**1. Introduction**

Pharmaceutical and personal care products (PPCPs) have been classified as emerging pollutants because of their environmental impact. Long-term exposure to these substances may affect a wide range of species, even at low concentrations [1]. Furthermore, the high persistence of PPCPs and their low removal from wastewater may increase their impact on aquatic ecosystems [2]. Traces of PPCPs have been found in drinking water supplies, which is a public health concern [3]. The implications of the toxicity of PPCPs in water include endocrine disruption (ED), which affects the equilibrium of hormone levels and leads to metabolic, neurological, and reproductive consequences in exposed species [4].

Levonorgestrel (LNG) is a pharmaceutical product widely used in Colombia as a contraceptive method; thus, complex effects similar to those of other PPCPs were found [5].

**Citation:** Ríos-Sossa, R.; García-Londoño, J.J.; Gil-Ramírez, D.; Patiño, A.C.; Cardona-Maya, W.D.; Quintana-Castillo, J.C.; Narváez-Valderrama, J.F. Assessment of Levonorgestrel Leaching in a Landfill and Its Effects on Placental Cell Lines and Sperm Cells. *Water* **2022**, *14*, 871. https://doi.org/ 10.3390/w14060871

Academic Editors: François Gagné, Stefano Magni and Valerio Matozzo

Received: 14 December 2021 Accepted: 8 March 2022 Published: 10 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

This substance is known to interact not only with progesterone receptors (PR), but also with another estrogen receptor, which may trigger complex pathways in non-targeted organisms [6]. LNG was detected in very low levels in water, leading to disruptions in the gonadotropin expression of aquatic species, such as teleost fish [7]. However, the information available on the occurrence and fate of this substance in aquatic ecosystems is limited. LNG is a steroidal molecule with strong molecular bonds, and thus, LNG is recalcitrant to degradation processes [8]. Therefore, LNG shows environmental persistence because natural conditions do not completely remove this substance. For instance, LNG is not removed by solar radiation at wavelengths close to 320 nm, and even energy radiation (UVC-265) reduces only 80% of this substance [9]. Therefore, more complex photodegradation should be applied for its removal in treatment plants.

Many PPCPs, including LNG, are disposed of in landfills, which could be a source of water pollution due to leaching [10]. The leaching that occurs in this landfill is a risk for long-term exposure in many people, but the effects are still unknown. For instance, some landfill leachates containing heavy metals induced genotoxic and cytotoxic effects in the peripheral blood erythrocytes extracted from Wistar strain rats, which showed the impact of landfills on biota [11]. Furthermore, the leachate may contain more complex substances, including PPCPs. However, leaching impacts may be reduced when the interaction of pollutants with a soil layer that is applied for covering waste residues in landfills (cover material) increases. Thus, the selection of an appropriate cover material decreases soilborne pollutants due to the soil layer working as a natural barrier. In previous research, Yang et al. found that hydrophobic partitioning is the main pathway for the sorption of progestins in soil [12]. For instance, the organic fractions in the soil layer may affect LNG leaching from landfills. The leached fraction of LNG may trigger ED in biota, among other complex implications in exposed organisms. For example, it has been found that levels between 3.3 and 40 ng·L−<sup>1</sup> of LNG can reduce fish reproduction by the masculinization of females, changes in gonad histology, and disruption to hormone levels [13]. However, LNG toxicity in human reproduction has not been deeply studied. In a previous report, we showed that LNG and its photodegraded fractions decrease human chorionic gonadotropin (β-hCG) hormone levels in placental cell lines. This may cause problems to occur during gestational development [9].

The Buenavista landfill, which is located in Antioquia, Colombia, has been found to contain expired PPCPs, including painkillers, contraceptives, and antibiotics, among others. Thus, this landfill could be a source of PPCPs leaching into the Piedras River, which plays an essential role in the supply of drinking water to the city of Medellín. However, an appropriate cover material to reduce the transport of leachate pollutants, as well as treatment, may reduce their impact in natural water. To this end, an upflow anaerobic sludge blanket (UASB) reactor is used for the treatment of leachates produced by the Buenavista landfill. However, the UASB reactor, among other conventional treatments, is not sufficient for persistent organic pollutant removal because they are extremely resistant to biological degradation processes, and more specific advanced oxidation processes should be included for the treatment of pollutants [14].

The aim of this study was to assay the levels of LNG from the effluent of a UASB reactor used in the Buenavista landfill to evaluate possible transport by leaching from cover materials. Additionally, we studied the effects of the cover material used in this landfill on LNG adsorption to approximate a better material selection for reducing leaching. Finally, we studied the endocrine disruptor effect of LNG on the BeWo cell line and human sperm cells by in vitro assay. The results may be relevant for future studies on the assessment of risk due to the long-term exposure of PPCPs disposed of in landfills and their impact on bodies of water.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Materials*

A stock solution of LNG was prepared at a concentration of 10.5 mg·L−<sup>1</sup> from the commercial pharmaceutical Cerciorat® containing 1.5 mg per pill (98–102% USP29). Then, seven pills were macerated and diluted in Milli-Q water. The stock solution was stored at −20 ◦C. Solvents, including methanol and acetonitrile (HPLC grade), fetal bovine serum (FBS), and Ham-F10 medium, were purchased from Sigma-Aldrich (San Luis, MO, USA). The water was purified in a Thermo Scientific® Barnstead 50131217 GenPure TM UV/UF for Type I Milli-Q water (deionized). The buffer solution for the mobile phase was prepared with 0.05% formic acid in deionized water. All solvents were filtered through cellulose nitrate (with pore sizes of 0.2 μm) and degassed in an ultrasonic bath for 10 min at 25 ◦C. Finally, the antibodies for β-hCG and progesterone analysis were provided by Medife S.A.S (the provider for Roche Colombia).
