# Phthalates in face masks and their effects on cellular energetics in human bronchial epithelial cells

Lyse Szymanski, Margueritta Al Zallouha, Clémence Bouzar, Edwige Votier, Christelle Monteil, Tiphaine Rogez-Florent

► To cite this version:

Lyse Szymanski, Margueritta Al Zallouha, Clémence Bouzar, Edwige Votier, Christelle Monteil, et al. Phthalates in face masks and their effects on cellular energetics in human bronchial epithelial cells. Ecotoxicology and Environmental Safety, 2025, 292, pp.117910. 10.1016/j.ecoenv.2025.117910. hal-05391596

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# Phthalates in face masks and their effects on cellular energetics in human bronchial epithelial cells

Lyse Szymanski, Margueritta Al Zallouha, Clémence Bouzar, Edwige Votier, Christelle Monteil ID, Tiphaine Rogez-Florent * ID

Univ Rouen Normandie, Université Caen Normandie, Normandie Univ, ABTE UR 4651, Rouen F-76000, France

## ARTICLE INFO

Edited by Dr Hyo-Bang Moon

**Keywords:**

Phthalates
Face mask
BEAS-2B
Mitochondrial dysfunction
Metabolic disruption
Glycolysis

## ABSTRACT

The widespread use of disposable masks has sparked concerns about potential exposure to harmful chemicals, including the already ubiquitous phthalates. It has been demonstrated that there is a causal relationship between the exposure to phthalates and the onset of respiratory diseases. Moreover, there is an increasing body of evidence, which suggests that mitochondria contribute to significant impact to the epithelial function in respiratory diseases. However, the specific effects induced by these compounds at the mitochondrial level in respiratory cells are unknown. The objective of this study was to identify phthalates in disposable face masks and to evaluate their effect on mitochondrial function in human bronchial epithelial cells (BEAS-2B). In order to identify and quantify the phthalates in face masks, an extraction method was developed and a phthalate quantification method using Gas Chromatography-Mass Spectrometry was employed. The analysis of the face masks sample revealed the presence of three major phthalates: di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP) and diisobutyl phthalate (DiBP). To evaluate the effects of these phthalates on mitochondrial function, BEAS-2B were exposed to the phthalates individually and in a mixture of the three at concentrations ranging from 0.5 to 10 mg/L for 24 hours. Then extracellular flux analysis (Seahorse analysis), adenosine levels (LC-DAD) and lactate levels (LC-MS) were evaluated. The observed effects at the mitochondrial level varied according to the specific phthalates that were tested. The major dysfunctions were identified following exposure to DEHP, which exerted inhibitory effects on the basal and maximal oxygen consumption rate of BEAS-2B cells, as well as an alteration in oxidative phosphorylation and a reduction in the ATP/ADP ratio. This was accompanied by an increase in both extracellular lactate production and extracellular acidification rate. A shift towards a glycolytic phenotype was noted. Similar, yet more pronounced effects, were observed following exposure to the mixture of the three phthalates, which suggest that DEHP may contribute to the overall effect of the mixture. This study is the first to provide knowledge on the adverse effects of a mixture of phthalates on mitochondrial function in bronchial epithelial cells, demonstrating the capacity of some phthalates to induce a shift toward a glycolytic phenotype. These results highlight the potential risks of inhaled phthalates on mitochondrial function, predisposing to the development of respiratory diseases.

## 1. Introduction

Face masks are crucial health care necessity against respiratory infections. Since the outbreak of the Coronavirus pandemic, the demand for face masks has increased, as a key measure for health protection to avoid the transmission of viruses. Between 2020 and 2021, nearly 60 % of the population wore a protective mask in public (Chen et al., 2022) with a global monthly consumption of approximately 129 billion masks during the early stage of the pandemic (Prata et al., 2020). The exact

composition of these masks is not fully documented. It is known that they are made of different polymers such as polystyrene, polypropylene, polyurethane, polyacrylonitrile or even polyester (Cao et al., 2023; Potluri and Needham, 2005). Different additives such as antioxidants, flame retardants, and phthalates are usually added to polymers. These latter improve their flexibility, elasticity and durability. Several studies have already highlighted the presence of phthalates in masks, however the results obtained between the various articles are very different, whether in terms of compounds found or in terms of content (Cao et al.,

<footnote>* Corresponding author.
E-mail address: tiphaine.rogez@univ-rouen.fr (T. Rogez-Florent).</footnote>
2023; Wang et al., 2022; Xie et al., 2022; Leoni et al., 2023; Vimalkumar et al., 2022). Phthalates are organic derived from phthalic acid, with a worldwide production of up to 15 million tons per year (Chen et al., 2018). They are mostly semi-volatile and non-covalently bounded to polymers of plastics therefore they can leach from the face mask and be released into the environment. Thus, masks constitute an important exposure source for humans through inhalation, dermal contact and ingestion.

Phthalates are ubiquitous compounds present in food, tape water, air and sediments (Dobaradaran et al., 2020; Arfaeinia et al., 2019; Mohammadi et al., 2022; Kanazawa et al., 2010). Epidemiological and experimental studies raised concerns for the adverse health effects associated with phthalate exposure. Among them, reproductive and developmental effects have been documented most extensively. Some phthalates such as DEHP, DBP, BBP and DiBP can act as endocrine disruptors causing many reproductive disorders in man and woman (Hlísnýková et al., 2020), therefore they have been classified as reproductive toxicants, carcinogenic, mutagenic or reprotoxic (CMR) category 1B or 1 A by European authorities (Lambré et al., 2022). It has been documented that the respiratory system is also susceptible to certain phthalates. These latter have been detected in indoor air and particulate matter, resulting in human respiratory exposure (Kanazawa et al., 2010; Alves et al., 2020). Associations have been established between exposure to phthalates and the development of diseases such as allergic rhinitis, asthma or chronic obstructive pulmonary disease (Li et al., 2017; Shen et al., 2023; Jaakkola and Knight, 2008). However, the underlying mechanisms remain to be elucidated.

Mitochondria are organelles that serve as the powerhouse of cells generating energy in the form of adenosine triphosphate (ATP) via oxidative phosphorylation (OXPHOS) carried out by electron transport chain and ATP synthase. In addition to energy production, mitochondria are involved in the regulation of metabolic activity, calcium signaling and storage, reactive oxygen species (ROS) signaling as well as apoptosis coordination (Tang et al., 2020). At the respiratory level, mitochondrial activities contribute to the morphology of lung airway and to epithelial functions. Therefore, mitochondrial dysfunction like disturbed OXPHOS capacity, overproduction of ROS or excessive mitophagy might contribute to the etiology of respiratory diseases like asthma, chronic obstructive pulmonary disease and lung cancer (Chellappan et al., 2022; Cloonan and Choi, 2016). Mitochondria are responsive to environmental stimuli. As a result, exposure to environmental factors like pollutants is believed to cause mitochondrial dysfunction. Phthalates, specially DEHP, have been shown to induce mitochondrial dysfunction in different cell types. Acute exposure to DEHP caused a decrease in ATP production in mice neuroblasts (Zhang et al., 2022). Modifications in oxidative stress status were also noted with an increase in mitochondrial ROS production after DEHP exposure in human embryonic kidney cells (Ashari et al., 2023) and ovarian cells (Tripathi et al., 2019). Mitochondrial dysfunction was also observed at the energetic levels with perturbations in cellular oxygen consumption, in acidification and a shift in cellular phenotype in rats cardiomyocytes (Posnack et al., 2012) and in endothelial cells (Poitou et al., 2022) exposed to DEHP. However, the effects of phthalates at the respiratory epithelia remain largely uninvestigated with no studies on the effects at the mitochondrial level. With the exception of one study (Poitou et al., 2022), all others evaluated the effects of exposure to one phthalate at a time. Although studying the effects of individual phthalate allows the understanding of its mode of action, this approach is not representative of real-life exposure where one is exposed to multiple phthalates at a time. The interaction of individual phthalates in a mixture may result in a different effect than that observed with individual phthalates.

In the light of current literature, it is crucial to conduct studies to look for a possible link between exposure to phthalates and dysfunction of the mitochondria at the respiratory cells level. The questions about the effects of mixed exposure to phthalates remain unanswered and should be further investigated. The objective of this study was to identify

the main phthalates present in a sample of masks and to investigate their effects, both individually and in mixtures, on bronchial epithelial cells in culture, with a specific focus on mitochondrial function.

## 2. Materials and methods

### 2.1. Chemicals

The phthalates were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France): dimethyl phthalate (DMP), diethyl phthalate (DEP), dipentyl phthalate (DPP), bis(2-propylheptyl) phthalate (DphP), benzyl butyl phthalate (BBP), diisobutyl phthalate (DiBP), dibutyl phthalate (DBP), di(2-ethylhexyl) phthalate (DEHP), di(2-ethylhexyl) terephthalates (DEHT) and diethyl phthalate-3,4,5,6-d4 (DEP-d4). Acetone, acetonitrile, dichloromethane, dimethyl sulfoxide (DMSO), n-hexane and cyclohexane were purchased from VWR (Fontenay-sous-Bois, France). HPLC-grade solvents were used. Ultra-pure water was supplied by Barnstead™ MicroPure™ system (Thermo Scientific™, Les Ulis, France). The characteristics of the different phthalates are listed in supplementary data (Table S.1).

### 2.2. Face mask samples

Four facemasks were purchased with different characteristics in terms of brand, country of manufacture, color and level of protection. Regarding the country of manufacture, three masks are made in France, and one mask is made in China. As for the color, there are two blue masks, one white and one purple. Finally, in terms of protection level, three of the masks selected are type IIR surgical masks, and one is a FFP2 mask. A detailed list with the characteristics of the four selected masks is provided in Table S.2. All samples were subsequently individually extracted and analyzed.

### 2.3. Identification and quantification of phthalates

The optimization and the validation of the analytical method using Gas Chromatography-Mass Spectrometry (GC-MS) have been previously described (Poitou et al., 2021). Chromatographic analyses were performed using an Agilent 7890 gas chromatography system coupled with an Agilent 5975 C mass spectrometer equipped with an electron impact ion source and a single quadrupole (Agilent Technologies, Santa Clara, CA, USA). Separation was carried out on a cross-linked poly 5 % diphenyl/95 % dimethyl siloxane HP-5-MS capillary column (30 m × 0.25 mm × 0.25 µm) from Agilent. A gradient separation mode was used to separate the plasticizers. The injector and transfer line temperatures were both at 280°C, and the mass spectrometer at 150°C. Helium was used as a carrier gas at a flow rate of 1 mL/min. One microliter was injected in splitless mode. The acquisition was performed on selected ion monitoring (SIM) mode, for which acquisition parameters are detailed in Table S.3. Visualization and analysis of the results were performed using the software MSD Chemstation E.02.02.1431 (Agilent Technologies). Stock and work solutions for GC-MS experiments were prepared according to a previous study (Poitou et al., 2021). Only glass materials were used to avoid plastic contamination. Cleaned glass materials were rinsed twice with the containing organic solvent. All stock solutions were prepared at a concentration of 10 g/L in acetone and were stored at 4°C. For method validation, calibration standards were prepared in acetone, with concentrations ranging from 0.05 to 1.2 mg/L for DMP, DEP, DPP, DphP, BBP, DiBP, DBP, DEHP, and DEHT; and from 0.5 to 12 mg/L for DiNP.

### 2.4. Extraction protocol

From each of the four brands, each mask was cut into small pieces (approximately 1 cm × 1 cm, around 40 mg) and placed in a glass tube with 2 mL of n-hexane. Only the center of the mask (the part in contact
with the nose and the mouth) was used. The glass tube was sonicated for 20 minutes at a constant temperature of 20°C. The supernatant was then transferred into another glass tube, and the mask was again suspended in 1 mL of n-hexane and the extraction steps were repeated twice. A volume of 3 mL of the extracts was evaporated under nitrogen flow. A blank sample underwent the same extraction procedure. Then, all samples were spiked with the internal standards DEP-d4 (0.1 ppm) and injected into the GC-MS device. For each condition, 4 replicates (N = 4) were conducted per mask.

## 2.5. Cell culture and phthalate treatment

Human bronchial epithelial cell line BEAS-2B was purchased from the American Type Culture Collection (ATCC®, USA). BEAS-2B cells were seeded in cell-bind 75 cm² culture flasks and maintained at 37°C with 5% CO₂ in serum-free LHC-9 medium (Gibco™; Fischer Scientific, France). When confluent, cells were detached enzymatically with 0.05% Trypsin-EDTA (Gibco™; Fischer Scientific, France), counted using the exclusion colorant trypan-blue 0.1% (Sigma-Aldrich®, France), and then sub-cultured into the appropriate plate according to each test. All stock solutions of phthalates were prepared by gravimetry at a concentration of 10 g/L in dimethyl sulfoxide (DMSO). Intermediates solutions were prepared at 2 g/L, 1.5 g/L, 1 g/L, 0.5 g/L and 0.1 g/L in DMSO. All solutions were stored at 4°C until use. Working phthalate solutions were prepared by diluting the intermediate solutions with the culture medium immediately before exposure. BEAS-2B cells were exposed to each of three phthalates DEHP, DBP, DiBP alone or to a mixture of the three (1:1:1 DEHP/DBP/DiBP) at different concentrations: 0.5; 2.5; 5; 7.5 and 10 mg/L for DEHP and the mixture and at 5; 7.5 and 10 mg/L for DiBP and DBP. Since DMSO was used as a solvent at the final concentration of 0.5% in all the tested conditions, cells exposed to culture medium at 0.5% DMSO without phthalates were considered as the control group.

## 2.6. Cell viability

To analyze cell viability, MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed after BEAS-2B exposure to phthalates. Briefly, cells were seeded in a 96-wells microplate at a density of $6 \times 10^3$ cells/cm² with 100 µL of LHC-9 medium. The day after seeding, cells were exposed for 24 hours to the different conditions listed in Section 2.5. To study the MTT reduction potential, cells were treated with 100 µL of MTT solution (Sigma-Aldrich, France) at 0.5 mg/mL diluted in growth media for three hours at 37°C and 5% CO₂. Then, formazan crystals were solubilized with 100 µL of DMSO. The absorbance was measured at 570 nm using the microplate reader CLARIOstar Plus (BMG LabTech, Champigny-sur-Marne, France). Cell viability is expressed in percentage and compared to the control 0.5% DMSO (100% viability).

## 2.7. Measurement of cellular adenosines

To determine intracellular adenosine content (ATP, ADP and AMP), $5 \times 10^4$ cells/cm² were seeded in 12 wells plate. After 24 hours of exposure, the supernatant was removed, cells were rinsed with PBS and 500 µL of perchloric acid (PCA, 1 N) were added to harvest cells. Then, the samples were centrifuged (15,000 rpm, 5 minutes, 4°C) and the pellets were resuspended in 1 N NaOH to determine protein content by the Bradford assay. Potassium carbonate ($K_2CO_3$, 1 N) was added to neutralize the supernatant and then samples were centrifuged again. Supernatants were used for the quantification of intracellular adenosines using High Performance Liquid Chromatography - Diode Array Detector (LC-DAD, Infinity II, Agilent Technologies, Santa Clara, CA, USA), method previously described (Juarez-Facio et al., 2021). Briefly, chromatographic separation was achieved on a Poroshell 120 EC-C18 (3 mm × 150 mm, 2.7 µm) with a temperature of 20°C. One microliter

of each sample was injected. The mobile phase was 50 mM potassium hydrogen phosphate (pH = 6.8). The flow rate was 0.6 mL/min. Detection of analytes was performed at 254 nm. The system control and data processing were performed using an OpenLab CDS ChemStation from Agilent.

## 2.8. Determination of cellular phenotype

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Agilent Seahorse XFp Cell Energy Phenotype Test Kit and analyzed with the Seahorse XF24 extracellular flux analyzer (Agilent technologies) according to the instructions of the user guide. BEAS-2B were seeded with a starting cells density of $2 \times 10^4$ cells per well in XF24 culture microplates for 24 hours at 5% CO₂ atmosphere at 37°C then treated with the different concentrations of phthalates or with the culture medium at 0.5% DMSO (Section 2.5.) for 24 hours (triplicates per condition). Prior to analysis, cells were rinsed 3 times with DMEM Seahorse XF medium (pH = 7.4, 100 mM pyruvate, 200 mM glutamine and 2.5 M glucose) and incubated for 1 hour at 37°C in a CO₂ free incubator. During OCR and ECAR monitoring, cells were treated with two inhibitors to induce high energy demand: 10 µM of oligomycin A (Sigma-Aldrich, France) and 10 µM of FCCP (carbonyl cyanide p-trifluoromethoxy phenylhydrazone) (Sigma-Aldrich, France). Therefore, immediately after measurement, assay media were discarded and 50 µL of NaOH 1 N were added in each well for one hour at room temperature in order to extract the proteins for quantification using Bradford assay.

Data analysis was done using Wave Software (Version 2.6.3.5, Agilent Technologies). Protein content was used to normalize OCR and ECAR values from each well. Basal OCR and ECAR were calculated as the average of OCR or ECAR (three time points) before inhibitors injections.

## 2.9. Protein dosage

The quantification of proteins was realized using the Bradford colorimetric test then the absorbance of each well of the 96-wells microplate was read using the Plus microplate reader (BMG LabTech) at two wavelengths: 595 nm and 465 nm.

## 2.10. Extracellular lactate measurement

Cells were seeded in 24-wells microplate at a density of $6 \times 10^4$ cells/cm² with 500 µL of LHC-9 medium. The day after seeding, cells were exposed for 24 hours to the different conditions of phthalates (Section 2.5.) or to 100 µM of 2-deoxy-D-glucose (2-DG) (Fisher Scientific, France). Cells exposed to 0.5% DMSO were used as control. After exposure, the culture medium was collected and heated at 100°C for 10 minutes. Then, 500 µL of PCA (1 N) were added to 500 µL of the culture medium and solutions were centrifuged (15,000 rpm, 5 minutes, 4°C). The supernatant was neutralized with 120 µL of $K_2CO_3$ (1 N), centrifuged and stored at -80°C until metabolites measurement. Chromatography and mass-spectrometric detection were performed using a 1260 Infinity II LC coupled to an Ultivo QQQ LC-MS (Agilent Technologies, Germany) using an electrospray ionization (ESI) source negative mode. Chromatographic separation of lactate was achieved using Nucleoshell HILIC column (150 x 4 mm, 2.7 µm, Macherey Nagel, Hoerdt, France) and a mobile phase consisting of 60% of acetonitrile and 40% of water with 0.1% of acetic acid. An isocratic elution was performed at 0.8 mL/min. The column was thermostated at 40°C, with an injection volume of 5 µL. Mass parameters were optimized for lactate using a positive or negative multiple reaction-monitoring (MRM) mode, as follows: *m/z* 89 → 43. The ion source parameters for the analysis were: 4500 V capillary voltage, 10 L/min gas flow, 325°C gas temperature, 20 psi nebulizer, 12 L/min sheath gas flow and 400°C sheath gas temperature. Standard stock solutions were prepared by dissolving each analyte in water.
## 2.11. Statistical analysis

Results are expressed as mean ± standard deviation (SD) of at least four independent experiments. Statistical analysis was performed using the non-parametric test Mann-Whitney (U test) using GraphPad Prism 9 Software (Version 9.5.1, GraphPad for Windows, San Diego, CA, USA). Data with p-value less than p < 0.05 were considered as statistically different.

# 3. Results and discussion

## 3.1. Phthalates concentrations in face masks

GC-MS is frequently employed for the analysis of phthalates in masks (Leoni et al., 2023; Xie et al., 2022; Chang and Wang, 2023). In this paper, a specific GC-MS method, for the quantification of thirteen phthalates in face masks was optimized based on our previous work (Poitou et al., 2021). To determine the optimal extraction conditions, different parameters were optimized: the mass of the mask, the volume and nature of the extraction solvent and the number and duration of the ultrasonic baths. Several organic solvents are described in the literature for the extraction of phthalates from masks (Vimalkumar et al., 2022; De-la-Torre et al., 2022; Idowu et al., 2023) with n-hexane being the most commonly used solvent (Jin et al., 2021; Wang et al., 2022; Cao et al., 2023; De-la-Torre et al., 2022). DCM, n-hexane, DCM/n-hexane (50/50), acetone, acetonitrile and cyclohexane were tested in our study. Extractions performed in n-hexane lead to a greater number of peaks in GC-MS (Fig. 1). Therefore, n-hexane was used to extract phthalates from the mask samples. Three phthalates were systematically quantified in the four masks studied: DEHP, DBP and DiBP (Fig. 2 and Table S.4), while DEP and DEHT were always below the limit of quantification (LOQ). Our results agree with previous studies showing the systematic presence of DEHP, DiBP and DBP in masks (Xie et al., 2022;

Vimalkumar et al., 2022; Leoni et al., 2023; Cao et al., 2023). The Σ phthalates mass load in our samples ranged from 1843 ng/g to 3872 ng/g. These values are similar than those reported in previous studies, ranging from 115 to 37,700 ng/g (Xie et al., 2022), 405–1060 ng/g (Vimalkumar et al., 2022) and 264–6804 ng/g (Cao et al., 2023). While masks 2 and 3 had similar contents, the maximum concentration was found in mask 4 for DiBP (1491.7 ± 132 ng/g), followed by DBP (1346.7 ± 55.2 ng/g) and DEHP (1033.3 ± 99 ng/g). The 4 masks used in the present study were purchased from different manufacturers. This could explain the difference in their composition in terms of phthalates content. The presence of phthalates could be attributed to the addition of phthalates to raw materials during manufacturing process, rather than to their migration from packaging materials (Min et al., 2021). DEHP is the most common phthalate used as additive to soften polyvinylchloride (PVC) plastic and other plastic materials. DBP and DiBP are used for polyester (PE) and polypropylene (PP) fabrics and as additifs for vinyl fabrics, food wrap, in adhesives and polyvinyl acetate polymers. This study together with previous ones revealed the presence of phthalates in masks, indicating that masks may constitute a potential source of exposure to phthalates by inhalation.

## 3.2. Modification of BEAS-2B viability after exposure to phthalates

To evaluate the influence of phthalates identified in mask samples on cell viability, BEAS-2B cells were exposed for 24 hours to each of the three phthalates DEHP, DiBP and DBP, individually as well as to a mixture of the three compounds at concentrations from 0.5 to 10 mg/L. Cells exposed to 0.5 % of DMSO in culture media were used as control. These exposure concentrations were chosen based on the literature review and solubility tests. Previous studies conducted on phthalates revealed very high exposure concentrations up to 390 mg/L of DEHP (Rafael-Vázquez et al., 2018). Only two studies used lower concentrations of 5 mg/L for DEHP and DBP (Shi et al., 2021) and 15 mg/L for

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** GC-MS chromatograms in SIM (Single Ion Monitoring) mode, *m/z* ratio = 149. (A) Chromatogram of a standard mixture of phthalates at a concentration of 0.4 mg/L, and (B) Chromatogram of an extract solution from the mask 3.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Phthalate content (ng/g) for the 3 phthalates quantified by GC-MS in each of the four masks, Data represents mean ± SD, N = 4 for each mask.</figcaption></figure>

DEHP (Poitou et al., 2022) for toxicological assays. A second criteria contributing to the choice of these exposure concentrations was the solubility of phthalates in the culture media. Solubility was verified by turbidimetry in order to obtain soluble solutions at a tolerable non-cytotoxic percentage of DMSO (0.5 % in culture media). After exposure to phthalates, cell viability was evaluated by the MTT assay (Fig. 3.A) and by the measurement of intracellular ATP levels (Fig. 3.B).

Cell viability was not affected after a 24 hours exposure to DBP or DiBP, regardless of the exposure concentration (Fig. 3.A). As for DEHP, a significant decrease in cell viability was noted starting at the lowest concentration of 5 mg/L with 78.5 % of viability when compared to the control group (100 %). Concerning the mixture of the three phthalates, a decreasing trend in cell viability was observed starting at 5 mg/L, but nothing significant compared to the control condition. In parallel, we measured intracellular ATP levels (Fig. 3.B), as a fast and sensitive cell viability assay (Riss et al., 2004). A significant decrease in cellular ATP levels was noticed after exposure to DEHP starting at the lowest concentration of 5 mg/L. This significant decrease was also observed in BEAS-2B cells exposed to the different concentrations of the mixture of the three phthalates. For the same concentration, the cytotoxicity was significantly higher in DEHP condition compared to DBP and DiBP for the MTT assay. ATP levels was significantly lower in DEHP and the mixture condition compared to DBP at the same concentration. This suggests that DEHP induces more cytotoxicity in BEAS-2B cells than DBP and DiBP.

Based on these results, lower exposure concentrations (0.5 and 2.5 mg/L) of DEHP as well as of the mixture were tested (Figures S1.A and S1.B). A significant reduction of 16 % in cell viability and 21 % in ATP levels were observed in BEAS-2B exposed to 2.5 mg/L of DEHP compared to the control. Our results agree with other studies. A decrease in cell viability was noted after exposure of alveolar epithelial cell line A549 to DEHP at 5 mg/L for 24 hours (Shi et al., 2021) and starting at 3.9 mg/L for 48 hours (Wang et al., 2017). Other *in vitro* studies also highlighted a decrease in cell viability however with very high concentrations of DEHP of 19.5–97.6 mg/L in human colon cancer cell line HTC116 exposed for 24 hours (Amara et al., 2020) and of 48.8 mg/L to 6.2 g/L in 16HBE respiratory cells exposed for 48 hours (Ma et al., 2018).

### 3.3. Phthalates induce mitochondrial dysfunction

We further assessed whether exposure to phthalates identified in this study could impair the mitochondrial function of BEAS-2B cells.

Extracellular flux analysis was first conducted to measure the oxygen consumption rate (OCR) as well as the extracellular acidification rate (ECAR) of BEAS-2B. Seeding cell density in the XF24 culture microplate was optimized as a first step. Four concentrations of 1.5 x 10⁴, 2 x 10⁴, 2.5 x 10⁴ and 3 x 10⁴ cells/well were tested. Microscopic observations as well as oxygen consumption rate measurements led to the choice of 2 x 10⁴ cells/well as the optimum number of cells/well. Thereafter, BEAS-

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Effects of DEHP, DBP and DiBP on BEAS-2B viability. Cells were exposed for 24 h to various concentrations of each of the three phthalates and to the mixture of the three. Cell viability was measured using (A) MTT test and (B) intracellular ATP levels. Results were compared to the solvent control condition (0.5 % DMSO). Data represents mean ± SD, N = 4 for each condition, * = p < 0.05 compared to the control, # = p < 0.05 compared to DEHP at equivalent concentrations, $ = p < 0.05 compared to the mixture at equivalent concentrations.</figcaption></figure>
2B cells were exposed to the different phthalates and OCR and ECAR were measured under baseline as well as stressed conditions by the addition of FCCP, a protonophore that acts as an uncoupler allowing a proton efflux across the mitochondrial membrane, and oligomycin, an inhibitor of mitochondrial ATP synthase (Fig. 4).

Under baseline conditions, the respiration was decreased after exposure to DEHP or the mixture of three phthalates at the three concentrations tested compared to the control (0.5 % DMSO) (Fig. 4.A). DBP exposure did not alter basal respiration. As for DiBP, although no significant changes were noted in basal OCR after BEAS-2B exposure to this phthalate, a general increasing trend was apparent compared to control. In parallel to these alterations in OCR levels, a significant increase in extracellular acidification rate ECAR was noted for all the phthalates and for all the tested concentrations compared to control (Fig. 4.B) with the highest ECAR noted in BEAS-2B cells exposed to the mixture.

OCR and ECAR were also evaluated after injection of the mixture of stressors to induce a high energy demand. Stressed OCR values were significantly lower in BEAS-2B cells exposed to DEHP and to the mixture compared to control (Fig. 4.A). For a same concentration, the decrease in basal and stressed oxygen consumption was significant in DEHP and the mixture conditions compared to DBP and DiBP. As for stressed ECAR, a significant increase was detected in cells exposed to the mixtures (Fig. 4. B).

In order to determine whether phthalates influence BEAS-2B bioenergetic profiles, cellular phenotype was represented including four bioenergetic states (Fig. 4.C). Exposure to DEHP and to the mixture of the three phthalates altered the metabolic phenotype of BEAS-2B cells. We noted a shift in the profile towards a more glycolytic phenotype compared with cells exposed to the vehicle alone (0.5 % DMSO) and this starting at the lowest concentration of 2.5 mg/L (data not shown). As for DiBP exposure, the trend to increase in both OCR and ECAR revealed an increase in cellular metabolism. This led to a shift from the basal phenotype (0.5 % DMSO) to a more energetic one (Figure S.2.A). Exposure to the different concentrations of DBP did not alter the metabolic phenotype of BEAS-2B (Figure S.2.B).

To confirm the effects of phthalates on cellular energetics, intracellular adenosine phosphate levels were measured in response to

phthalate exposure. ATP/ADP ratio, an indicator of the energy status of the cell, showed a drastic decrease in DEHP- and mixture-treated cells. Moreover, for the same concentration, the ATP/ADP ratio was significantly lower in DEHP and the mixture compared to DBP and DiBP (Fig. 5.A). This could be explained by the decrease in ATP levels and the increase in ADP noted after exposure (Table S.5). Adenosines levels were also measured at lower exposure concentrations of DEHP and the mixture. A significant decrease in ATP/ADP ratio was observed starting 2.5 mg/L compared to control condition (Table S.6).

We therefore investigated extracellular lactate content in BEAS-2B exposed to the different phthalates alone or in a mixture (Fig. 5.B). A significant increase in lactate content compared to the control was noted for all the tested conditions and starting at the lowest exposure concentrations (Table S.6). Variations in extracellular lactate levels were also significant between DEHP and all the other conditions mainly at 10 mg/L.

To validate that phthalates-induced increase in lactate was glycolysis dependent, extracellular lactate was measured after a combined exposure to both phthalates and to 2-deoxy-D-glucose (2-DG), a competitive inhibitor of the glycolytic enzyme hexokinase. Phthalate-induced lactate accumulation in the extracellular matrix observed in Fig. 5.B was inhibited by the exposure to 2-DG. Similar lactate content to that found in the control group was noted (Fig. 5.C). These results indicate that lactate was produced via glycolysis from glucose.

A literature review shows that only few *in vitro* studies were conducted to evaluate the acute effects of phthalates on mitochondrial energy status despite its important role in cell function. It was reported that DEHP could decrease mitochondrial respiration (OCR) and increase extracellular acidification rate (ECAR) in endothelial cells HMEC-1 exposed to a concentration of 15 mg/L for 24 hours. A decrease in ATP/ADP ratio was noted after a 72 hours exposure (Poitou et al., 2022). To our knowledge, the effects of phthalates on respiratory cell mitochondria have not been evaluated.

The three phthalates selected based upon our identification in face masks samples seem to have different effects on BEAS-2B mitochondria. Our results highlighted a metabolic reprogramming towards a glycolytic phenotype after exposure to DEHP and to the mixture of three phthalates. It is well known that glycolytic turnover is associated with

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Cellular energetic of BEAS-2B exposed for 24 h to the three phthalates DEHP, DBP and DiBP alone or in a mixture measured by Seahorse analyzer: (A) basal and stressed oxygen consumption rate (OCR); (B) basal and stressed extracellular acidification rate (ECAR); (C) Cellular phenotype. Data represents mean ± SD, N = 4 for each condition, \* = p < 0.05 compared to the control 0.5 % DMSO, # = p < 0.05 compared to DEHP at equivalent concentrations, $ = p < 0.05 compared to the mixture at equivalent concentrations.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** (A) ATP/ADP ratio and (B) extracellular lactate level in BEAS-2B exposed for 24 h to the three phthalates DEHP, DBP and DiBP alone or in a mixture; (C) percentage of extracellular lactate content in the case of a combined exposure to 5 mg/L of the phthalate and 100 µM of 2-DG. Data represents mean ± SD, N = 4 for each condition, \* = p < 0.05 compared to the control 0.5 % DMSO, \# = p < 0.05 compared to DEHP at equivalent concentrations, $ = p < 0.05$ compared to the mixture at equivalent concentrations.</figcaption></figure>

extracellular medium acidification. During glycolysis, glucose is no
longer transformed preferentially into pyruvate but into lactate
explaining the high levels of lactate obtained in our results. However,
lactate anion is not the source of extracellular acidification. A rise in
lactate production is a byproduct of increased glycolytic turnover. A
correlation between changes in basal ECAR in several adherent cell
types and end-up measurement of extracellular lactate has been reported
in literature. Cell types included cancer cell models (Blouin et al., 2010),
myocytes (Teperino et al., 2012), immune cells (Everts et al., 2014) and
adipocytes (Keuper et al., 2014). These two parameters can qualitatively
track glycolytic conditions. During this oxidation reaction, ATP mole-
cules are hydrolyzed into ADP. This could explain the significant
decrease in ATP levels in BEAS-2B cells exposed to DEHP and to the
mixture and the tendency to increase in ADP. Hydrolysis of ATP is
acidifying causing the release of protons to the medium and therefore
possibly leading to an increase in ECAR. On the other hand, glucose
breakdown can also contribute to acidification. As a consequence of
breakdown of a glucose molecule, CO₂ is formed and can later undergo a
hydration reaction and dissociate into bicarbonate and a proton.
Therefore, respiratory CO₂ should be considered since it can contribute
to the rate of medium acidification and over-report glycolysis contri-
bution. It is important to assess whether mitochondrial derived CO₂
contributes to ECAR. Baseline OCR/ECAR ratio has been identified as a
marker of cell susceptibility to this CO₂ effect according to the manu-
facturer (Agilent Seahorse XF Cell Energy Phenotype Test Kit guide,
Agilent Technologies) (Divakaruni et al., 2014). This ratio was calcu-
lated in our study (Table S.7) and revealed that cells exposed to DEHP
and to the mixture present an OCR/ECAR ratio lower than 4 suggesting
that CO₂ makes a negligible contribution to ECAR. On the contrary,
control cells and those exposed to DBP and DiBP had a ratio that was
higher than 4 meaning that the stressed ECAR parameter could include
both mitochondrial respiration and glycolysis. Taking all together, these
results strengthen the hypothesis that the acidification noted after
BEAS-2B exposure to DEHP and to the mixture of the three phthalates is
attributable to a glycolytic turnover leading to the formation of lactate.
The decrease in ATP content in exposed BEAS-2B compared to control

cells could also suggest a metabolic alteration in the process of cellular
energy production mainly at the oxidative phosphorylation step high-
lighted by the reduced oxygen consumption. A measurement of mito-
chondrial ATP production could give us more insigne into the effects of
phthalates on the oxidative phosphorylation.

As for DBP, it appeared to have no effect on cellular energetics and
adenosine levels supporting its non-cytotoxicity to respiratory epithelial
cells. DiBP seems to have a different effect on BEAS-2B cells compared to
DEHP increasing their mitochondrial oxygen consumption and their
extracellular acidification rate. It seems that cells are shifted towards an
energetic phenotype where both oxidative phosphorylation and
glycolysis contribute to the cellular respiration.

Although each of the three phthalates tested did not affect the
mitochondria in the same way, they did alter cellular metabolism.
Among them, the effects of DEHP were the most pronounced, which is
why it is the main contributor to the effects observed with the mixture.

4. Conclusion

In this study, we were able to develop and validate a technique to
detect and quantify phthalates in a sampling of masks. The methodology
developed can be applied to the analysis of other matrices. We
confirmed that face masks contain phthalates in their composition, with
DEHP, DiBP and DBP representing the predominant components.
Thereafter, an *in vitro* approach was used to assess the effect of these
phthalates on mitochondrial function of bronchial epithelial cells BEAS-2B.

Taking together, our results demonstrated that phthalates can induce
mitochondrial dysfunction at the respiratory level, thereby confirming
the mitochondria as a target of phthalates. A possible pathway of
toxicity of these phthalates is through a phenotypic change towards a
more glycolytic one. Mitochondrial dysfunction associated with meta-
bolic shift in epithelial cells metabolism in favor of glycolysis has been
observed in lung pathologies. Consequently, further research is required
to gain a deeper understanding of the underlying mechanisms respon-
sible for this disruption in energy metabolism following phthalate
exposure. To our knowledge, this study is the first one to provide in-
formation on the adverse effects of phthalates present in a mixture on
mitochondria of respiratory epithelial cells. Finally, this study suggests
that the identity and the levels of phthalates should be evaluated in the
process of face masks production in order to reduce the health risk of
human exposure via inhalation.

Funding sources

Lyse Szymanski is funded by fellowships from the Normandie Doctoral School (EdNBISE). This work was financially supported by the University of Rouen Normandie.

CRediT authorship contribution statement

**Szymanski Lyse:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis. **Al Zallouha Margueritta:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Conceptualization. **Monteil Christelle:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Conceptualization. **Rogez-Florent Tiphaine:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Conceptualization. **Bouzar Clémence:** Methodology, Investigation. **Votier Edwige:** Methodology, Investigation.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2025.117910.

Data Availability

Data will be made available on request.

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