Ecological Risk Assessment of Five Endocrine-Disrupting Compounds in Wastewater Treatment Plants from Monterrey, Mexico

The potential ecological risk of �ve residual endocrine-disrupting compounds (EDCs) in four wastewater treatment plants (WWTPs) was studied. The wastewater samples were collected in WWTPs of the Metropolitan Area of Monterrey, Mexico (designed as Monterrey City hereinafter) and 17β-estradiol (E2), 17α-ethinylestradiol (EE2), bisphenol A (BPA), 4-nonylphenol (4NP), and 4-tert-octylphenol (4TOP) were studied by SPE/GC-MS method. Results showed that all EDCs are widely distributed in WWTPs, �nding high concentrations of BPA (450 ng/L) and EE2 (407.5 ng/L) in in�uents, while EE2 and 4TOP were the most abundant in e�uents at levels from 1.6–26.8 ng/L (EE2) and < LOQ – 5.0 ng/L (4TOP), which corroborate that the wastewater discharges represent critical sources of EDCs to the aquatic environments. The potential ecological risk of residual EDCs was evaluated through risk quotients (RQs), and results indicated that the e�uents of the WWTPs represent a high risk to exposed aquatic species, mainly due to the effect of residual estrogens E2 and EE2 which were considered as the most hazardous compounds among the studied EDCs, with RQ values up to 49.1 and 1165.2, respectively.


Introduction
Endocrine-disrupting compounds (EDCs) belong to a subclass of so-called emerging organic pollutants and their presence in the environment represents a potential risk to public health (Sauvé and Desrosiers 2014). According to the United States Environmental Protection Agency (USEPA) and the European Chemicals Agency (ECHA), EDCs are exogenous substances or a mixture of substances that alter the structure or function(s) of the endocrine system and cause adverse effects at the level of the organism, its progeny, populations or subpopulations of organisms (EPA 1998; ECHA and EFSA 2018). These compounds are widely distributed in the environment, mainly in aquatic ecosystems, and have attracted special attention due to their potential risk to the health of aquatic organisms and humans, even at very low concentrations such as ng/L (Adeel et al. 2017). Several studies have reported that EDCs may generate different adverse effects on exposed organisms, including feminization and masculinization, de ciencies in the sexual, prostate, brain, and immune development, gonadal atrophy, infertility, precocious puberty, and different types of cancer (prostate, testicles, breast, ovaries, among others) (Roby 2013; Kabir et al. 2015; Wang et al. 2019; Jackson and Klerks 2020). Moreover, it has been reported that EDCs may be associated with a high incidence of obesity and the development of diseases such as diabetes in humans (Hatch et al. 2010).
Among the different EDCs, the estrogens E2 and EE2, the alkylphenols 4NP and 4TOP, as well as the plasticizer BPA, are the most frequently EDCs detected in various aquatic environments such as rivers, lakes, and coastal areas  Čelić et al. 2020); due to their high estrogenic potential, these substances are being regulated by the European legislation through the European Water Framework Directive (2000/60/EC). In this sense, 2.0 µg/L has been de ned as the maximum admissible concentration for 4NP and 4TOP in surface water (European Parliament 2008). In addition, BPA and E2 were recently proposed for inclusion as priority hazardous substances, recommending 0.01 µg/L and 0.001 µg/L as the maximum allowable concentration in surface water, respectively (European Parliament 2018). Regarding EE2, has been included in the watch list to monitor it and evaluate its possible incorporation as a priority hazardous substance for human health (European Parliament 2013).
In another way, it is known that e uents from WWTPs are one of the main sources of EDCs emissions to aquatic environments, and several studies have reported the detection of EDCs in WWTP e uents around the world, revealing that these substances are partially removed during wastewater treatment processes ( Manickum and John (2014) reported the detection of E2 (4.0-107.0 ng/L) and EE2 (1.0-8.0 ng/L) in the e uent of a WWTP in South Africa. On the other hand, BPA (3.31 ng/L), 4NP (4.62 ng/L), and 4TOP (4.44 ng/L) were detected in a WWTP e uent in Portugal (Carvalho et al. 2016). Furthermore, 4NP (420-2120 ng/L) and 4TOP (5.3-54.8 ng/L) were detected in e uents of nine WWTPs in Iran (Bina et al. 2018). Therefore, the incomplete removal of EDCs in WWTPs contributes to their frequent detection in surface water, which may subsequently impact on the essential activities such as agriculture, livestock, sheries, drinking water, and domestic and recreational activities (Čelić et al. 2020).
In Mexico, some studies have reported the detection of E2, EE2, and BPA in several WWTPs in a wide concentration range from < LOD -16,600 ng/L ( In these studies, the risk quotients (RQ) have been calculated for several aquatic organisms (i.e. algae, crustaceans, and sh), and it is considered a useful strategy for the evaluation of water resources, which could contribute to improve the wastewater treatment processess, implementing effective strategies of management and control of wastewater discharges, surface, and drinking water, for the health protection of human and aquatic organisms (Xu et al. 2016). Therefore, this study aims to determine the potential environmental risks associated with ve dissolved EDCs in four WWTPs from Monterrey City, which to our knowledge, constitutes the rst report of this type in WWTPs from Mexico.
Standard stock solutions (1000 µg/mL) of individual EDCs and Chrysene-d12 used as internal standard (1500 µg/mL) were prepared in acetone. These solutions were stored in amber vials at 4°C until use.

Sample collection and instrumental analysis
In this work, four WWTPs that receive urban and industrial wastewater were studied, the geographical location of WWTPs is shown in Fig. 1, and detailed information of each WWTP and the main quality parameters of the wastewater samples were included in Table 1.

Data analysis
Daily mass Where MEC is the measured concentration in in uents and e uents of WWTPs (ng/L), and PNEC is the predicted noeffect concentration. PNEC values for each EDCs in wastewater were calculated by Eq. 4: Where HC5 is the hazardous concentration for 5% of the exposed species, and AF is the assessment factor which was set to 3 in order to protect other more sensitive organisms no considered in the toxicological studies (Tamis and Jongbloed 2019). HC5 values for E2, EE2, BPA, 4NP, and 4TOP were taken from scienti c literature and are shown in Table 5. Further, the criteria for establishing the potential ecological risk were the following: RQ ≥ 1, high risk; 0.1 ≤ RQ ≤ 1, medium risk, and RQ < 0.1, low risk (Huang et al. 2018). In addition, regarding RQs for surface water, a dilution factor of 10 was used for MEC values (Jiang et al. 2020), assuming that e uents of WWTP would be diluted after being discharged.

Concentrations of EDCs in in uents
The frequency of detection of the ve EDCs in the in uents was variable, ranging from 62.5% (4NP) to 100% (EE2) as shown in Table 2. Notably, EE2 was detected in all samples collected during the two sampling campaigns reaching high levels up to 407.5 ng/L (Fig. 2). These EE2 levels   As shown in Fig. 2, EE2 as well as BPA, were the most abundant EDCs in the in uents of WWTPs, and its wide concentration range re ects the high consumption rates and its constant emission in urban and industrial wastewater discharges from Monterrey City. Furthermore, in this study, low levels of 4NP and 4TOP compared to those detected levels in other research suggests a moderate use of these alkylphenols contained in surfactants and detergents, mainly used in domestic and commercial activities (Bina et al. 2018). In addition, Table 3 shows a summarized comparison between EDC levels detected in this research and those reported in other regions, where the EDCs amounts are often widely detected of the order of ng/L.

Concentrations of EDCs in e uents
The detection frequency and concentrations of the ve selected EDCs in the e uents were lower than those detected in the in uents (Table 2 and Fig. 2), indicating that the treatment processes in WWTPs have a notable effect on the remotion of these substances. In e uents, levels of estrogens E2 and EE2 ranged from < LOQ -9.5 and 1.6-26.8 ng/L, respectively. The synthetic estrogen EE2 standing out for its frequent detection (100%) in all WWTPs e uents, which reveal its high persistence to treatment processes, which is associated with the ethinyl functional group that makes it highly resistant to biodegradation (Ting and Praveena

Mass balance of EDCs dissolved in WWTPs
The mass balance for the ve EDCs in the soluble phase was determined based on the concentrations detected in the WWTPs and the volume of water treated per day ( Table 1). The results are shown in  Fig. 3 shows the daily mass of EDCs in the e uents of the WWTPs, and it was observed that the emission rate of EDCs is directly related to the number of the population served and the volume of wastewater treated (see Table 1). Also, it is observed that EE2 is the major constituent (73.7%) of the dissolved EDCs in the four e uents, which are directly discharged to La Silla, Sabinal, Santa Catarina, and Pesquería rivers (Fig. 1), suggesting a potential health risk to exposed aquatic ecosystems and human health. It is worth noting that some negative values were observed for E2, EE2, and 4TOP in WWTP A (-493.5, -462.5, and − 162.3%, respectively), and this phenomenon was also observed for 4TOP in WWTP C (-15.5%). These negative values indicate that levels of these EDCs were higher in e uents than in uents, and can be attributed to the likely transformation of conjugated estrogens to their active forms, and the biodegradation of polyethoxylated alkylphenols to give rise to metabolites such as 4TOP by the action of the microbial consortia in the WWTPs, which increase their concentration in the e uents, and it is consistent with that described by Yu et al. (2013) and Zuo et al. (2006).

Risk assessment
The occurrence of EDCs caused for the discharge of treated and untreated wastewater may represent a potential ecological risk to both aquatic species and humans. Therefore, the risk quotients (RQ) were calculated from the in uent and e uent data of the four WWTPs using the MEC values, and the PNEC values for each EDC were taken from the scienti c literature. The results are shown in Table 5 and Fig. 4 suggesting a medium risk for exposed species. Likewise, the treated water in WWTPs e uents were also considered as a high risk for aquatic organisms, mainly attributed to the abundance of E2 (RQ = 49.1) and EE2 (RQ = 1,165.2), which are considered as potent endocrine disruptors due to their high estrogenic potential, even at trace levels such as pg/L (European Commission 2012; Brion et al. 2019). In addition, as can be seen in Fig. 4, BPA, 4NP, and 4TOP represent a low risk in WWTPs e uents (RQs < 0.1 for each); however, this was not signi cant because they constitute a minimal part of the remaining EDCs in the e uents (Fig. 3). Finally, the possible ecological risk was estimated for the receiving waterbodies impacted by the e uents of WWTPs (Fig. 4), and results suggest that the organisms in these aquatic ecosystems may be exposed to high risk, attributed to the abundance of remaining E2 (RQ = 4.9) and EE2 (RQ = 116.5) in the e uents of WWTPs (Fig. 3). Additionally, in the worst-case scenario, the results obtained in this study could underestimate the potential ecological risk related to EDCs since in this study only two sampling campaigns were carried out. Moreover, the suspended phase of EDCs in the WWTPs and the fraction of untreated wastewater directly discharged to the environment were not considered. In addition, the emission of pollutants such as EDCs may be a function of population consumption and excretion rates, as well as urban and industrial activities which vary over time. Also, in the environment, EDCs are regulated to some extent by environmental factors such as temperature, precipitation, solar radiation, microbial activity, among others.

Conclusions
This study revealed that EE2 and BPA were the most frequently detected EDCs in the in uents of WWTPs, likely associated to the extensive use of EE2 as a contraceptive method and the use of BPA as a plasticizer in industrial activities. In addition, the detected levels of 4NP and 4TOP suggest their moderate use in urban activities in Monterrey City. Furthermore, EE2 was detected in all WWTP e uents ranging from 1.6 to 26.8 ng/L being the most recalcitrant compound in the treatment processes. Also, the mass balance in the dissolved phase revealed that the WWTP e uents constitute critical sources of EDCs emission to the environment, despite that these compounds present high remotion percentages in some WWTPs. Moreover, through the RQ values, it was estimated that the wastewater in in uents and e uents of the studied WWTPs represent a high risk to exposed aquatic species, due to the abundance of dissolved E2 and EE2. Also, a preliminary assessment of the risk quotient in receiving water bodies indicated a potential environmental risk for La Silla, Santa Catarina, Sabinal, Topo Chico, and Pesqueria rivers. However, ecotoxicological studies and extensive monitoring are needed to protect the biodiversity of these aquatic environments. Additionally, it is necessary pay more attention to the possible public health risk associated with the occurrence of EDCs in WWTPs in densely populated and industrialized zones such as Monterrey City. Declarations