.
In a study of in vitro bioassays to assess wastewater treatment, Escher et al. (2014) observed that 5 out of 25 nuclear receptors were activated when exposed to e uent extracts; including the pregnane X, PPARγ, liver X and glucocorticoid receptors. Based on the results from the 10-Pathway Reporter Array described in the current study, it is apparent that the estrogen and liver X receptors showed the greatest upregulation in treatments with wastewater extracts. Note that upregulation of these pathways occurred in treatments with samples of in uent, e uent and surface water. Signi cant upregulation was also observed for the retinoid X receptor.
Upregulation of liver X and retinoid X pathways is of particular interest as the two receptors form heterodimers that can then regulate genes associated with a range of cellular processes, such as lipid metabolism and in ammation. (Gage et al. 2016). The regulation of PPAR receptors by wastewater extracts is also of interest as these receptors are targeted by cholesterol-regulating drugs (Roberts et al. 2015), including the gem brozil drug selected for analysis in this study. Metcalfe et al. (2013) detected PPAR-agonists in extracts prepared from wastewater using an in vitro assay, but these responses were not correlated with the concentrations of cholesterol-reducing drugs targeted for analysis. A variety of other compounds that can be present in wastewaters have the capacity to bind with PPARs, including anti-in ammatory drugs (Gijsbers et al. 2011), and phthalates, per uorinated compounds and bisphenol-based compounds (Desvergne et al. 2009, Riu et al. 2011, Chamorro-Garcia et al. 2012. Synthetic glucocorticoids such as prednisone and hydrocortisone are drugs that are widely prescribed for suppression of in ammation. Synthetic progestins are the active ingredients for hormone therapies (e.g., for endometrial hyperplasia) and in many birth-control formulations. Future monitoring of wastewater using analytical techniques could include analysis for glucocorticoid and progesterone agonists used for therapy (Schriks et al. 2010, Wu et al. 2019).

Results of ERα CALUX Assay
The activation of the estrogen receptor observed in all samples tested with the 10-Pathway Reporter Array highlighted the need for additional tests of estrogenicity using the ERα CALUX assay. The results from the ERα CALUX assay in the present study, expressed as ng L − 1 E2 equivalents, demonstrated differences in the estrogenic potency of in uent and e uent extracts, as there was a decrease in the estrogenicity in all e uent samples collected following wastewater treatment ( Table 5). The mean estrogenic response to extracts from WWTP 1 in uent ranged from 27 to 72 ng L − 1 E2 equivalents, while the mean estrogenic response to extracts from WWTP 1 e uent ranged from 1 to 10 ng L − 1 E2 equivalents ( Table 5). The highest estrogenic activity was observed in samples collected in the months of June and August (Table 5). For samples collected from WWTP2, the mean estrogenic response to in uent samples ranged from 34 to 59 ng L − 1 E2 equivalents, while the mean estrogenic potency of e uent samples ranged from 2 to 14 ng L − 1 E2 equivalents (Table 5).
Similarly to WWTP 1, the highest estrogenic responses from WWTP 2 were observed in treatments with samples collected in May, June and August (Table 5).
No signi cant difference in estrogenic activity was observed between e uent samples from WWTP1 and WWTP2. Previously, it was suggested that nitri cation may enhance the degradation of steroid estrogens (Servos et al. 2005, Khanal et al. 2006). The treatment train for WWTP2 includes a nitri cation step, but since the mean concentration of nitrate plus nitrite in the e uent of WWTP 2 (i.e. 16.1 mg L − 1 ) over the monitoring period was only marginally higher than the mean concentration of nitrate and nitrite in the e uent of WWTP 1 (i.e. 14.8 mg L − 1 ), it is di cult to speculate on whether nitri cation is an important parameter for reducing estrogenic activity. The treatment train in WWTP 2 includes tertiary treatment by ltration, whereas there is only secondary treatment at WWTP 1, but the additional treatment step in WWTP 2 did not seem to enhance the reduction in estrogenicity. For grab samples collected from the wastewater treatment lagoon (WL), the mean estrogenic responses to extracts from in uent ranging from 56 to 215 ng L − 1 E2 equivalents were higher than the estrogenic responses observed in treatments with in uent from WWTPs 1 and 2 (Table 6). Nonetheless, the estrogenicity of e uents from WL was comparable to the estrogenicity of the e uents from the WWTPs, with mean values ranging from 4 to 13 ng L − 1 E2 equivalents. These values are higher than the 1-216 pg/L E2 equivalent values reported previous for wastewater (Kase et al. 2018). This indicates that treatment of wastewater in lagoons that are properly managed can be equally e cient as conventional WWTPs.
Finally, surface water samples collected downstream of the WL discharge were also estrogenic, with mean potencies of 5 to 17 ng L − 1 E2 equivalents ( Different values have been proposed as effect-based trigger (EBT) values for wastewater, 100-500 pg/L E2-equivalent (EEQ), and based on the discussion presented in Kase et al. (2018), the use of an EBT of 400 pg /L EEQ seems justi ed. Considering that the values measured in the treated wastewater (e uent) were much higher, in the range of 1-14 ng/L E2 equivalents, the results indicate a potential ecological risk associated with the discharge of the e uent. In the EU, an EBT value for the ERα CALUX assay of 3.8 ng L − 1 E2 equivalents has been proposed for drinking water and source waters , Brand et al. 2014). Since estrogenic activity was detected in surface waters downstream of the lagoon discharge (5 to 17 ng L − 1 E2 equivalents), it may be advisable to monitor drinking water for estrogenic activity using the ERα CALUX assay or another sensitive in vitro assay. Table 5 Mean (± %SD) responses in the ERα CALUX assay (ng L -1 E2 equivalents) and NFR2 assay (mg L -1 tBHQ equivalents) in treatments with extracts prepared from wastewater collected from WWTP 1 and WWTP 2.  Table 6 Mean (± %SD) responses in the ERα CALUX (ng L -1 E2 equivalents) and NFR2 (mg L -1 tBHQ) in treatments with extracts prepared from wastewater collected from WL and surface water downstream of the discharge from the lagoon.

Results of Nrf2 Assay
The Nrf2 assay is an indicator of oxidative stress in cells. More speci cally, the bioassay measures induction of the Nrf2-Keap-ARE pathway, which protects cells against oxidative damage resulting from spontaneous cellular processes or exposure to contaminants. (Jia et al. 2015) observed that the Nrf2-Keap-ARE pathway responds to "a very wide range of chemicals" but did not specify which classes of compounds were active. Martin et al. (2010) reported that 165 of the 309 chemicals tested in the Phase I ToxCast survey conducted by the US EPA induced oxidative stress, as detected by Nrf2 activation. After testing 19 compounds for responses in various in vitro assays, van der Linden et al. (2014) reported that 2,4-dichlorophenol, curcumin, ethyl acrylate, p-nitrophenol and propyl gallate, in addition to tBHQ, gave a positive response in an Nrf2 bioassay. Tang et al. (2013) detected signi cant Nrf2 activation in treatments with extracts from urban storm water and commented that, "further chemical analysis is required to identify the causative agents for the underlying toxicity".
Overall, the responses to wastewater extracts in the Nrf2 assay used in the present study indicate that there was a signi cant decrease in the capacity to induce oxidative stress in e uent extracts relative to in uent extracts from samples collected at WWTP 1 and WWTP 2 ( Table 5) and in WL (Table 6). In treatments with extracts from WWTP 1, exposure to untreated wastewater samples induced mean responses of t-BHQ equivalents ranging from 0.26 to 0.34 mg L − 1 , while mean responses to e uent samples did not exceed 0.12 mg L − 1 and were as low as 3.5×10 − 3 mg L − 1 ( Table 5). Likewise for samples from WWTP 2, exposures to extracts from in uent samples resulted in mean t-BHQ equivalents ranging from 0.41 to 0.56 mg L − 1 , while exposure to e uent samples resulted in signi cantly lower responses, with mean t-BHQ equivalents ranging from 0.17 to 0.24 mg L − 1 (Table 5). Jia et al. (2015) also reported that activity was reduced in extracts from samples collected after wastewater treatment in an Nrf2 Luciferase Luminescence Assay with a different cell line.
The data for WL showed similar trends, with responses to treated e uent samples indicating reduced capacity to induce oxidative stress ( Table 6). The responses to extracts from surface water samples collected downstream of WL indicated that there were compounds present that induce oxidative stress, but the responses to extracts from surface water showed no apparent correlation with the activity in the corresponding lagoon e uent samples. For example, while mean t-BHQ equivalent values for e uent and surface water samples collected in May of 2014 were 0.25 and 0.14 mg L − 1 , respectively, treatments with extracts from June 2014 e uent and surface water samples showed mean responses of 0.12 and 0.28 ± mg L − 1 t-BHQ equivalents, respectively (Table 6). However, exposures to extracts from e uent and surface water samples collected in September resulted in very similar responses (Table 6).