*3.3. Assessment of Leaching Processes for LNG in Soil Layers Used as Cover Materials in the Buenavista Landfill*

To approximate a possible explanation as to why LNG may be found in the effluent of the UASB reactor located at the Buenavista landfill, a leaching experiment was carried out. First, physicochemical analysis of the soil layers was performed. See Table 2 for details.


**Table 2.** Soil properties. CEC: cation exchange capacity.

Overall, the soil layers showed high clay and silt content, which is related to the high soil sorption of PPCPs [23]. Although low amounts of organic material were found in the soil layers, lipophilic interactions may occur (Table 2). For instance, the humic acid in organic matter may increase the soil–pollutant interaction [24]. Additionally, the cation exchange capacity (CEC) in the soil allows for electrostatic interactions with pollutants due to the charges and ionizable states of molecules.

The water infiltration in the soil was found to be between 31% and 75.6%. The highest level of infiltration was found in L1, which may play an essential role in the transport of pollutants by soil–water partitioning. For instance, intensive rainfall events in the landfill may increase the leaching of pollutants [10].

Second, the leaching of LNG was tested in all soil layers. According to Figure 2a, more than 90% of LNG pollutants were adsorbed in all layers used as a cover material, but a higher level of soil sorption of LNG was found in L2 and L3. Some physicochemical characteristics in soil layers may increase the LNG–soil interaction. For instance, the lipophilic properties of LNG may induce more sorption in organic fractions and decrease the water interaction. Thus, higher amounts of LNG may remain for a long period of time in cover material. For instance, according to Ahmed et al., nonpolar substances are adsorbed into soil more than polar substances due to lipophilic affinity [25]. To understand the influences of the soil physicochemical properties of pollutant sorption, a correlation

plot is provided in Figure 2. Some properties, such as the CEC, water infiltration, and silty fraction showed a good correlation (Pearson *r* > 0.99, *p*-value = 0.1910).

**Figure 2.** Analysis of soil sorption of LNG by leaching analysis: (**a**) leaching of LNG from soil layers used as cover materials; (**b**) effects of water infiltration on soil layer sorption; (**c**) effects of the silty fraction on soil layer sorption; (**d**) effects of CEC on soil layer sorption.

The water infiltration measured in the soil layers was indirectly correlated with the soil sorption of LNG (Figure 2b). Therefore, the leaching of pollutants depends on water infiltration, which occurs due to hydrogen bonds, polar interactions, and even liquid–solid partitioning. LNG shows a hydroxyl group that may interact with water by hydrogen bonds, but due to the high *Log Kow*, only a lower amount can remain in water at equilibrium, which explains why less than 8% of the LNG was transported through the column. For instance, some LNG ketone and hydroxy groups may interact in soil, increasing LNG uptake in soils, and inducing hydrogen bonding and pore-filling [12]. Unfortunately, desorption of synthetic progestin may be induced by rainfall, and thus, a low amount may be transported by leaching. Therefore, selecting an appropriate cover material and ensuring adequate operating conditions is critical to reducing LNG leaching from landfills.

The silty fraction showed a direct relationship with the soil sorption of LNG (see Figure 2c). Therefore, a higher silty fraction may increase the soil sorption of steroid molecules because the size particles may uptake substances with a higher molecular weight, operating as size exclusion chromatography. According to Yang et al., the relatively high surface area of soils and sediment favored the uptake of some estrogens with similar molecular structures of LNG, such as altrenogest, drospirenone, and medroxyprogesterone acetate [26]. However, the soil layers studied in this paper contain small amounts of clay, which may play an important role in potentializing the soil sorption of pollutants due to the small grain size [27].

Similarly, the CEC showed a direct relationship with the soil sorption of LNG (see Figure 2d). Thus, higher values of CEC increased its soil sorption. According to Tang et al., the equilibrium time for the soil sorption of LNG took a shorter amount of time at lower CEC values because LNG may fill the vacant sites in soils more quickly in order to reach saturation [2].

According to our results, the grain size, the CEC, and water infiltration may play essential roles in the leaching fractions of LNG. Some soil-borne LNG levels may induce chronic toxicity with long-term exposure in aquatic organisms and possibly overexposed humans. However, more data for the correlation between the soil sorption of LNG in cover materials and the soil properties should be included in future studies.

#### *3.4. Assessment of Leachate Sample in Placental Cell Lines (BeWo) and Sperm Cells*

Both the LNG and leached LNG from soil layers under laboratory conditions did not show a change in the conventional sperm parameters, including progressive motility (Figure 3a), non-progressive motility (Figure 3b), immotile sperm cells (Figure 3c), and viability (Figure 3d) compared to the negative controls.

**Figure 3.** Sperm motility test and viability. Experimental data (*n* = 5): (**a**) Percentage of sperm progressive motility; (**b**) Percentage of sperm non-progressive motility; (**c**) percentage of immotile sperm cells; and (**d**) Percentage of sperm viability.

The findings of this study show that the sperm motility and viability of seminal samples were not affected when they were in contact with LNG. However, chronic exposure to this substance, even at low concentrations, may alter some species' reproductive capacities. For instance, low amounts of LNG may result in reproductive disorders in male zebrafish in progestin-contaminated aquatic environments due to LNG causing a significant decrease in the plasma concentrations of 11-ketotestosterone (11-KT) or estradiol (E2) in males exposed to between 10 and 100 ng·L−<sup>1</sup> of LNG [28]. Additionally, previous studies have shown

a reduction in sperm kinematics and capacitation in ovine spermatozoa when exposed to estrogens such as 17β-estradiol and progesterone [29]. On the other hand, more longterm exposure to LNG may affect sperm kinematics in male fathead minnow (Pimephales promelas) [30].

The BeWo cell line was applied to assess possible changes in β-hCG production because this hormone is related to corpus luteum rescue in trophoblastic cell implantation. Significant differences (SD) between the negative control and the stock solution of LNG or the leachate sample were found by applying an unpaired *t*-test. Our results show that LNG and leached fractions decreased β-hCG in the cell medium after BeWo cell line exposure (see Figure 4). This could be a result of the ED effect because the MTT assay did not show differences in viability during the test. This effect is related to changes in β-hCG production and not to acute toxicity due to the reduction in cell numbers.

**Figure 4.** Study of LNG reproduction effects and leached LNG from different soil layers by analysis of β-hCG levels in the medium of the placental cell line. The MTT viability test is presented in the small box. Significant differences (SD; *p*-value < 0.05); non-significant differences (NSD; *p*-value > 0.05).

In a previous study, we found that low levels of LNG and its degradation products may induce a similar reduction in β-hCG levels after the exposure of placental lines to those fractions [9].

On the other hand, previous reports have shown that LNG may work as an ED in the reproductive functions of fish and amphibians, which causes the gene expression of pituitary gonadotropins and gonadal steroidogenic enzymes [7,31]. Furthermore, these alterations induce estrogenic effects in fathead minnows (males and females) exposed to different concentrations of LNG (up to 3124 ng·L<sup>−</sup>1) for 28 days [32].

According to the biological activity of progestins, it is known that they act intracellularly through the binding and activation of the progesterone receptor (PR), which serves as a transcription factor to induce genomic effects [33]. These nuclear receptors are evolutionarily conserved in vertebrate animals and, thus, indirect exposure in water at low levels could induce similar pathways [4].

#### *3.5. A Possible Implication of Leached LNG in Medellín, Colombia*

The Buenavista landfill is located 600 m away from the Piedras River. This landfill has a slope steeper than 30% and, thus, runoff and leaching may occur. Therefore, the quality of the river may be impacted by pollutants transported from the landfill (for more details, see Supplementary Materials SM4). For instance, we detected LNG in effluents of the UASB reactor in the Buenavista landfill, and this substance may reach the water body. Therefore, the levels of LNG should be monitored in this river. A percentage of water from the Piedras River is pumped to the La Fe reservoir, one of the most important drinking water reservoirs for the Valle del Aburrá in Antioquia (Colombia), supplying potable water to more than two million people. The natural water is purified in La Ayurá, a drinking water treatment plant (DWTP) located in Medellín before its distribution. Thus, the exposure risk for people may depend on five factors, including: (1) the final disposal of PPCPs in the landfill; (2) the cover material sorption; (3) the leaching treatment at the UASB reactor; (4) natural degradation; and (5) purification in the DWTP (La Ayurá). However, regarding the first factor, we found many empty pharmaceutical blisters and plastic containers in the Buenavista landfill, which shows that this place is the final destination for many PPCPs. Secondly, we found that cover materials may allow for the leaching of LNG at values lower than 8% (under laboratory conditions). Furthermore, the levels of LNG in the effluents of the UASB reactor indicate that the cover material interaction and even the leaching treatment do not sufficiently reduce the levels of LNG. Recalcitrant pollutants, such as PPCPs, are not efficiently removed from wastewater treatment plants (WWTPs) by conventional processes. For instance, Botero-Coy et al. detected levels of antibiotics above <sup>1</sup> <sup>μ</sup>g·L−<sup>1</sup> in the effluents of WWTPs in cities such as Bogotá, Medellín, and Florencia, which means that these pollutants are not completely removed by conventional methods [34]. Thus, the design of treatment plants for leaching water and wastewater should include advanced oxidation, among other tertiary treatment processes for the removal of pollutants.

Natural degradation is not efficient enough to remove LNG in natural water. In a previous report, we found that solar energy for the photolysis of LNG does not have a strong enough effect to enable its degradation under natural conditions [9]. Thus, LNG fractions may remain in the water for a long time. However, biodegradation should be considered in future studies. Although information about the biodegradation and biotransformation of LNG as well other contraceptives is still unknown, its half-life in non-sterile soil is ten times higher than in sterile soil and, thus, microorganisms may play an important role in its natural degradation [35]. According to our results, LNG was detected in the effluent of the UASB reactor; through runoff, this fraction could reach the natural water in the Piedras River. Thus, the exposure risk for people in Medellín may depend on the DWTP, which applied coagulation and chlorination during treatment. However, these conventional methods are not efficient for the removal of pollutants, as discussed above. Thus, LNG exposure could have some endocrine effects on the biota and exposed population. Although the effects of short-term exposure were not found in sperm cells by in vitro assay in this study, long-term exposure to LNG should be considered because of the risks of continuous exposure at low levels. According to Elisabeth Carlsen et al., the quantity of semen has decreased by fifty percent in the last 50 years from <sup>113</sup> × <sup>10</sup><sup>6</sup> mL−<sup>1</sup> [36]. Although the exact causes of this decrease have not been confirmed, complex xenobiotic exposure may be related to the reduction in sperm count. For instance, recently, more PPCPs have been found in bodies of water, and reproduction disorders in exposed aquatic species have increased [1]. According to the US Environmental Protection Agency (EPA), steroidal hormones, including LNG, may induce feminization, among other reproductive dysfunctions, in non-target organisms [37].

Finally, in this study, we found that leached fractions of LNG decreased the levels of β-hCG in a placental cell line, which indicates possible effects on implantation pathways. Therefore, more studies should be carried out on human ED due to low levels of unintentional exposure to contraceptives that may leach from landfills. Expired products may be disposed of in the Buenavista landfill, and soil sorption may be the final barrier

preventing pollutants from ending up in surface water. However, we found that the cover material allowed the passage, through the soil layer, of small amounts of freely dissolved concentration of LNG, which may affect the endocrine systems of exposed species, even in humans. Future studies should focus on reducing the disposal of PPCPs in landfills and developing an efficient cover material for the sorption of pollutants to avoid exposure to soil-borne LNG.

#### **4. Conclusions**

Many expired or partially consumed PPCPs are disposed of in landfills. In these places, the soil layers used as cover materials influence the sorption capacity of these pollutants. This interaction may affect the transport of these substances, allowing them to reach water bodies, inducing imbalances in exposed ecosystems and, consequently, increasing the risk to public health. The soil layers used in the Buenavista landfill contain high amounts of clay and silt, which increase LNG–soil interactions. However, LNG is not completely adsorbed and, as a result, it was detected in leachate samples. The samples that contained LNG and the LNG stock solution affected the production of β-hCG in placental cell lines. Therefore, indirect exposure to LNG may cause problems during gestational development.

On the other hand, the levels of LNG detected in the leachate samples did not affect the seminal parameters of sperm cells. However, studies on the effects of chronic toxicity on the andrological pathways should be carried out to assess the outcomes of long-term exposure to LNG in drinking water. This was a preliminary study carried out to understand the influence of the Buenavista landfill as a source of PPCPs in the Piedras River due to leaching.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/w14060871/s1, SM1. Normal sperm values, SM2. MTT assay, SM3. UV spectrum of LNG, SM4. Analysis of PPCPs in the landfill.

**Author Contributions:** Conceptualization, A.C.P. and J.C.Q.-C.; methodology, R.R.-S., W.D.C.-M. and J.J.G.-L.; validation, D.G.-R.; formal analysis, writing—original draft preparation and supervision, J.F.N.-V. All authors have read and agreed to the published version of the manuscript.

**Funding:** The materials and methods were supported by grants from Ministerio de Ciencia y Tecnología MINCIENCIAS (Project code: 136577757707). Additionally, this paper was supported by Corporación Universitaria Remington and Universidad Cooperativa de Colombia (Project code: 4000000098).

**Institutional Review Board Statement:** Informed consent was obtained from all subjects involved in the study.

**Informed Consent Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethic Committee of the Faculty of Medicine-University of Antioquia (Acta de aprobación No. 003, date: 14-02-2019)." for studies involving humans. Written informed consent has been obtained from the patients to publish this paper.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank the Ministerio de Ciencia y Tecnología MIN-CIENCIAS for funding the project entitled "Potencial de bioacumulación de agroquímicos y contaminantes persistentes en una cuenca del oriente antioqueño: Evaluación de un problema de salud pública". The authors also thank the authors of the research project entitled "Ensayo de disrupción endocrina de micro-contaminantes acumulados en prótesis mamarias de silicona por medio de passive dosing y expresión de la hormona β-hCG en la línea celular BeWo" which was funding by Universidad Cooperativa de Colombia and Corporación Universitaria Remington. Additionally, the authors thank the Hospital Marco Fidel Suarez (Bello-Antioquia) for analyzing the levels of β-hCG in the samples and for their support.

**Conflicts of Interest:** The authors declare that there is no conflict of interest with regard to the published results.
