*2.8. Ethical Statements*

The BeWo cell line experiments do not require ethical review and approval because this line is obtained as a commercial immortalized from placental choriocarcinoma (ATCC® CCL-98) which did not include human experimentation. However, ethical approval for the semen analysis was obtained from the Bioethics Committee of the School of Medicine at Universidad de Antioquia (F-CBI-012, 14 February 2019), and the patients signed an informed consent form.

#### **3. Results**

#### *3.1. Validation Parameters for LNG Analysis by uHPLC–DAD*

The chromatography method allowed for the separation of the LNG at a retention time of 17.63 min. The quality parameters, for quantification purposes, are presented in Table 1.

**Table 1.** Data on LNG for qualification and quantification purposes.


The UV wavelength for LNG detection and quantification was 254 nm. The *n*-octanol-water partition coefficient (*Log Kow*) is included.

The linearity was analyzed by homoscedasticity and *r*2, which produced a good fit in a linear model, and the slope was significantly non-zero (*p*-value < 0.05) For more details, see Supplementary Materials SM3.

According to the uHPLC–DAD method, the LOQ appeared to be sensitive. However, some integrated techniques for sample extraction may increase its sensitivity. For example, Huang et al. applied dispersive liquid–liquid microextraction for LNG analysis in natural water, obtaining a sensitivity 100 times higher [18].

#### *3.2. Analysis of LNG in the Effluent of the UASB Reactor from the Buenavista Landfill*

The UV spectrum obtained at a retention time of 17.63 was in agreement with the spectrum found in the literature; therefore, the sample showed the presence of LNG (see Supplementary Materials SM3). Additionally, the standard addition appeared to increase at the same time in the spiked samples. Therefore, the leachate sample from the Buenavista landfill showed small amount of LNG at 315 <sup>μ</sup>g·L−1, which was calculated by linear regression (see Figure 1). A similar method was developed to determine the levels of LNG in tap water, lake water, and river water samples using uHPLC–DAD [18]. However, the authors applied dispersive liquid–liquid microextraction for analysis to increase the sensitivity of the methods. In our work, we analyzed samples by direct injection because we consider that more polluted water (leached water), and thus higher levels of pollutants, do not require a more difficult extraction process.

**Figure 1.** LNG analysis by uHPLC–DAD. The dashed line shows the analysis of the LNG standard. The black line shows the analysis of the leachate sample, and the horizontal dashed line shows the spiked sample.

According to the detected levels, this pharmaceutical product may be transported through the cover material used in landfills. This substance has been detected in wastewater and drinking water treatment plants worldwide at levels between 3.6 and 78.95 ng·L−<sup>1</sup> [13,19]. Although the *Log Kow* presented for LNG in Table 1 indicates a possible lipophilic interaction, the soil is the final barrier that can trap molecules inside. The soil sorption and interaction are presented in Section 3.3 in order to explain the leaching process.

Consequently, the risk assessment for LNG exposure may be considered for public health studies because people can ingest low levels of these substances that are not entirely removed, as was previously found [20]. Progestins may accumulate in exposed organisms because of their lipophilic properties. For instance, LNG was reported to accumulate in the blood plasma of rainbow trout, reaching levels of 8.5–12 ng·mL−<sup>1</sup> when the species was exposed to treated wastewater effluent (at a concentration of 1 ng·L−<sup>1</sup> of LNG) for 14 days [21]. This concentration exceeds the plasma concentrations of the therapeutic doses used in humans (2.4 ng·mL<sup>−</sup>1).

On the other hand, LNG may bioaccumulate and biomagnify in all tropic chains. For instance, this substance can cause severe consequences in the gestation process and embryonic development of zebrafish by bioaccumulation [22].
