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. Author manuscript; available in PMC: 2021 Nov 15.
Published in final edited form as: Sci Total Environ. 2020 Jul 6;743:140401. doi: 10.1016/j.scitotenv.2020.140401

Steroid Hormones and Estrogenic Activity in the Wastewater Outfall and Receiving Waters of the Chascomús Chained Shallow Lakes System (Argentina)

Anelisa González 1,+, Kevin J Kroll 2, Cecilia Silva-Sanchez 2, Pedro Carriquiriborde 3, Juan I Fernandino 1, Nancy D Denslow 2,*, Gustavo M Somoza 1,*
PMCID: PMC7492445  NIHMSID: NIHMS1610924  PMID: 32653700

Abstract

Natural and synthetic steroid hormones, excreted by humans and farmed animals, have been considered as important sources of environmental endocrine disruptors. A suite of estrogens, androgens and progestogens was measured in the wastewater treatment plant outfall (WWTPO) of Chascomús city (Buenos Aires province, Argentina), and receiving waters located downstream and upstream from the WWTPO, using solid phase extraction and high-performance liquid chromatography mass spectrometry. The following natural hormones were measured: 17β-estradiol (E2), estrone (E1), estriol (E3), testosterone (T), 5α-dihydrotestosterone (DHT), progesterone (P), 17-hydroxyprogesterone (17OHP) and the synthetic estrogen 17α-ethinylestradiol (EE2). Also, in order to complement the analytical method, the estrogenic activity in these surface water samples was evaluated using the in vitro transactivation bioassay that measures the estrogen receptor (ER) activity using mammalian cells. All-natural steroid hormones measured, except 17OHP, were detected in all analyzed water samples. E3, E1, EE2 and DHT were the most abundant and frequently detected. Downstream of the WWTPO, the concentration levels of all compounds decreased reaching low levels at 4500 m from the WWTPO. Upstream, 1500 m from the WWTPO, six out of eight steroid hormones analyzed were detected: DHT, T, P, 17OHP, E3 and E2. Moreover, water samples from the WWTPO and 200 m downstream from it showed estrogenic activity exceeding that of the EC50 of the E2 standard curve.

In sum, this work demonstrates the presence of sex steroid hormones and estrogenic activity, as measured by an in vitro assay, in superficial waters of the Pampas region. It also suggests the possibility of an unidentified source upstream of the wastewater outfall.

Keywords: Aquatic Pollution, Endocrine disruption, Estrogens, Androgens, Progestogens, Municipal wastewater treatment

Graphical Abstract

graphic file with name nihms-1610924-f0001.jpg

1. Introduction

The occurrence of endocrine disrupting compounds (EDCs) has received widespread attention around the world because they are able to upset the endocrine system of animals because of their effects at low concentrations, leading to the dysfunction of important processes such as early development and reproduction (Colborn et al., 1993; Mills and Chichester, 2005; Matthiessen et al., 2018). Natural and synthetic steroid hormones, and their metabolites, excreted by humans and farmed animals have been considered as important sources of environmental EDCs, causing adverse effects on aquatic organisms, even at very low concentrations (Adeel et al., 2017; Orlando et al., 2004; Tyler et al., 1998; Windsor et al., 2018). Synthetic hormones are commonly used in daily life for human health and livestock production for contraception, hormonal therapies and the promotion of growth (Blackwell et al., 2014; Patel et al., 2019). Additionally, these compounds and their metabolites, are continuously discharged into receiving waters due to their incomplete removal in Municipal Wastewater Treatment Plants (MWWTPs, Verlicchi et al., 2012).

Estrogenic steroids have been found and widely studied in the aquatic environment and their detrimental impact on wildlife has been observed (Baronti et al., 2000; Kidd et al., 2007; Kiyama et al., 2015; Yu et al., 2019). Moreover, in a previous study high levels of E2 and EE2 were found in the Chascomús, Argentina, wastewater effluent (Valdés et al., 2015), prior to renovation of the treatment plant. Much less information is available on the occurrence of androgens or progestogens in the aquatic environment worldwide (Chang et al., 2008; 2009; Fent el al., 2015; Weizel et al., 2018) and particularly no previous studies measuring these steroids simultaneously have been conducted in Argentina.

In this context, the aim of this study was to analyze the occurrence of sex steroid hormones in the wastewater treatment plant outfall (WWTPO) and receiving waters of the Chascomús Chained Lake System. The targeted steroids were: Estrone (E1), 17β-Estradiol (E2); Estriol (E3), Ethinylestradiol (EE2), Testosterone (T), 5α-dihydrotestosterone (DHT), P (Progesterone) and 17-hydroxyprogesterone (17OHP). In addition, the estrogenic endocrine activity in water was assessed by the in vitro transactivation bioassay ER-GeneBLAzer.

2. Materials and Methods

2.1. Materials

High-purity organic solvents, methanol and dimethylsulfoxide (DMSO), were purchased from Fisher Scientific (Pittsburgh, PA, USA). 17β-estradiol (E2, CAS # 50–28-2, > 98% purity), 17α-ethinylestradiol (EE2, CAS # 57–63-6, > 98% purity), testosterone (T, CAS # 58–22-0, >98% purity) and 5α-dihidrotestosterone (DHT, CAS # 521–18-6, >98% purity) were purchased from Sigma Aldrich. Cell kits, reagents and media for in vitro cell bioassays were obtained from Life Technologies-Thermo Scientifics.

2.2. Sampling Area

The Chascomús shallow lake (35°36´ S 58°02´ W) is one of several shallow lakes of the “Pampa” region (Buenos Aires province, Argentina) and the second of the Chascomús Chained Lakes System, emptying into the left bank of the Salado River that in turn flows into the Atlantic Ocean (Torremorell et al., 2007). Chascomús shallow lake is connected upstream with Vitel shallow lake and downstream with Adela shallow lake, through the Girado stream. The surface of the Chascomús shallow lake is around 3,000 hectares and the average depth is 2.12 meters. This shallow lake has five major natural tributaries (La Tambera, Brown, Vitel, Valdes, and San Felipe streams) and empties into the Girado stream through a 50-meter-wide concrete water gate (WG) that controls the water levels. The eponymous Chascomús City, with a population of approximately 35,000, is located on the northeast shore of the lagoon (Dirección Provincial de Estadística de Buenos Aires, 2020; Torremorell et al., 2007). The city sewage is collected and conducted to the wastewater treatment plant (with conventional primary and secondary treatments) and the effluent is emptied through a 6 Km long pipe. Effluent from the WWTPO is discharged into a 200 m artificial channel that empties into the Girado stream (E200), 300 m downstream from the WG (Fig. 1).

Figure 1:

Figure 1:

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Concentrations of assessed steroid hormones: estrogens (E1, E2, E3 and EE2), androgens (DHT and T), and progestogens (P and 17OHP) measured in surface water samples. A) The map shows the region sampled and points the location of the Municipal Wastewater Treatment Plant (MWWTP) and the different sampling points. (B) steroid hormone levels downstream and upstream from the WWTPO.

2.3. Sample collection and processing

Receiving and surface water samples were collected during May 2017 located downstream and upstream from Chascomús WWTPO. The sampled sites were: the WWTPO (35°37’53.63”S, 58° 0’23.76”W); the WWTPO effluent discharge site on the Girado stream, E200 (approximately 200 m from WWTPO, 35°37’57.9”S 58°00’27.5”W); half-way between Chascomús and Adela shallow lakes, D3000 (approximately 3,000 m downstream from WWTPO, 35°39’03.2”S 58°00’02.5”W), and at the river mouth of the Girado stream into the Adela shallow lake, D4500 (approximately 4,500 m downstream from WWTPO, 35°40’01.7”S 57°59’50.6”W). Samples were also taken upstream from the discharge site and before the water gate, U300, (approximately 300 m upstream from WWTPO, 35°37’48.8”S 58°00’28.9”W); across from the water gate, U600, on the Chascomús shallow lake (approximately 600 m upstream from the WWTPO, 35°37’45.4”S 58°00’28.0”W) and in the center of Chascomús shallow lake, U1500 (approximately 1,500 m from the WWTPO, 35°37’22.4”S 58°00’28.4”W). Measurements of depth, water temperature (T°), pH (VWR Scientific 2000), conductivity (Hach), Secchi disk readings, nephelometric turbidity (Turner Design, SCUFA) and dissolved oxygen (DO) (YSI 5000) were performed in situ (Supplementary Table 1). At each site, two 1-L samples (with and without a spike with a mixture of E2, EE2, T, and DHT, each at 50 μg/L) were collected in pre-cleaned amber glass bottles. The samples were kept on ice, transported to the laboratory and processed within 24 h. Two hundred mL of the unspiked and the spiked samples were filtered using Whatman 45 μm nylon membrane filters and passed through pre-conditioned solid phase extraction cartridges (C18 Oasis HLB) following the manufacturer’s protocol. In brief, the cartridges were preconditioned with methanol and distilled water, loaded, washed, and eluted with 5 mL of methanol. The eluates were then concentrated under vacuum, dried under gentle nitrogen flow, and re-dissolved in 0.5 mL of methanol. Then, 0.1 mL were re-dissolved 1:10 in DMSO for the transactivation in vitro assay and 0.4 mL were used for measuring steroid levels by HPLC-MS/MS.

2.4. Cell-based bioassays

Commercially available GeneBLAzer® (Invitrogen, Life Technology) cell bioassays were used to evaluate estrogen receptor (ER) activity in samples that did not receive hormonal spikes as described in Mehinto et al. (2016). Briefly, division-arrested HEK 293T cells (GeneBLAzer® ER alpha) were diluted in assay media and seeded into black-wall clear bottom 96-well plates and exposed to serial dilutions of sample extracts or different E2 concentrations as a reference chemical. The final DMSO concentration in each well was 0.5%. After incubation for 16 hours at 37 °C in a 5% CO2 atmosphere, a loading substrate solution was added to each well, the cells were incubated for 2 hours at room temperature, and bioactivity was measured using a fluorescence microplate reader with bottom reading capabilities. Excitation and emission wavelengths for the ER transactivation assay were 460 and 530 nm, respectively, and cell viability was measured at 590 nm with Presto Blue. Sample extracts, which were already diluted 1:10 in DMSO were further diluted 1:20, 1:40, 1:80, and 1:160 in DMEM media (containing carbon stripped FBS, pyruvate, nonessential amino acids, penicillin/streptomycin, and 0.5% DMSO) in glass in a cell culture hood. The samples were further diluted 1:10 when they were placed in the plate wells resulting in a final dilution of 1:2,000, 1:4,000, 1:8,000 and 1:16,000. A standard curve of 17β-estradiol was generated in the same buffer at the following final concentrations in the wells: 1 × 10−8, 3.3 × 10−9, 1.1 × 10−9, 3.7 × 10−10, 1.2 × 10−10, 4.1 × 10−11, 1.4 × 10−12, 4.5 × 10−12, 1.5 × 10−12 M. All sample extracts were analyzed in triplicate. Data quality was validated against assurance/quality control (QA/QC) criteria for calibration, blank, DMSO control, cytotoxicity (cell viability) and sample dose response as detailed in Mehinto et al. (2016). The dose-response curves were utilized to quantify the bioactivity in the water samples. Results were expressed as bioanalytical equivalent concentrations (ER-BEQ ng/L). The anti-estrogenic activity was analyzed by adding the same E2 concentration, 2 × 1−10 M (70% of maximum ERα response), to all extract samples and then following the above-mentioned protocol.

2.5. Chemical analysis

Each sample was analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) using a 6500 QTRAP (AB SCIEX, Palo alto, CA) coupled to a Nexera X2 UPLC (Shimadzu, Japan) system. Chromatographic separation was achieved on an Eclipse plus C18 column, 2.1 × 100 mm, 3.5 μm (Agilent, CA), using 0.1 mM ammonium fluoride in water (A) and 100% methanol (B) buffers. Eight microliters of sample extract in methanol, out of a final extract volume of 0.4 mL, was injected. The column was kept at 40 °C for a better separation and eight microliters were injected at a flow rate of 300 nL/min. The column was equilibrated with 10% B and increased to 98% B in 12.5 min, held for 1 min at 98% B and brought back to 10 % B in another minute. The instrument was run under scheduled multiple reaction monitoring (MRM). The instrument was operated in positive mode for 17OHP, DHT, P and T; and in negative mode for E1, E2, E3, EE2 (Supplementary Table 2). The following source parameters were used; curtain gas (CUR) 25 psi, source temperature (TEM) 500 °C, nebulizer gas (GS1) 35 psi, heater gas (GS2) 50 psi, collision gas (CAD) 11 psi. Ion spray voltage in positive mode was set at 5250 V, and in negative mode at −4500 V. There were at least two transitions monitored for each hormone, using the strongest one for quantitation and the second one as a qualifier (Supplementary Table 2). An eleven-point standard curve was prepared for each hormone using standards for each compound. All samples were spiked immediately before chromatography with a combination of heavy isotope standards including Progesterone-d9, 17OH-Progesterone-13C3, Estradiol-d5, and Ethinyl estradiol-d4 that were used as specific and surrogate standards to correct for ion suppression. Data acquisition and data analysis was done using Analyst v1.6.2.

The machine limits of detection and quantitation (LOD/LOQ) were derived for each hormone using 3 or 10 times the standard deviation of the response divided by the slope of the calibration curve, respectively.

Final concentrations were calculated using whole recoveries estimated from spiked samples with E2, EE2, T and DHT. Then, an average of the observed recovery of spiked samples was used to quantify steroid hormones in environmental samples.

2.6. Data analysis

The limit of detection (LOD) of the GeneBLAzer® cell bioassays test was determined as the minimum calibration point showing a response higher than two standard deviations of the mean response above background. The equivalent concentration of E2 (EEQ) was obtained for each sample using a linear regression of the standard curve (using E2 as reference chemical) between EC50 and the LOD.

3. RESULTS

The machine limits of detection and quantitation (LOD/LOQ) were derived respectfully for each hormone: 17β-estradiol (E2), 32 and 106 ng/L; 17α-ethinylestradiol (EE2), 29 and 98 ng/L; estriol (E3), 19 and 63 ng/L; estrone (E1), 44 and 146 ng/L; 5α-dihydrotestosterone (DHT), 3 and 10 ng/L; 17-hydroxyprogesterone (17OHP), 2 and 8 ng/L; T, 1.9 and 6 ng/L; P, 5 and 15 ng/L. Because the water extracts were concentrated 400-fold, we were able to measure final concentrations in the ng/L range for all of them. The recoveries for the spiked hormones were 81%, 42%, 96% and 144% for EE2, E2, T and DHT respectively, in accordance with other studies (Chang et al., 2008; Pedrouzo et al., 2009; Valdés et al., 2015).

3.1. Steroid hormone concentrations in the WWTPO and surface waters

3.1.1. Estrogens

All estrogens, E1, E2, E3 and EE2, were detected in the WWTPO, with most of them at their highest level and then decreasing with increasing distance downstream from WWTPO (Figs. 2 and 3). The estrogenś concentrations in WWTPO and at the closer sampling point, E200, were higher for E3 (384 and 176 ng/L respectively), than for E1 (85 and 56 ng/L), followed by EE2 (64 and 48 ng/L), and finally E2 (5 and 7 ng/L). Downstream from the WWTPO, at the D3000 location, high levels for E1 (13.7 ng/L), E3 (12.8 ng/L) and EE2 (3.2 ng/L) were detected. Lower levels were observed at the Adela shallow lake at location D4500, and in all samples upstream from the outfall (U300, U600 and U1500) for the natural estrogens: E1 (1.63 ± 0.09 ng/L), E2 (1.62 ± 1.1 ng/L) and E3 (1.1 ± 0.29 ng/L), but not for EE2, which was not detected at any point upstream or beyond 3000 m downstream from the source (Table 1; Figs. 1 and 2).

Figure 2:

Figure 2:

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Concentration of each steroid hormone and the estrogenic activity in relation to the distance from the WWTPO and the sampled area. The red dot marks the WWTPO site (effluent discharge. See Figure 1).

Figure 3.

Figure 3.

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Percentage contribution of each steroid hormone on the total concentration measured, expressed with a pie chart for each sample site. Estrogens: E1, E2, E3 and EE2; Androgens: DHT and T, and Progestogens: P and 17OHP. MWWTP: Municipal Wastewater Treatment Plant. WWTPO: Wastewater Treatment Plant Outfall. Downstream from WWTPO: D3000 and D4500. Upstream from WWTPO: U300, U600 and U1500. The red solid arrow in the map indicates the path of the effluent from the plant to the Girado stream in a 6 km sewage pipe, the black dashed arrow indicates the direction of water runoff downstream from the WWTPO.

Table 1.

Steroid hormones concentration and estrogenic activity.

SAMPLING SITE CONCENTRATION (ng/L)
 ESTROGENIC ACTIVITY (EEQ-ng/L)
E1 E2 E3 EE2 DHT T P 17OHP MEAN ± SE
WWTPO 85,15 4,72 384,43 64,24 33,32 16,39 12,73 6,85 151 ± 114
E200 56,11 7,06 176,48 47,55 16,59 5,61 5,64 5,64 39.1 ± 34.1
U300 1,54 0,94 0,40 0,00 1,13 2,89 7,52 0,45 7.54 ± 5.10
U600 1,65 1,01 0,48 0,00 1,29 1,46 5,72 0,32 4.85 ± 4.08
U1500 1,71 2,90 0,94 0,00 5,70 2,28 7,50 0,50 7.24 ± 5.96
D3000 13,73 2,68 12,82 3,18 1,34 1,78 2,55 2,94 8.60 ± 6.46
D4500 2,65 2,02 1,91 0,00 1,16 2,32 3,62 0,00 7.19 ± 6.28
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Sampling sites of grab sampled of Wastewater Treatment Plant Outfall (WWTPO); 200 m from WWTPO (E200); downstream at 3000 m from WWTPO (D3000); downstream, in the Adela lake, at 4500 m from WWTPO (D4500); upstream at 300 m from WWTPO (U300); upstream at 600 m from WWTPO (U600) and upstream, in the Chascomús lake, at 1500 m from WWTPO (U1500). Estrogenic activities are expressed as equivalent concentration of E2 (EEQ) of the mean and the standard deviation (MEAN ± SE) of three replicate analytical samples.

3.1.2. Androgens

The androgens, DHT and T were detected at the WWTPO. DHT and T were at their highest levels at this site and then decreased with downstream distance from the discharge. The concentrations of androgens in WWTPO and in the closer sampling point, E200, were highest for DHT (33 ng/L and 17 ng/L) followed by T (16 ng/L and 6 ng/L). The values downstream seem to plateau for DHT, ranging from 1.1 to 1.3 ng/L, and for T, ranging from 1.8 to 2.3 ng/L. However, upstream from WWTPO, the situation is different. Starting in Chascomús shallow lake at location U1500, somewhat increased levels for DHT and T were also detected (5.7 and 2.3 ng/L, respectively). These levels decreased to a plateau of about 1.2 ng/L for DHT and 2.2 ng/L for T at U600 and U300. (Table 1, Figs. 1 and 2).

3.1.3. Progestogens

The highest concentrations of P and 17OHP were also detected at the WWTPO. Progesterone showed its highest concentration at the WWTPO sampling point (13 ng/L) and dropped to 6 ng/L at E200 and then continued to decrease downstream with an average of about 3.1 ± 0.8 ng/L. Concentrations of 17OHP were 7 ng/L at the WWTPO and 6 ng/L at the E200 level but then decreased with distance, falling to 3 ng/L at D3000 and 0 ng/L at D4500. Immediately upstream from the WWTPO, the values of 17 OH P were low and averaged 0.42 ± 0.09 Table 1, Figs. 1 and 2).

3.1.4. Relative distribution of steroid hormones downstream and upstream from the WWTPO sampling point

The analysis of the steroid hormone concentrations, as a percentage of the total steroid concentration at each sampling site, showed that the frequency of detection of some of the hormones varied depending on the sample location (Fig. 3). At the WWTPO, we observed that estrogens, except E2, were the main compounds, while androgens and progestogens were found at lower concentrations and in similar proportions, resulting with the following concentration profile: E3 > E1 > EE2 > DHT > T > P > 17OHP ~ E2 (E3= 63 %; E1= 13 %; EE2 = 11 %; DHT= 6 %; T= 3 %; P= 2 %; 17OHP= 1% E2= 1%; Fig. 3). A little different profile was also found at the Girado stream downstream from the WWTPO (D3000; E1 > E3 > EE2 > 17OHP ~ E2 > P > T > DHT, E1= 34 %; E3= 31 %; EE2= 8 %; 17OHP= 7 %; E2= 7 %; P= 6 %; T= 4 %; DHT= 3 %). However, in Adela Lagoon (D4500), we observed a more homogeneous relationship among steroid hormones (Fig. 3), with the following concentration profile P > E1 > T > E2 > E3 > DHT > EE2 = 17OHP (P= 27 %; E1= 19 %; T= 17 %; E2= 15 %; E3= 14 %; DHT= 8 %; EE2= 0 %; 17OHP= 0 %). Unlike this pattern, upstream from WWTPO, towards the Chascomús shallow lake, we observed that the relative proportion of compounds varied over a short distance (Fig. 2). While in WWTPO and E200 estrogens predominated, on both sides of the water gate (U300 and U600) and in Chascomús shallow lake (U1500), androgens and progestogens predominated with the following order of detection frequency: P > T > E1 > DHT > E2 > E3 ~ 17OHP > EE2 (U300: P= 51 %; T= 19 %; E1= 10 %; DHT= 8 %; E2= 6 %; E3= 3 %; 17OHP = 3 %; EE2= 0 %; U600: P= 48 %; E1= 14 %; T= 12 %; DHT= 11 %; E2= 8 %; E3= 4 %; 17OHP =3 %; EE2= 0 %) and, at U1500: P > DHT > E2 > T > E1 > E3 > 17OHP (Fig. 3; P= 35%; DHT= 27 %; E2= 13 %; T= 11 %; E1= 8 %; E3= 4 %; 17OHP= 2 %; EE2= 0 %).

3.1.5. Estrogenic/antiestrogenic activity at the WWTPO and surrounding superficial waters

Dilutions of water sampling extracts showed no apparent cytotoxicity, with less than 20 % of cell mortality observed compared to the control vials (with DMSO) and a low cell-free background (culture medium only). The comparison between only-cells and cells with DMSO responses indicated that this solvent presented a non-measurable impact on the fluorescence quantification. All collected samples screened for ER-related activity were above the detection limit and the EC10 for E2 (Fig. 4). The WWTPO site exceeded the EC50 concentration for E2, with ER-BEQ levels of 150 ng- E2/L, corresponding to 63 % of the estrogenic activity found in the area (Fig. 4). The next sampling point with high estrogenic activity was E200, with 32 ng-E2/L, corresponding to 17 % of the estrogenic activity in the area and the rest of the samples showed ER-BEQ levels averaging 7 ± 1 ng-E2/L (Fig. 4). No anti-estrogenic activity was found in any of the collected samples (Supplementary Fig. 1).

Figure 4.

Figure 4.

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Total estrogenic activity expressed as equivalent concentrations of E2 (EEQ ng/L) of the superficial waters sampled downstream and upstream from WWTPO, E200, D3000, D4500, U300, U600 and U1500. The red dotted line points to the 50% effective concentration for E2 (EC50), the green dotted line points to the 10% effective concentration (EC10) and the blue dotted line points to the limit of the technical detection. Each column represents three replicate analytical samples.

4. Discussion

Sex steroid hormones, acting as EDCs, are continually discharged into aquatic environments, producing pleiotropic effects on biota that are not entirely well understood. Although there is not much information available, the occurrence of natural and synthetic steroids has also been documented in Latin American surface waters (Ternes et al., 1999; Peña-Guzmán et al., 2019; Spindola-Vilela et al., 2018; Valdés et al., 2014; 2015). Specifically, in Argentina, different steroid compounds were detected in the wastewater treatment plants discharge and surface waters of two different provinces (Valdés et al., 2014; 2015). In the present study we analyzed the presence of eight steroid hormones and the estrogenic activity at the WWTPO of Chascomús city and surrounding superficial waters. Six out of these eight steroids were ubiquitous in all water samples analyzed. Estrogens were the dominant steroids detected, followed by androgens and progestogens. However, it is not possible to determine whether this concentration profile is a consequence of a higher concentration of estrogens in the influent or is a product of differential removal efficiency in the plant, as already reported in other places (Chang et al., 2011; Fan et al., 2011; Manickum and John, 2014).

It is important to note that the concentrations of E1, E3 and EE2 found in this study were between 2 and 100 times higher than those reported for WWTPOs around the world (Adeel et al., 2017). In the same way, the observed estrogenic activity was up to 10 times higher than previous reports from Japan and Australia, tested with similar in vitro technologies (Ihara et al., 2015; Leusch et al., 2010). As in other studies, E1 and E3 appeared in the highest concentrations (Adeel et al., 2017). In this sense, it is known that E1, E2 and E3 can be metabolized with a specific increase of E1, as a product of the degradation of E2 and EE2 and E3 by microorganisms living in aerobic conditions (Adeel et al., 2017; Carballa et al., 2004; Casey et al., 2003; D’Alessio et al., 2014; Duncan et al., 2015; Goeppert et al., 2014; 2015). Therefore, it is expected that metabolites, such as E3 and E1, could be found in greater proportion at the WWTPO. On the other hand, it is possible that estrogen metabolite profiles could reflect the estrogen source, describing the environmental characteristics of the sampled place (Adeel et al., 2017). In this sense, the profile of estrogen concentrations found at the WWTPO in this study is similar to those found in WWTPOs from other South American countries, such as Brazil and Chile, which could be related to a similar wastewater treatment in this region of the world (Adeel et al., 2017). In relation to this fact, Valdés et al. (2015) using LC-MS/MS, tried to measure E2, EE2 and E1 in the same effluent but obtained a different profile: E2 > EE2 > E1 (not detected). Although E3 was not analyzed by Valdés and collaborators (2015), the low levels of E1 could be a consequence of an increased microbial degradation rate in Chascomús WWTPO at the time of the first measurement or owing to different sensitivity of methodologies used. In addition, EE2, one of the main components in contraceptive pills, was already detected by Valdés, et al (2015) in the same location. These results, together with those of Brazil by Adeel et al. (2017), show that high environmental estrogen levels are not only a worrying problem for developed countries, but it is also a problem for developing nations and even for small cities, such as Chascomús.

Androgens and progestogens are generally poorly studied in the aquatic environment. In Argentina, there is a single prior investigation that reported the presence of the androgen dihydrotestosterone (DHT) in superficial waters of Córdoba province (Valdés et al., 2014). Our study is the first report for progestogens for this country. In the Chascomús shallow lake among the androgens and progestogens analyzed, we found concentrations higher than 7 ng/L of DHT and P. Also, with respect to androgens, the concentrations found in our study were higher than those found previously by Valdés and collaborators (2015) in superficial waters. It is important to highlight that if we compared our study with WWTPO effluents from other parts of the world, our results exceed from 2 to 100 times the androgen levels found elsewhere (Chang et al., 2011; Liu et al., 2011; Manickum and John, 2014; Vulliet et al., 2007). This could be probably linked to an ineffective sewage treatment in the area, as already suggested by Kookana et al. (2014) for low- and middle-income countries. Further studies will be necessary to measure the anthropogenic impact of the high level of androgen in biota.

Progestogens were detected in most of the sampled sites (WWTPO, U300, U600, D4500), and P levels were on average 4.5 times higher than their metabolite 17OHP. In a previous study of a Chinese WWTPO (Chang et al., 2011), P levels were also always up to 10 times higher than 17-OHP in different sampling sites; however, in all cases, the concentrations were lower than those from this current study. However, progestogen concentrations are usually similar between effluents and surface waters, the levels commonly found in other parts of the world vary between 0.07–10 ng/L (Chang et al., 2008; 2009; 2011; Manickum and John, 2014; Fent et al., 2015; Liu et al., 2011; 2014; Velicu et al., 2009; Vulliet et al., 2007). Compared to previous studies, progestogens, androgens, and estrogens were found in relatively higher levels in Argentina, highlighting the potential high impact of small cities on lentic environments.

In addition to the previous study, focused on the WWTPO effluent and its impact on receiving waters downstream from the source, here we also analyzed the occurrence and dynamic of a broader number of steroid hormones not only downstream but also upstream of the WWTPO. Six out of eight analyzed steroid hormones (DHT, T, P, 17-OHP, E3, and E2) were detected in the Chascomús Lake (U1500), farthest site upstream of the WWTPO, even presenting higher levels than at closer points to the WWTPO (U300 and U600). A similar pattern was found for T and P, showing similar values at D3000, and D4500, far downstream of the WWTPO. These results are indicating that the WWTPO effluent is not the only source of sex steroid hormones in this area. These results suggest that measured androgen and progestagen levels could be showing natural background levels. For example, high levels of P coming from the conversion of plant phytosterols have been reported (Carson et al., 2008; Janeczko, 2012). However, other potential diffuse sources of pollution such as cattle manure used as fertilizers or the septic tanks from small settlements (i.e. Girado neighborhood) should not be discarded. On the other hand, EE2 was the only studied steroid exclusively detected downstream of the WWTP outfall, showing this compound as a specific marker of sewage-driven pollution.

The in vitro estrogenic transactivation bioassay was consistent with the chemical analysis, higher at the WWTPO and E200, indicating that the measured estrogens or other contaminants (such as Bisphenol A, Nonylphenol, etc.) are relevant in explaining the total estrogenic activity of the samples. In addition, the bioassay was useful to understand that those estrogens are bioavailable to interact with biological systems. The problems associated with the use of in vitro estrogenic bioassays in risk assessment have been discussed elsewhere (i.e. overestimation because of interference of natural estrogenic compounds, Safe et al., 2002), but the utility of these bioassays still is rewarding and represents an integrated amount of all estrogenic compounds present in the water. Although no regulations are based on estrogenic equivalence in Argentina, this bioassay is a quick and easy tool for predicting the estrogenic effects of complex environmental mixtures as the ones assessed in this study, and therefore, a useful approach for lower-tier ecological risk assessment (Leusch et al 2010; Wagner et al., 2013). In addition, bioanalytical assays complement analytical methods used to measure individual compounds, as they integrate the response across chemicals, that are almost always found in complex mixtures, that target similar adverse biochemical action (Escher et al., 2020; Neale et al., 2017). Validation of these types of assays vis a vis normally accepted analytical methods and in vivo animal models of hormonal action is gaining traction around the world with several studies beginning to develop “trigger values,” for water quality assessment, i. e. benchmark concentrations that above which a more in depth environmental analysis would be recommended (Brand et al., 2013).

According with the previous mentioned, this study shows that steroid hormones, especially estrogens and estrogenic activity, were highest close to the discharge site (WWTPO), which corresponds to the intersection of the Girado stream with Chascomús shallow lake (E200, U300, U600). Although seasonal and serial sampling over time are necessary to predict changes in the levels of steroid hormones from the sewage effluent, considering previous work at the same sampling site (Valdés et al., 2015) and ours, it is possible to propose that the continuous flow of steroid hormones in the same area could promote high and stable concentrations and high estrogenic activity in this environment. Consequently, the organisms that inhabit this area would potentially have further risks of being exposed to chronic concentrations of these compounds, which could exert harmful effects on individuals, on their progeny or on populations (Milla et al., 2011). According to this, similar levels of estrogen activity found in our study have been able to induce vitellogenin (Vtg) production in sexually immature males (Ihara et al., 2015). In addition, our laboratory studies with pejerrey (Odontesthes bonariensis), a native fish of the Pampean area, showed that 45 ng/L of EE2 altered the regulation of key endocrine-reproductive axis genes (gnrhIII and fshr), inducing the occurrence of degenerated germ cells and the absence of spermatocytes in the germinal epithelium of adult testes that consequently could lead to sterility (Gárriz et al., 2017). Moreover, another study with a native fish inhabiting Chascomús Lagoon, Cnesterodon decemmaculatus, showed that concentrations between 30–100 ng/L of EE2 induced the appearance of testis-ova in males (Young et al., 2017). Therefore, the steroid levels measured in the present study should be used in future studies to measure the impact of these substances on sex determination, sex reversal and reproduction of fish and other animals living in a lentic water environment impacted by a sewage treatment plant.

5. Conclusions

Concluding, this work demonstrates the presence of estrogens, androgens and progestogens, considered as EDCs, in surface waters in the Pampas region in Argentina at higher levels than reported in other parts of the world. Also, our results suggested that these concentrations may not only be exclusive to the discharge of sewage, but possibly also of other sources, which keep the background concentrations and estrogenic activity of these compounds high even upstream of the main source of contamination.

Supplementary Material

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Highlights.

  • Surface waters of the Chascomús Lagoon, Argentina, show high levels of steroids.

  • E3, E1, EE2 and DHT were the most abundant and frequent steroids found.

  • EE2 was the only steroid exclusively detected downstream from the WWTP outfall, and so it shows up as a specific marker of sewage-driven pollution.

  • Wastewater effluent is not the only source of sex steroid hormones in this area.

  • These waters show high estrogenic activity compared to other parts of the world.

Acknowledgements

This project was made possible by a scholarship of the Bunge & Born and Fulbright Foundations to AG, by a grant of ANPCyT (PICT2015-2783) to GMS and by U.S. National Institutes of Health (NIH) Shared Instrumentation (Grant 1S10OD018141-01A1 to N.D.D.). We appreciate the help of Javier Herman (INTECH) for his kindness and willingness to collaborate in water sampling.

Abbreviations

E1

Estrone

E2

17β-estradiol

E3

Estriol

T

Testosterone

DHT

5α-dihydrotestosterone

P

Progesterone

17OHP

17-hydroxyprogesterone

WWTPO

Wastewater Treatment Plant Outfall

Footnotes

The authors declare no competing financial interest.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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