*2.6. Effects of Leachate Sample from the UASB Reactor on BeWo Cells and Their Production of β-hCG*

The BeWo cell line was supplied by ATCC® CCL-98™, and it was cultured using Roswell Park Memorial Institute (RPMI)-1640 medium with 2% fetal bovine serum under a 5% CO2 atmosphere at 37 ◦C and 95% humidity (confluence > 70% and viability > 90%). A negative control was prepared with 100 μL of PBS in 5 mL of medium containing <sup>5</sup> × <sup>10</sup><sup>5</sup> cells and similar conditions. It was tested with 100 <sup>μ</sup>L of the leachate sample from the UASB reactor. Additionally, 100 μL of the LNG stock solution was assayed. All experiments were carried out in duplicate for 24 h. A colorimetric assay (MTT assay) was carried out for cell viability according to Lomonte et al. [16]. For more details, see the Supplementary Materials (Supplementary Materials SM2). The analysis of the levels of β-hCG hormone in the medium was assayed after 24 h by ElectroChemiLuminescence using the COBAS® 411 Integrated System (HITACHI) analyzer for the immunoassay test. A total of 1 mL of the medium was taken out from each experimental well and added to a conical Eppendorf tube for the analysis of progesterone and β-hCG using the COBAS® 411 device. The UV–vis absorption and photoluminescence were recorded on the COBAS® 411 by the HITACHI provider. This device was supplied by the Hospital Marco Fidel Suarez (Bello, Colombia).
