Research articleRemoval of pharmaceutical’ estrogenic activity of sequencing batch reactor effluents assessed in the T47D-KBluc reporter gene assay
Graphical abstract
Introduction
Access to fresh water in South Africa is challenging, as the country receives average annual rainfall of 450 mm which is lower than the global annual rainfall of 860 mm (Benhin, 2015). Therefore, the implementation of micropollutant bioremediation management strategies for water bodies is necessary. Numerous pharmaceutical compounds (PhCs) are recalcitrant, are not effectively removed by conventional wastewater treatment plants (WWTPs) and are regarded as emerging environmental pollutants (Lloret et al., 2010). In addition, several emerging pollutants including PhCs found in the environment, have been revealed to be able to mimic or to inhibit the actions of the natural estrogen 17-β estradiol (E2) and as such have been named environmental estrogens (Kiyama and Wada-Kiyama, 2015). These have been detected in WWTP effluents, rivers, groundwater and drinking water (Lloret et al., 2010; Van Zijl et al., 2017).
Considering the complex structures and low bioavailability of most of these pollutants, their partial biodegradation from conventional biological or physicochemical treatment processes remains a challenge. Incomplete removal from conventional wastewater treatment processes necessitates investigation of alternative processes. Chemical/physical treatment such as advanced oxidation processes including ozonisation or ultraviolet (UV) exposure, have demonstrated variable degradation yields (Lloret et al., 2010). Another treatment option seems to be bioremediation and in this field, fungal strains and their ligninolytic enzymes have been found a valuable potential alternative for the removal of these recalcitrant pollutants and the reduction of their estrogenicity from the environment (Mao et al., 2010).
One of the challenges encountered in the application of advanced and biological treatment processes is the formation of intermediate compounds which display even higher estrogenicity than the parent compounds (Noguera-Oviedo and Aga, 2016; Rivera-Jaimes et al., 2018). Several in vitro assay methods have been used to assess estrogenic activity in aquatic environmental samples including yeast-based reporter assay and mammalian-based reporter gene assays. These assays include the yeast estrogen screen (YES), luciferase-transfected human breast cancer cell line (MELN) reporter gene assay, estrogen receptor chemically activated luciferase gene expression (ER-CALUX), and the T47D-KBluc reporter gene bioassay (Könemann et al., 2018; Van Zijl et al., 2017; Wilson et al., 2004).
In the present study which intended to evaluate the estrogenic and antiestrogenic activities of the sequencing batch reactor (SBR) effluents, the T47D-KBluc reporter gene bioassay was used. (Wilson et al., 2004).
The underlying principle of the method is that compounds that bind to and activate the ER will result in the ER dependent production of the luciferase enzyme. Agonist compounds induce luciferase expression and are compared to the vehicle control (media plus ethanol) or to the positive control 17β-estradiol (E2, Fig. 1A). The synthetic estrogen receptor antagonist ICI 182,780 (Fig. 1B) is used in the bioassay as the positive control for anti-estrogenic activity. In the bioassay using the T47D-KBluc cells, an estrogen is defined as a chemical that induces dose dependent luciferase activity that can be specifically inhibited by the anti-estrogen ICI 182,780 (Wilson et al., 2004).
Based on their chemical structures, a number of estrogenic compounds are found to be organic, in particular phenolics or carbon ring structures of varying structural complexity (Darbre, 2006). A list of phenolic compounds as reported by Kiyama and Wada-Kiyama (2015) is provided by Kasonga et al. (2019). However, the majority of endocrine-disrupting compounds found in effluents, especially PhCs that show limited removal in wastewater treatment processes are polar compounds with complex aromatic chemical structures. These compounds have molecular masses normally ranging from 200 to 1000 Da. Therefore, a minor modification in the chemical structure might have an important impact on physicochemical properties that govern their environmental fate to some extent, e.g. their potential to interact with ERα and ERβ (Hui et al., 1998; Kiyama and Wada-Kiyama, 2015). When these compounds end up in drinking water or food, they may have adverse health effects in humans (Aneck-Hahn et al., 2009; Mahomed et al., 2008). The three selected PhCs in this study, namely carbamazepine (CBZ), diclofenac (DCF) and ibuprofen (IBP), also have aromatic chemical structures as shown in Fig. 2. Their environmental concentrations range from a few ng/L to 100 μg/L (Agunbiade and Moodley, 2016; Ferrari et al., 2003). Higher drug concentrations can be found discharged in the aquatic environment from pharmaceutical factories ≥1 mg/L (Cardoso et al., 2014). Several studies have reported the estrogenic activity of PhCs, especially their intermediates rather than the parent compounds. Chronic exposure may lead to toxic effects (Efosa et al., 2017; Gröner et al., 2017; Jelic et al., 2012; Yu and Chu, 2009).
Because of the poor removal of the selected PhCs CBZ, DCF and IBP from conventional WWTPs, the present study intended to use a consortium of five previously isolated and identified South African indigenous fungal strains in the SBR. This led to evaluate the fungal ligninolytic enzyme activity, as well as the ability of the fungal consortium to degrade these recalcitrant compounds at a retention time of 24 h in the SBR. Moreover, the bioassay was conducted to evaluate the risk involved in the emission of the treated SBR effluents in reducing the production of intermediates and therefore, reduce their estrogenic activity.
Section snippets
Sequencing batch reactor configuration and operational conditions
An SBR system was designed using a single glass cylindrical tank with a conical base and was filled with a working volume of 2 L of medium under non-sterile conditions out of a total volume of 3 L. The SBR had an inner diameter of 9 cm and a height of 47 cm, of which the conical base had a height of 11.5 cm (Fig. 3). The SBR driven by a fungal consortium of five isolated South African indigenous fungi inoculated at 30% (v/v) of mycelium solution, was run at room temperature under continuous
Results
Despite the agitations due to the continuous air supply and the stirrer shear force, the SBR operating in continuous mode appeared to be suitable for the fungal consortium of isolated fungi. Even though the renewal of the medium (inflow synthetic wastewater) was increasing the pH to around 3.9 to 4.6, the pH in the SBR was found to be decreasing below 3 in the samples after 24 h, indicating that the fungal consortium was active. In addition to that, the increase recorded in the fungal dry
Discussion
The goal of the current study was to evaluate the efficiency of the SBR driven by a fungal consortium of five isolated South African indigenous species in the removal of the estrogenic activity of PhC transformation metabolites using the T47D-KBluc gene reporter bioassay.
Conclusions
The present study revealed that the mixture of CBZ, DCF and IBP did not exhibit any estrogenic or antiestrogenic activities. The ligninolytic enzymes Lac, MnP and LiP produced by the fungal consortium appeared to contribute to the removal of PhCs by producing known corresponding intermediate transformation compounds. No quantifiable levels of E2 EEq were detected in the SBR effluent samples. Therefore, the SBR designed in the present study, and setup with the five isolated South African
Availability of data and materials
The data generated in this paper, tables and figures are found inside the paper. Supplementary data related to this article can be found in Data in Brief article.
Authors’ contributions
Teddy Kabeya Kasonga, Martie A. A. Coetzee and Maggy Ndombo Benteke Momba conceived and designed the experiments; Teddy Kabeya Kasonga performed the experiments and wrote the paper; Catherina Van Zijl conducted the T47D-KBluc bioassay and reviewed the paper, Martie A. A. Coetzee reviewed the paper; Martie A. A. Coetzee and Maggy Ndombo Benteke Momba contributed reagents/materials/analysis tools.
Competing interests
The authors declare that there are no competing interests.
Ethics approval
The human cells used in the present study were not harvested by ourselves. An established and modified cell line from the ATCC® CRL-2865™ was used.
Funding
The project “Developing a fungus granulation process for the removal of Endocrine Disrupting Chemicals (EDCs) from wastewater (COE2016/2)” has received financial support from the Department of Environment, Water and Earth Sciences of the Tshwane University of Technology. This work was also supported by grants from the South African National Research Foundation (NRF) through the South African Research Chairs Initiative in Water Quality and Wastewater Management (SARChI, grant number 87310) at
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