*2.3. Leaching Analysis of LNG from the Soil Layers under Laboratory Conditions*

The experiment was carried out inside packed columns of polyvinyl chloride (PVC) using each of the soil layers in duplicate (with a diameter of 2.54 cm and a length of 30 cm). Then, 50 mL of the LNG stock solution was passed through the columns of PVC. The volume of the standard solution was calculated according to the average precipitation around the sampling site per area. Next, 50 mL of deionized water was used in the soil layers as a negative control. All leachate fractions were collected after 24 h in conical flasks to measure the volume, and they were centrifuged at 3000 rpm for 10 min at 25 ◦C for cleaning. Finally, 1 mL of the sample was injected into the uHPLC–DAD system using the recommended method.

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

We analyzed 10.5 mg.L−<sup>1</sup> of the stock solution of LNG by uHPLC–DAD (Thermo Scientific Dionex UltiMate 3000) at a wavelength of 245 nm. The LNG was identified using the UV spectrum at its retention time. The LNG was separated on a C18 Hypersil Gold column (5 μm × 150 × 4.6 mm). A volume of 20 μL of the LNG solution was injected and separated using a mobile phase with a ratio of 85:15 (*v:v*) of buffer–acetonitrile, by applying a flow of 0.450 mL/min<sup>−</sup>1. After 10 min, the mobile phase was modified at a ratio of methanol–acetonitrile of 50:50 (*v:v*), under a flow of 0.500 mL/min<sup>−</sup>1. The mobile phase was set back to initial conditions (85:15 (*v:v*) of buffer–acetonitrile). Finally, a calibration curve was plotted between 1 and 10 ppm, and the validation parameters, such as the limit of detection (LOD), the limit of quantification (LOQ), and recovery were estimated. See the Supplementary Materials (SM) for more details.

#### *2.5. Assessment of Leached Fractions in Human Sperm Cells*

The experimental in vitro assays included five fresh semen samples from healthy individuals, collected by masturbation in a sterile container after sexual abstinence for 3 to 5 days. The normozoospermia for semen parameters is presented in Supplementary Materials SM1. The sperm motility and viability were quantified for each seminal sample following the guidelines established in the manual for seminal analysis published by the World Health Organization [15] before and after incubating an aliquot of 4–5 × 106 sperm cells with 100 μL of the leachate samples from the effluents of the UASB reactor for three hours. Both sperm parameters were quantified at the initial time (0 h) and 3 h after incubation (once every hour). The negative control was incubated with only 100 μL of PBS, while the leachate sample was incubated with 100 μL of samples from the UASB reactor. Additionally, the direct effect of the LNG was tested with 100 μL of the stock solution.
