# Air cyclone samplers for assessing the toxicological potential of particulate matter: A case study in railway environments

Ambre Delater, Manon Plumail, Gaëlle Uzu, Laurent Meunier, Sébastien Fable, Maxime Floreani, Christelle Gamez, Takoua Mhadhbi, Jessica Queron, Olivier Le Bihan, et al.

► To cite this version:

Ambre Delater, Manon Plumail, Gaëlle Uzu, Laurent Meunier, Sébastien Fable, et al. Air cyclone samplers for assessing the toxicological potential of particulate matter: A case study in railway environments. Aerosol Science and Technology, 2025, pp.1-17. 10.1080/02786826.2025.2568698. ineris-05322712

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# Air cyclone samplers for assessing the toxicological potential of particulate matter: A case study in railway environments

Ambre Delater<sup>a,b</sup> ID, Manon Plumail<sup>a</sup>, Gaëlle Uzu<sup>c</sup> ID, Laurent Meunier<sup>a</sup>, Sébastien Fable<sup>a</sup>, Maxime Floreani<sup>a</sup>, Christelle Gamez<sup>a</sup>, Takoua Mhadhbi<sup>c</sup>, Jessica Queron<sup>a</sup>, Olivier Le Bihana ID, Isabelle Coll<sup>b</sup> ID, Brice Berthelot<sup>a</sup>, and Ghislaine Lacroix<sup>a</sup> ID

<sup>a</sup>INERIS, Parc Technologique ALATA, Verneuil-en-Halatte, France; <sup>b</sup>Univ Paris Est Creteil and Université Paris Cité, CNRS, LISA, Créteil, France; <sup>c</sup>Univ. Grenoble Alpes, CNRS, INRAE, IRD, Grenoble INP, IGE, Grenoble, France

## ABSTRACT

Evaluating the toxicological impact of atmospheric particulate pollution (aerosols) is challenging. Several parameters of sampling need to be controlled to provide reliable evaluations. Notably collecting sufficient sample mass and preserving sample properties from collection to testing (cells for *in vitro* tests, lung fluid for reactive oxygen species (ROS) activity) is key. Sampling on a filter is the most widely used approach, but it has a major limitation. The extraction of aerosols trapped in the filter still requires the use of solvent and/or sonication, that may modify the size and/or the composition of the sampled aerosols. Here we explore alternative methodologies with cyclone air samplers to minimize alterations to the properties of the sample by collecting aerosols in a cone. The results show that the use of sonication remains necessary with a dry cyclone (no liquid of collection), whereas the use of a liquid with a surfactant avoids this. The associated toxicological tests show that 10 min of sampling in a moving train is sufficient to discriminate stations based on aerosols-induced effects. Higher oxidative stress levels are observed in underground stations than in overground stations for the intracellular Reactive Oxygen Species (1.87 vs 1.31 exposed cells/control), dithiothreitol activity (0.12 vs 0.09 nmol min⁻¹) and ascorbic acid activity (0.52 vs 0.03 nmol min⁻¹). Cyclone sampling thus represents a promising method for collecting a sufficient mass of aerosols in a short duration and limiting the modification of their properties for direct toxicological evaluation.

## GRAPHICAL ABSTRACT

New sampling protocols to study aerosol toxicity.

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## ARTICLE HISTORY

Received 11 February 2025
Accepted 22 September 2025

## EDITOR

Akua Asa-Awuku

## 1. Introduction

Exposure to atmospheric particulate pollution can have a multidimensional impact on health such as effects on the cardiovascular system (e.g., increased

blood pressure, heart rate, death from stroke), development of psychological (e.g., depression), neurodegenerative (e.g., Alzheimer's and Parkinson's disease) or chronic illnesses (e.g., insulin resistance leading to the

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development of diabetes, increased risk of asthma from the prenatal period, increased risk of obesity), and lung cancer (Hamra et al. 2014; Liu et al. 2014; Heusinkveld et al. 2016; Buoli et al. 2018; Gładka et al. 2018; Hamanaka and Mutlu 2018; Hayes et al. 2020; Yang et al. 2020; Peden 2021; Lin et al. 2022; Yuan et al. 2022).

Toxicological testing can provide relevant information to evaluate the relationship between human exposure to aerosols and health effects. Toxicological experiments require direct contact between aerosols and biological models (e.g., cells, lung fluid), which is achieved through active sampling of the aerosols and studying biological endpoints (e.g., cell viability, oxidative stress, inflammation). The most widely used method for sampling aerosols is active collection on a filter using a pump (Raynor et al. 2011). The aerosols are subsequently extracted from the filter. This step implies using solvents (submerged filter) as well as spin-down and/or sonication (ultrasonic energy) to maximise the extraction of aerosols trapped in the filter (Bein and Wexler 2015). However, all these methods do not allow for preserving the properties of the aerosols. Solvents can modify the aerosols' induced effects, such as their Oxidative Potential (OP) (Calas et al. 2018; Pietrogrande et al. 2019). Sonication at high energy (e.g., direct sonication) can generate free-radical species that can react biologically *in vitro* (Miljevic et al. 2014). Moreover, it can decrease the size of aerosols (due to their disaggregation), which may alter the dose delivered to the cells, as the small particles are less likely to settle (Schneider et al. 2020). The extraction of the aerosols is also non-quantitative and depends on the extracted species (metals, carbons, Polycyclic Aromatic Hydrocarbons -PAH-) and the extraction methods (solvent, ultrasound, spin-down, mix of these methods) (Roper et al. 2019). All these issues can lead to biased effects, such as studying only a part of the biological induced effects by the sample (in concentrations and/or species) and the inability to compare studies since there is no reference protocol for extraction (Goulaouic 2009; Bein and Wexler 2014; Roper et al. 2015; Van Winkle et al. 2015; Kaur et al. 2019; Hadei et al. 2020; Kumar et al. 2021).

The nature of the collection site (environment) can also contribute to the lack of toxicological data regarding airborne aerosols. For example, the railway environments are of great interest, since high concentrations of particles have been measured there (Mugica-Álvarez et al. 2012; Minguillón et al. 2018; Smith et al. 2020), but the induced effects of these particles are still too insufficiently documented to

determine their impacts on health (Loxham and Nieuwenhuijsen 2019; Anses 2022). This may be because collecting enough mass of particulate matter in bulk for toxicological studies can be challenging, especially in some microenvironments. In some cases, sampling must be carried out without access to electricity (e.g., on a curb), in small spaces (e.g., in a cabin car) and/or in motion (e.g., on a moving train). These constraints require the use of a portable sampler. In addition, certain microenvironments necessitate sampling over a short period of time (e.g., in a train passing through railway stations). Therefore, it is also recommended for such approach to use a high-volume sampler (at least 16 L min⁻¹) to collect a sufficient mass of particles for toxicological assays (Bachoual et al. 2007; Rivas et al. 2014; Tavera Busso et al. 2020; Kumar et al. 2021).

Cyclones can meet all these requirements. The cyclone collection imparts a circular motion to the entering air, moving particles toward the walls of a collection cone, then to the bottom of the cone (Kulkarni et al. 2011; Watson and Chow 2011). As a result, particles are not impacted within any collection medium. Furthermore, a wide range of cyclone models exist, with different sizes and flow rates, and which can be battery-operated. Cyclones have already been deployed in numerous studies to collect aerosols in several types of environments (e.g., railway platform, tunnel, household) and study their toxicological impacts (Walters et al. 2002; Wang et al. 2010; Sussan et al. 2014; Tirkkonen et al. 2016; Miyashita et al. 2022).

However, the smallest (and thus portable) cyclones have a flow rate too low (around 3 L/min) to collect particles over a short period of time, while some high-volume cyclones can suffer other limits (power supply availability, large-volume pumps that cannot be housed in certain microenvironments). Designing and manufacturing a cyclone, even if it is a prototype for research, is extremely expensive and time-consuming and requires accurate skills. Studies do not necessarily highlight the commercial references of the cyclone system used, nor do they describe the protocol for recovering particles in the cones (e.g., scraping, suspension in a liquid) despite the fact that these steps can modify their properties. Similarly, when a physicochemical characterization of samples is performed, the protocol for separating and preparing the sample is not always described. For all these reasons, a new evaluation of high-volume and portable cyclones, with different collection media (dry or liquid), would complement the literature to study the induced effects of aerosols in microenvironments.
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The main issue of cyclones is that they generally have a rather high cutoff of 1 µm upwards, which means that they do not fully sample the finest fraction of particle size (Marple and Olson 2011). The sampling efficiency of particles depends on the cyclone design (e.g., air inlet shape) and the sampling settings (e.g., flow rate, sampling duration) (Moore and McFarland 1993; Karagoz et al. 2013; Ma et al. 2020). Thus, the collection of the fine fraction can be improved by studying these parameters. For illustration, Nishita-Hara et al. (2024) and Rule et al. (2010) showed that by modifying the design of a cyclone, a 0.2 and 0.1 µm cutoff diameter could be achieved respectively.

In this context, our study aimed to evaluate mid-volumes and portable cyclones with different collection media, as a complement to the existing studies, to investigate the induced effects of aerosols in micro-environments like railway environments. The improvement of their sampling efficiency will then be investigated in another study. The evaluation of the cyclone was focused on two parameters: (1) the recovery of particles in a cone followed by their separation for toxicological tests and chemical analyses with minimal alteration of sample properties (without or with limited use of sonication, solvent or spin-down), and (2) the reproducibility of the results of the toxicological tests. The evaluation was made with different collection media, either in a dry cone or in a cone with liquid, through 5 toxicological tests (3 *in vitro* assays and 2 acellular assays derived from oxidative potential tests that were not mass-normalized).

# 2. Materials and methods

## 2.1. Cyclone air samplers

The two selected devices are both commercially available cyclones, a liquid cyclone (namely Coriolis µ) and a dry cyclone (namely Coriolis Compact) from Bertin Technologies (Montigny-le-Bretonneux, France). The cyclones measure 25.5 × 13.5 × 13 and 22 × 33 × 36 cm³ (Length × Width × Height) and weigh 1.2 and 2 kg for the dry and liquid cyclones respectively, allowing them to be positioned in small spaces. Both cyclones sample particles (aerosols) in a plastic cone, which can be capped immediately after sampling to protect from contamination. Their cutoff diameter is 500 nm. The liquid cyclone collects the particles in a volume of liquid of 10–15 mL. The autonomous sampling duration is 8 h for the dry cyclone. The liquid cyclone was designed by the manufacturer to work at a maximum of 10 min duration in “stand-alone mode” regardless of the flow duration. This mode is intended to avoid excessive liquid

evaporation at the end of the collection. This duration can be extended to 6 h by adding liquid with a peristaltic pump, called the Long Time Monitoring (“LTM mode,” Bertin Instruments). The LTM refills the sampling cone every minute to compensate for liquid evaporation. The liquid cyclone has a flow rate of 100–300 L min⁻¹ in 50 L min⁻¹ steps. The dry cyclone has a single flow rate of 50 L min⁻¹.

## 2.2. Railway environments

Two railway environments with different typologies were studied. During the first field campaign, both cyclones were installed in an underground railway station. During the second field campaign, only the liquid cyclone was implemented in a moving train, a passenger car, as the dry cyclone encountered a technical problem and could not be used for this campaign.

Samples were collected on weekdays in Paris region (France) for both campaigns. Samples were collected in the underground railway station in March 2020, between the 4th and 13th and in the passenger car in September 2020, between the 15th and 24th.

The underground railway station has no mechanical ventilation. Daily traffic starts at around 4:00 am and stops at around midnight. On one sampling day, traffic was interrupted between 10:00 am and 16:00. During this interruption, only work trains ran through the station. In this station, we investigated the influence of the time of collection on the toxicological profile of the samples collected. We also compared normal traffic situations and the passage of work trains through the station. Cyclones were installed less than 3 m from the platform, one next to the other (around 1 m), in a position where a passenger could wait for a train. Both cyclones were positioned as much as possible at the same height, on a tripod at 80 cm and on a trolley at 50 cm from the ground, respectively for the liquid and dry cyclone.

The passenger car has no mechanical ventilation. In this train, we compared the effects induced by aerosols collected when the rolling stock passed through overground stations with those collected 20 min later when the train passed through underground stations only. The liquid cyclone was installed on a passenger seat.

## 2.3. Strategy of sampling and sample preparation

As mentioned in the introduction, the aim was to determine whether samples can be recovered from
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cones and split (to perform several analyses) with
minimal alteration of the properties of the sample for
a liquid and dry collection. To this end, we identified
two main targets: (1) to collect a sufficient mass of
particles and (2) to put in suspension the sample in
the liquid used for the toxicological tests. For the first
target, the required mass is considered to be at least
1 mg according to the literature (Kumar et al. 2021).
For the second target, we aimed to perform cellular
(in vitro) and acellular assays (Ascorbic Acid (AA)
and Dithiothreitol (DTT) activities). The liquid com-
monly used during the cellular assays is a culture
medium, RPMI 1640 1x (Gibco) with 1% (v/v) of
Fetal Bovine Serum -FBS- (Gibco), 1% (v/v) of antibi-
otics (Penicillin/Streptomycin) and 1% (v/v) of anti-
fungal agent (Amphotericin B) to prevent bacteria or
fungi contaminating samples during sampling. For the
acellular assays, the liquid is a lung fluid surrogate
solution, a Gamble's solution.

To reach the targets, we tested 4 protocols (sam-
pling parameters and collection media) for setting up
the cyclones. Protocols were named based on the col-
lection media used: Culture medium, Water, Water-
surfactant and Dry collection. The Culture medium,
Water and Dry collection were carried out during the
first campaign in the railway stations. The results led
to the development of a new protocol, the Water-
surfactant collection, which was carried out during the
second campaign in the passenger car. The protocols
are described below.

We were unable to determine the mass of the par-
ticles collected in each cone (given the small mass of
particles compared to the mass of the cone).
Therefore, this study cannot be used to assess a dose-
response relationship. However, the air volume col-
lected is known, which makes it possible to assess the
air volume/response of an environment. Hence, we
propose here to compare the results of samples col-
lected in the same conditions (air volume sampled,
sampling parameters, chosen cyclones) between them,
to determine the environments with the highest toxi-
cological response, regardless of mass.

**2.3.1. Dry collection (dry cyclone) in the under-
ground railway station**

2.3.1.1. For mass collection. We operated the cyclone at its single flow rate, 50 L min⁻¹. The mass concentration of PM₁₀ (particles with an aerodynamic diameter of less than 10 µm) averages between 140 and 480 µg/ m³ during the day in about 2/3 of railway platforms and underground stations in Paris region (France) (Airparif 2024). Thus, to achieve the collection of

around 1 mg in the station, we operated the dry cyclone for 2 and 4 h. Collecting samples for 4 h secures the recovery of a sufficient mass, while collecting samples for 2 h provides a larger number of samples to characterize the induced effects of particles over a day.

**2.3.1.2. For toxicological testing.** Dividing a dry sample from the cone may be non-quantitative, as aerosols adhere to the cone. To avoid scraping the walls of the cone, we chose to suspend the sample in culture medium with surfactant (FBS) before splitting it to perform the various tests. This helps to recover particles, but a layer of particles was still visible on the cone walls. Therefore, we placed cones in an ultrasonic bath (37 kHz, FB150055, Fisher Scientific) for 2 min to detach these particles. As mentioned in the introduction, sonication can lead to changes in sample properties. To minimize/avoid this effect, we used an ultrasonic bath at:

* a lower duration than Miljevic et al. (2014), who found a generation of radical species in the culture medium after 15 minutes of sonication with an ultrasonic bath of the same energy as the one used in our study;

* with less energy than Schneider et al. (2020), who found that high-energy ultrasound (8 J/s) reduced aerosol size and prevented them from settling on the cells.

Finally, we divided the samples with a graduated pipette in Falcon™ tubes. To perform the acellular assay, we diluted samples suspended in the culture medium in Gamble's solution.

**2.3.2. Culture medium collection (liquid cyclone) in**
**the underground railway station**

**2.3.2.1. For mass collection.** When the flow rate increases, it can lead to losses of particles from the liquid collection, as already observed for a liquid impinger (Su et al. 2020). For this reason, the liquid cyclone was operated at its lowest flow rate (100 L.min⁻¹). The sampling duration was set at 120 min (2 h) according to public air pollution data (see Section 2.3.1) to collect at least 1 mg (Airparif 2024).

**2.3.2.2. For toxicological testing.** The strategy was to sample aerosols directly in the liquid for *in vitro* assays. During the sampling, we hypothesized that some compounds may undergo preferential evaporation, favoring the concentration and precipitation of inorganic salts included in the culture medium. To
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avoid this phenomenon, we compensated for evaporation with a diluted culture medium (1:10 in water) during the sampling. Then, we froze the samples at -80 °C before the tests. To prevent particle agglomeration after freezing, as observed in the literature, we placed the cones in an ultrasonic bath for 1 min (Marvanová et al. 2018). Then, we vortex mixed them for 30 s. Considering that high sonication or solvent was not required to recover the aerosols, we assume that the sample alteration is minimize. Finally, we divided the sample into FalconTM tubes. To perform the acellular assay, we diluted samples collected in the culture medium in Gamble's solution.

### 2.3.3. Water collection (liquid cyclone) in the underground railway station

The Water collection method was set up as an alternative to the Culture medium collection. The aim was to carry out acellular tests and chemical analysis (see Section 2.6) in water.

**2.3.3.1. For mass collection.** Similarly, as with the Culture medium collection, we operated the liquid cyclone at 100 L min⁻¹ for 2 h.

**2.3.3.2. For toxicological testing.** We sampled aerosols only in water and compensated for evaporation by refilling with fresh water. Then, we wanted to vortex mix the samples in cones, prior to diluting them in Gamble's solution for acellular tests. However (see Section 3.1 for results), particles adhered to the walls of the cones during the collection at the vortex mixing phase.

### 2.3.4. Water-surfactant collection (liquid cyclone) in the passenger car

The Water-surfactant collection was developed following the results of the field campaign 1, presented in Section 3.1.

**2.3.4.1. For mass collection.** We tested a sampling time of 10 min to evaluate the effects of particles in a short period (e.g., here in a train passing through stations), even though the collection of a mass of 1 mg might not be achieved. To collect a large amount of mass, the cyclone was operated at its highest flow rate, 300 L min⁻¹.

**2.3.4.2. For toxicological testing.** We selected Milli-Q water supplemented with 0.001% of surfactants (Tween®20, P2287, Sigma-Aldrich) as the liquid of collection based on the results of the first campaign (see Section 3.1). Prior to the field campaign, we characterized the cytotoxicity of the surfactant on cells at different concentrations. Results showed that the 0.001% concentration had no significant impact on cellular metabolic activity (CMA) and LDH release from cells, i.e., 95 ± 9% of CMA and 0.3 ± 6% of LDH release. Similar to the Culture medium, after freezing at -80 °C, we placed the cones in an ultrasonic bath for 1 min, then vortex mixed them for 30 s. To make the samples comparable with each other, we adjusted them to the same volume of liquid, 6 mL with Milli Q water. This avoids any induced effects on cells being attributable to volume rather than sample concentration (smaller sample volumes may be more concentrated than larger ones). Then, we divided the samples into FalconTM tubes and diluted one fraction in culture medium for the *in vitro* tests and one fraction in Gamble's solution for acellular tests. To minimize sample dilution, we chose concentrated culture medium (RPMI 10x, Sigma). We diluted the samples at 9:10 in culture medium. Before testing, we added additives (baking soda, L-Glutamine, folic acid) to the RPMI 10x solution. Finally, we added FBS at a final concentration of 10%, which does not alter the sample dilution, since the volume of FBS is negligible in comparison to the total volume.

### 2.3.5. Summary of the four collection media tested

A description of the parameters of the 4 collection media and their deployment is summed up in Table 1.

<table><caption>**Table 1.** Types of cyclones, collection medium tested and conditions applied for sampling aerosols in different environments.</caption><thead><tr><th>Names</th><th>Cyclones</th><th>Environments investigated</th><th>Samples</th><th>Sampling duration</th><th>Flow rate</th><th>Media collection</th></tr></thead><tbody><tr><td>Culture medium</td><td>Liquid</td><td>- Passage of work trains versus normal traffic<br/>- Normal traffic depending on time</td><td>n = 6</td><td>2 hours</td><td>100 L min<sup>-1</sup></td><td>Culture medium<br/>RPMI 1x with 1% of FBS, antibiotics and antifungal agent, filled with 9:10 of distilled water and 1:10 of RPMI 1x</td></tr><tr><td>Water</td><td>Liquid</td><td></td><td>n = 1<br/>n = 3</td><td>2 hours<br/>4 hours</td><td>100 L min<sup>-1</sup></td><td>10 mL of water, filled with water</td></tr><tr><td>Dry</td><td>Dry</td><td></td><td>n = 6<br/>n = 3</td><td>2 hours<br/>4 hours</td><td>50 L min<sup>-1</sup></td><td>Dry sampling in a cone</td></tr><tr><td>Water-surfactant</td><td>Liquid</td><td>- Overground versus underground stations</td><td>n = 2</td><td>10 minutes</td><td>300 L min<sup>-1</sup></td><td>Water with 0.001% of surfactants, filled with water</td></tr></tbody></table>
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## 2.4. Elemental analysis

We analyzed samples of the Dry and Water-surfactant collection to determine the elemental composition of the samples. Samples of the Dry collection are suspended in culture medium for cells (see Section 2.3.1) and samples of the Water-surfactant collection are suspended in water with 0.001% of Tween®20 (see Section 2.3.4). We also analyzed a blank of the culture medium to account for potential background contamination.

We performed semi-quantitative screening analysis. We used Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) (Agilent, 5110 model) after mineralization of the samples by an acid digestion (HNO₃; H₂O₂) and according to standard NF EN 14385 (2004). The semi-quantitative screening method enables rapid identification of 67 elements. As the sample volumes available differed from one sample to another (depending on the tests carried out on them), the dilution required for performing the analysis also differed between samples. After screening, 4 elements were found in some but not all samples: barium (present in 21% of samples), chromium (present in 3% of samples), copper (present in 39% of samples) and titanium (present in 11% of samples). To ensure that these results were not due to differences caused by sample dilutions, we analyzed these 4 elements using ICP-MS (Mass Spectrometry), a quantitative method which enabled reducing the quantification limit by a factor of 10.

## 2.5. In vitro assays

### 2.5.1. Preparation of the co-culture

In vitro tests were carried out using a co-culture of human cells (A549 cell line, type pneumocyte II, ATCC CCL-185TM) and macrophages (PMA-differentiated THP-1 cell line, ATCC TIB-202TM). A549 cells were seeded in a 96-well plate (25,600 cells/well). The same day, THP-1 cells were differentiated by adding phorbol myristate acetate (PMA, P1585, Sigma) at a final concentration of 30 ng/mL. After 24 h of incubation at 37°C and 5% CO₂ in culture medium (RPMI 1640 1x (Gibco)), THP-1 cells were added on top of A549 cells at a ratio of 10 A549 cells to 1 THP-1 macrophage. Then, the co-culture model was incubated for another 24 h before exposure to the collected samples.

Samples at different dilutions were deposited on co-cultures in triplicate. To maximize the mass of particles that would sediment upon contact with the cells, we chose to deposit a large volume (200 µL) of suspension into the wells of the 96-well plates. The exposure duration was 24 h at 37°C with 5% CO₂.

The dilutions in the culture medium were: 1:1 (no dilution), 1:2 and 1:10 dilutions (sample volume/total volume). Three *in vitro* tests were then performed to assess the cytotoxicity, the activity of the intracellular Reactive Oxygen Species (ROS) and the inflammation.

### 2.5.2. Cytotoxicity

Cytotoxicity was assessed using two parameters, the cellular metabolic activity (CMA), and the cell integrity.

CMA was determined using a fluorogenic probe (Prestoblue®, Invitrogen, ref. A13262). This probe passively penetrates cells in its non-fluorescent form and is reduced to a soluble fluorescent product after metabolization by living cells. After 1 h incubation with Prestoblue®, fluorescence intensity is then measured using a plate-reading spectrophotometer (TECAN infinite® 200). The fluorescence is proportional to the number of living cells. Results are expressed as a ratio of exposed to unexposed cells (the control is at 100% viability). The control consists of cells exposed only to culture medium (RPMI 1640 1x Gibco) with 1% (v/v) of Fetal Bovine Serum (FBS) (Gibco).

The supernatant from each well is then transferred to the well of a new plate to determine the cell integrity by measurement of lactate dehydrogenase (LDH) release in cell culture medium. Measurement of the enzyme LDH provides an indication of cell integrity since it is released by cells into the culture medium in the event of cell membrane damage. LDH release was measured using a commercial kit (Cytotox One, Promega) by measuring the fluorescence intensity using a plate-reading spectrophotometer (TECAN infinite® 200). Results are expressed as a percentage of cytotoxicity calculated against the maximum LDH release obtained in cells lysed by triton 10%.

Based on cytotoxicity results, we selected sample dilution factors for ROS and inflammation measurements, which are sublethal effects. These tests must therefore be carried out in living cells (i.e., with no significant cytotoxicity). The dilution used is the one that allows cell viability to exceed 90%.

### 2.5.3. Intracellular ROS measurement

ROS detection was done using a cell-permeable probe, 2, 7-dichlorofluorescein-diacetate (DCFH-DA, Sigma-Aldrich). It was brought into contact with the cells for 45 min. The cells were then exposed to the samples for 15 min to 24 h. If ROS are produced, the DCFH-DA probe is broken down into DCF, a fluorescent compound that can be quantified. By measuring this
<figure><img src="image_13.png" /></figure>

fluorescent molecule, ROS production can be determined. Results are expressed as a ratio of fluorescence between exposed and unexposed cells (the control is at a ratio of 1 of ROS production). ROS production by cells was measured at different times, corresponding to 6 exposure durations, to evaluate ROS production as a function of time. This experiment was done 3 times (i.e., on 3 different weeks) to study the reproducibility. A positive control, TBHP (tert-Butyl hydroperoxide, 458139, Sigma-Aldrich) was used.

### 2.5.4. Pro-inflammatory responses

Inflammation was assessed with an ELISA kit by quantifying the 4 pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 in the cell culture medium of the cells after 24h of exposure to the collected samples (V-Plex Proinflammatory panel, human, MSD Meso Scale Discovery). These 4 cytokines can be released by the co-culture in the event of inflammation. To compare the pro-inflammatory responses induced by samples, the results are expressed as a ratio of exposed cells compared to unexposed cells (the control is at a ratio of 1). This experiment was done 3 times (i.e., on 3 different weeks) to study the reproducibility. A positive control, LPS (lipopolysaccharide, L2880, Sigma), was used.

The level of endotoxins was also determined in samples to interpret the results, as they can induce strong pro-inflammatory responses. Endotoxin concentrations were evaluated using a kit (Pierce LAL chromogenic endotoxin quantitation kit, Thermofischer).

## 2.6. Acellular tests: AA and DTT loss into a lining fluid

The study quantified the consumption rate of DTT in a cell-free system containing particles collected from aerosols to assess the oxidative potential. We used DTT and AA activities instead of mass-normalized activities, because the cyclone sampling did not allow obtaining the mass of the samples.

The measurement of DTT consumption rate, which is directly proportional to the concentration of redox-active compounds in particulate matter (PM) samples, is observed under standardized conditions. The corresponding redox activity is expressed in nanomoles per

minute (nmol min⁻¹), corresponding to the amount of DTT consumed per unit of time. DTT loss is obtained after titration by DTNB (5,5'-Dithiobis 2-Nitrobenzoic Acid) and by monitoring TNB (1,3,5-Trinitrobenzene) formation at 412 nmol at 0, 10, 20 and 30 min. The AA loss is directly monitored at 285 nm every 4 min over 30 min.

## 2.7. Statistical analysis

Statistical analysis was performed using R code.

To evaluate the reproducibility of the toxicological tests, we focused here on the results of the intracellular ROS measurements, as this test showed the greatest variability. We calculated a coefficient of variation (CV) for each sample (as mentioned in Section 2.4.3, the test was performed three times per sample). The CV is the ratio of the standard deviation to the mean, expressed as a percentage. Since the intracellular ROS test was conducted at 6 different exposure durations of samples on cells, 6 CV were thus calculated per sample.

To compare cells exposed to samples versus control cells (Section 3.3), normality was first verified by a Shapiro-Wilk test. Then, a one-tailed t-test or a Wilcoxon test was performed for data following or not a normal distribution respectively, using a 95% confidence interval. Differences were considered statistically significant when p-values were less than 0.05. The quality of the p-values is expressed with *, as follows: * $p < 0.05$, ** $p < 0.01$, *** $p < 0.001$.

# 3. Results

## 3.1. Evaluation of the 4 protocols

Table 2 summarizes the results of the evaluation of the 4 protocols. For each protocol, the method to recover the particles from the cone is given, as well as the number of samples. Also, a reproducibility indicator of the intracellular ROS measurements test (see Section 2.7) is provided, which is the mean and standard deviation of all the CV.

We have recovered the particles from the cones of the Culture medium collection only by vortex mixing the cones. Nevertheless, the sample reproducibility is poor, since the mean CV is close to 50%. The CV

<table><caption>**Table 2.** Summary of the evaluation of the 4 protocols.</caption><thead><tr><th>Name</th><th>Cyclones</th><th>Number of samples</th><th>Recovery of particles</th><th>Reproducibility indicator</th><th>Status</th></tr></thead><tbody><tr><td>Culture medium</td><td>Liquid</td><td>6</td><td>Vortex mixing and low-energy sonication if frozen</td><td>49.5 ± 14.7%</td><td>Discarded</td></tr><tr><td>Water</td><td>Liquid</td><td>Not analyzed</td><td>Scraping and high sonication</td><td>Not analyzed</td><td>Discarded</td></tr><tr><td>Dry</td><td>Dry</td><td>9</td><td>Suspension in culture medium and vortex</td><td>7.3 ± 3.1%</td><td>Retained</td></tr><tr><td>Water-surfactant</td><td>Liquid</td><td>2</td><td>Vortex mixing and low-energy sonication if frozen</td><td>9.7 ± 4.2%</td><td>Retained</td></tr></tbody></table>
<figure><img src="image_14.png" /></figure>

ranges from 13.5% to 73.5%. This means there was variability between intracellular ROS measurements (for the same sample and at the same exposure time on the cells). The lack of reproducibility of the results with the culture medium suggests potential medium alteration using this medium, influencing ROS activity. This could be due to a change in the concentration of media constituents due to uncontrolled evaporation. A change in the culture medium would cause the cells to have insufficient nutrients to survive, which would alter the test. At this stage, we do not know which compounds in the culture medium have been modified and how they can be restored. Consequently, the Culture medium collection was discarded for the remainder of the study. It does not allow for conducting *in vitro* assays with good reproducibility.

For the **Water collection**, a layer of particles was observed on the cone's wall, and we failed to recover them only with vortex mixing. Only aggressive methods for aerosol properties, such as high sonication (5451 J with Q700 Cup Horns, QSonica) and scraping, were useful for taking them off. Thus, the Water collection was discarded. In comparison, the **Culture medium collection** (image on the right in Figure 1) enables the recovery of aerosols in the liquid of cones (on Figure 1, culture medium is pink, visible aerosols are black). In the Culture medium collection, we used FBS at a concentration of 1%. As FBS contains surfactants, we can

assume that they help prevent aerosols from sticking to the walls.

Based on the observations of these 2 protocols evaluated in an underground railway station, we carried out a new protocol with surfactant in a passenger car, the **Water-surfactant collection**. The results showed that the addition of surfactant (Tween®20) at a concentration of 0.001% was sufficient to prevent particles from sticking to the walls in visible layers. Considering the role of surfactants, ultrasound is not necessary to detach particles from the walls. The CV of the intracellular ROS measurements show a good reproducibility of the test, since the average is around 10%, with values ranging from 4.9% to 18.4%. Thus, the **Water-surfactant collection** was retained to study the effects induced by aerosols present in a passenger car.

We have recovered the samples from the cone of **Dry collection** just by vortex mixing and a few minutes of sonication (see Section 2.3.1). The CV of the intracellular ROS measurements show a good reproducibility of the test, since the average is less than 10%, with values ranging from 1.9% to 15.9%. Hence, the **Dry collection** was retained for the toxicological assays.

Therefore, in the following Sections 3.2. and 3.3, we present the results using the 2 retained protocols: the Dry collection, which was set up in an underground railway station and the Water-surfactant collection, which was set up in a passenger car. Data

<figure><img src="image_15.png" /><figcaption>**Figure 1.** Pictures of cones of liquid cyclone. Water collection: during the scraping (scraper in yellow) of a layer of particles on the walls of the cone. Culture medium collection: after the sonication in the ultrasonic bath.</figcaption></figure>
<figure><img src="image_16.png" /></figure>

are summarized in Table S1 (see the online supplementary information (SI)).

## 3.2. Results of elemental analysis

Table 3 presents the concentration of the elements (in µg/mL) analyzed in samples of the Dry collection in the underground railway station. The analysis showed that the concentrations of Ca, K, Mg, Na, P, S, and Si in samples are close to those of the blank (see Section 2.4), which proves that they come from the culture medium and FBS. In addition, there is a major contribution of Fe in the samples. It is the most concentrated element in all samples, with concentrations ranging from 21 to 26 µg/mL.

Table 4 presents the concentration of the elements (in µg/mL) analyzed in both samples collected in a passenger car with the Water-surfactant collection. Summing up the concentrations of each element, the most concentrated sample was the one collected during the passage of trains through underground stations, with 91.95 µg/mL versus 18.54 µg/mL for the one collected during passage through overground stations.

## 3.3. Results of in vitro assays and acellular tests

### 3.3.1. In an underground railway station (Dry collection)

Figure 2 shows the results of the toxicological tests as a function of the sampling method and of the type of trains passing through the station.

The values of the cellular metabolic activity (CMA) (graph at the top of Figure 2) for each undiluted sample tested (ranging from 68% to 82%) are very close

to the CMA of 1:2 diluted samples, except for the single sample collected during the passage of work trains through the station. At 1:10 dilution, CMA is above 92% for all samples. Hence, these results reveal a nonlinear effect of dilutions on CMA. It is interesting to note that there is no impact of the sampling time on the CMA results. For the same dilution (1:1, 1:2, or 1:10), similar effects are observed between samples collected at different times. Importantly, the undiluted samples collected during the passage of the work trains had a lower CMA than the others (51% versus at least 68% for other samples), which suggests a potential increased biological impact for working trains compared to normal trains. Although we have no repetition of this experiment, these results are interesting and deserve further investigation.

The results of LDH release (data not shown) indicate that there is no significant LDH release in all samples and for the 3 dilutions tested, meaning there are no cytotoxic effects induced by the exposure to the 6 samples. These results converge with those of another article, where no significant LDH release was observed in two underground railway stations in France too (Bachoual et al. 2007). Therefore, particles in railway environments may not significantly alter the physical integrity of the cells but rather impact their metabolic activity.

As the 1:10 dilution of samples induced the lowest mortality and no LDH release, we selected this dilution to perform the other *in vitro* tests (cell ROS activity and pro-inflammatory response), as these tests require low-cytotoxic conditions for functioning properly.

For all samples, the intracellular ROS production factor (graph in the middle of Figure 2) increases as a

<table><caption>**Table 3.** Concentration of the elements (µg/mL) found in air samples, by semi-quantitative and quantitative methods. The blank is made of culture medium RPMI 1x with 1% FBS.</caption><thead><tr><th rowspan="2">Sampling hours</th><th colspan="8">Semi-quantitative concentration (µg/mL)</th><th colspan="4">Quantitative concentration (µg/mL)</th><th rowspan="2">Total without blank elements</th></tr><tr><th>Al</th><th>Ca</th><th>Fe</th><th>K</th><th>Mg</th><th>Na</th><th>P</th><th>S</th><th>Si</th><th>Ba</th><th>Cr</th><th>Cu</th><th>Ti</th></tr></thead><tbody><tr><td>Blank</td><td>&lt;LQ</td><td>20.75</td><td>&lt;LQ</td><td>15.61</td><td>10.24</td><td>1076</td><td>157</td><td>76.08</td><td>3.56</td><td>&lt;LQ</td><td>&lt;LQ</td><td>&lt;LQ</td><td>&lt;LQ</td><td>–</td></tr><tr><td>09:22–11:23*</td><td>0.84</td><td>27.15</td><td>12.48</td><td>18.18</td><td>12.54</td><td>1202</td><td>171</td><td>84.93</td><td>5.85</td><td>0.21</td><td>0.02</td><td>0.65</td><td>0.05</td><td>14.25</td></tr><tr><td>07:32–09:32</td><td>1.94</td><td>23.63</td><td>25.58</td><td>16.03</td><td>13.56</td><td>1164</td><td>164</td><td>83.57</td><td>4.92</td><td>0.85</td><td>0.09</td><td>1.38</td><td>0.13</td><td>29.98</td></tr><tr><td>09:37–11:37</td><td>1.44</td><td>23.77</td><td>21.10</td><td>15.76</td><td>12.71</td><td>1136</td><td>162</td><td>83.30</td><td>7.45</td><td>0.54</td><td>0.08</td><td>1.24</td><td>0.09</td><td>24.49</td></tr><tr><td>09:46–11:47</td><td>1.53</td><td>23.02</td><td>26.36</td><td>16.86</td><td>12.56</td><td>1118</td><td>158</td><td>80.15</td><td>5.41</td><td>0.63</td><td>0.09</td><td>1.50</td><td>0.04</td><td>30.15</td></tr><tr><td>13:57–15:57</td><td>1.46</td><td>23.80</td><td>23.70</td><td>16.79</td><td>13.39</td><td>1162</td><td>166</td><td>85.71</td><td>4.75</td><td>0.53</td><td>0.09</td><td>1.42</td><td>0.04</td><td>27.24</td></tr><tr><td>16:06–18:05</td><td>0.37</td><td>22.96</td><td>25.74</td><td>16.10</td><td>13.10</td><td>1115</td><td>155</td><td>77.89</td><td>3.93</td><td>0.74</td><td>0.02</td><td>2.10</td><td>0.04</td><td>29.01</td></tr></tbody></table>

The sample with * has been collected during the passage of work trains.

<table><caption>**Table 4.** Concentration of the element (µg/mL) analyzed in samples, by a semi-quantitative and a quantitative method.</caption><thead><tr><th rowspan="2">Sampling hours</th><th colspan="5">Semi-quantitative concentration (µg/mL)</th><th colspan="4">Quantitative concentration (µg/mL)</th><th rowspan="2">Total</th></tr><tr><th>Ca</th><th>Fe</th><th>K</th><th>Na</th><th>S</th><th>Si</th><th>Ba</th><th>Cr</th><th>Cu</th><th>Ti</th></tr></thead><tbody><tr><td>Overground stations</td><td>0.93</td><td>0.93</td><td>6.58</td><td>6.69</td><td>2.09</td><td>0.93</td><td>0.27</td><td>0.03</td><td>0.06</td><td>0.03</td><td>18.54</td></tr><tr><td>Underground stations</td><td>0.93</td><td>16.98</td><td>30.00</td><td>29.28</td><td>10.69</td><td>3.23</td><td>0.61</td><td>0.12</td><td>0.12</td><td>0.04</td><td>91.95</td></tr></tbody></table>
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**Figure 2.** Results of the toxicological tests on samples collected using the Dry collection and depending on the sampling time. The samples were collected during the passage of passenger or work trains at the stations. Top: the CMA at 3 dilutions of samples before the deposition on cells. The dashed line represents the control signal. Middle: the mean intracellular ROS production after several exposure durations of cells for samples diluted at 1:10. The dashed line represents the control signal. The stars \* illustrate a significant difference between the results and the control. Bottom: results of mean AA and DTT losses.</figcaption></figure>

function of the exposure duration. It is significantly higher than the control cells after at least 2 h of cell exposure. ROS production factors for all durations of cell exposure tested ranged from 1 (no ROS production) to 1.94. At 2 h of cell exposure, the mean ROS production factors are between 1.2 and 1.5. As these values are close, we can consider that all samples, regardless of the type of trains that crossed the station or the sampling duration, significantly induced cellular ROS production. These results are in line with the literature studies, which showed ROS activity in railway environments after 2-3 h of cell exposure, for PM₁₀ concentration in samples ranging from 25 to 70 µg/mL (Karlsson et al. 2005; Loxham et al. 2013; Spagnolo et al. 2015).

The results of the pro-inflammatory responses of the cells after incubation with samples (Figure S1 in the SI) show that none of the samples or endotoxins

tested induced significant secretion of any of the 4 cytokines. Endotoxin concentrations were close between samples (averaging 3.44 ± 0.05 endotoxin/mL in all 5 samples). The test was validated as the positive control (LPS) induced a significant increase in cytokine secretions. There is no consensus in the literature regarding the cytokine secretion of samples collected in railway environments. A secretion of cytokines was observed with aerosols collected at a station in London (Seaton et al. 2005), while no effect was observed for samples collected at a station in Sweden (Karlsson et al. 2006) for tests carried out on A549 cells. The results of this article are in line with those observed in Sweden.

In contrast to *in vitro* tests using cells, AA and DTT redox activity tests (graph at the bottom of Figure 2) differ from one sample to another. This is particularly true for AA consumption. The highest
<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

**Figure 3.** Results of toxicological tests on samples of the Water-surfactant method, depending on the crossed stations (over- or underground). Top: the CMA at 3 dilutions of samples before the deposition on cells. The dashed line represents the control signal. Middle: the mean intracellular ROS production after several exposure durations of cells for samples diluted at 1:2. The dashed line represents the control signal. The stars \* illustrate a significant difference between the results and the control. Bottom: results of mean AA and DTT losses.

AA consumption over time was obtained with the sample collected during the afternoon (16:05-18:05), while the lowest was obtained with the sample collected during the passage of work and for the sample collected during the first hours of sampling (07:32-09:32). Values range from $8.80 \times 10^{-2}$ to $1.19 \times 10^{-1}$ nmol min$^{-1}$. For DTT activity, antioxidant consumption is close between all samples collected during the normal traffic, with values ranging from $1.33 \times 10^{-1}$ and $1.57 \times 10^{-1}$ nmol min$^{-1}$. During the passage of work trains, the consumption is lower, at $1.11 \times 10^{-1}$ nmol min$^{-1}$.

### 3.3.2. In a passenger car (Water-surfactant collection)

Figure 3 shows the results of the toxicological tests depending on the type of stations crossed (underground or overground stations).

The values of CMA (at the top of Figure 3) are greater than 96% for all dilutions and samples tested. This proves that none of the samples induced significant cell mortality. Furthermore, there was no significant release of LDH at all 3 dilutions (data not shown), indicating no damage to cell integrity. Importantly, under the tested conditions, the type of station where the aerosols were collected had no influence on the cytotoxicity results. Although the CMA was close to 100% for all dilutions (including when samples were undiluted), samples were diluted at 1:2 to perform the other *in vitro* tests (cell ROS activity and inflammation), as interferences were observed with other undiluted samples (data not shown).

The intracellular ROS measurement (graph in the middle of Figure 3) increases as a function of the cell exposure duration for both samples. However, the only sample that induced significant ROS production
<figure><img src="image_21.png" /></figure>

(after 3 h of cell exposure) compared to control cells was the one collected during the passage of the train through underground stations only. The ROS production factors ranged from 1.0 to 1.3 for overground stations versus 1.1 to 1.9 for underground stations. In contrast to the cytotoxicity tests, cell ROS activity showed discriminating results between the two samples, with significant ROS activity for the underground sample only.

With regards to pro-inflammation responses, none of the samples or endotoxins induced significant cytokine secretion for all 4 cytokines (Figure S2 in the SI). Endotoxin concentrations were similar between samples, i.e., at 70.04 and 69.41 endotoxin/mL for over- and underground stations respectively.

Regarding AA and DTT redox activities (graph at the bottom of Figure 3), DTT reactivity is slightly higher for the sample collected in underground stations ($1.16 \pm 0.03 \cdot 10^{-1} \text{nmol min}^{-1}$) than for the one sampled in overground stations ($8.82 \pm 0.05 \cdot 10^{-2} \text{nmol min}^{-1}$). For the AA reactivity, the gap between samples is greater. While AA reactivity is close to 0 for the sample collected in overground stations, AA activity reaches $5.24 \pm 0.12 \cdot 10^{-1} \text{nmol min}^{-1}$ for the one collected through underground stations. Thus, the acellular test together with the cell ROS production assay appear more efficient than cytotoxicity and inflammation assays for discriminating stations of different natures with respect to aerosol-induced effects.

## 4. Discussion

Preventing modifications of sample properties is important when running toxicological studies. We showed that it is essential to consider the collection medium to avoid sample alterations. The Watersurfactant collection appears more effective than the Dry collection by limiting sample modifications. As the samples are suspended in liquid, sonication is not required to recover the samples from the cones. Nevertheless, we decided to sonicate the samples for one minute to help particles deagglomerate after freezing. We suggest removing this step in future studies and only conserving the vortex mixing to minimize sample modifications (Miljevic et al. 2014).

Regarding the cyclone performance, only one study investigated the sampling efficiency of the Coriolis μ (the liquid cyclone) and found an efficiency between 40% and 50% for aerosol particle diameter between 0.7 and 2 μm at a flow rate of 200 L min$^{-1}$ (Carvalho et al. 2008). This lack of data requires determining the sampling efficiency of the dry and liquid cyclone.

Computational fluid dynamics combined with a test bench are complementary methods that can be used for this purpose. In a second step, the design of the cyclone could be modified to improve the inlet efficiency (the fraction of particles that can enter in the cyclone). It could also be interesting to test different types of surfactants in liquid collection. Chang et al. (2019) show that the collection efficiency of fungi can be improved depending on the surfactant used.

The liquid cyclones are also characterized by their retention efficiency, i.e., the ability of the cyclones to retain the particles collected in the liquid until the end of sampling. A study of impinger samplers showed that the longer the sampling duration, the lower the retention efficiency, especially for the finest particles, which are more likely to be aerosolized and lost, e.g., when sampling for 1 h, the retention efficiency is 86% for 10 μm particles versus 26% for 1 μm particles with AGI-30 impinger (Kesavan et al. 2010). Moreover, another study on the same liquid cyclone we used showed that particles (1 μm in diameter) reaerosolize after 30 min at 300 L min$^{-1}$ (Rufino de Sousa et al. 2020). To improve the retention efficiency, they replaced the collection cone with a new one after each 10-minute cycle, leading to a 50% improvement in collection. Since the sampling duration at 300 L min$^{-1}$ in our study was 10 min, we can assume that retention efficiency was good according to their observations. To go further, we can reproduce the same assessment as Rufino de Sousa et al. (2020) with our collection liquid (water and surfactant) and different flow rates to determine the limit of sampling duration that can be performed by cyclones before the sample loses representativeness. This would make it possible to obtain samples for a larger number of spaces, e.g., all the stations covered by a moving train.

The cyclone was selected for its applicability in railway environments. Its small size and high flow rate made it possible to study several railway environments, and it could also be used in other microenvironments, e.g., cabin cars, offices, schools. Estimating the induced effects of aerosols without the use of ultrasound or solvent can also be achieved with other samplers, such as a cascade impactor, as the high-volume Sierra model 235 (Sierra Anderson, Smyna, GA, USA) and a liquid impinger, as the BioSampler (SKC Inc.). In the first case, particles are collected on plates and then brushed off in a liquid before the toxicological assays (Billet et al. 2007). In the other case, they are collected directly in a liquid. These methods also have their limitations. The plates of the cascade impactor must be covered with grease to prevent
<figure><img src="image_22.png" /></figure>

particle bouncing and ensure size segregation, which is not compatible with toxicological testing (the impactor has to be operated without grease) (Pozzi et al. 2005). The BioSampler suffers from a decrease in retention efficiency over time, typical of the liquid samplers as mentioned before (Su et al. 2019). Nevertheless, in order to widen the scope of possibilities, it would be interesting to carry out a comparison of these methods to select the most appropriate for each type of environment to be studied. We could, for example, imagine that the method with the lowest flow rate might be more suitable for the cabin car, where the air volume is small, while the largest units can be easily installed in a railway station.

The protocols developed in this article, the Dry collection and the Water-surfactant collection, represent a holistic approach like sampling on a filter. They were enabled to build a profile of the induced effects of particles through the implementation of 3 *in vitro* tests, 2 acellular tests and 1 analysis to interpret the data. We showed in this study that the toxicological profiles of the collected aerosols were test-dependent, since not all toxicological test results point in the same direction. For example, in the same underground station, we observed a constant cell ROS production depending on the sampling time for all the samples, while AA reactivity (acellular test) differed from one sample to another. These observations are in line with the literature, where different conclusions have also been reached on the toxicity of aerosols collected in railway environments. For example, *in vitro* tests showed that these aerosols were more harmful than those recovered in ambient air, while *in vivo* results were not significant in this respect (Loxham and Nieuwenhuijsen 2019).

The results of the elemental analysis of samples show that the metals present in both railway environments studied are typical of those found in other railway areas, according to the literature. The Fe is most likely emitted by train brakes through abrasion on the rails (Seaton et al. 2005; Salma et al. 2007; Perrino et al. 2015; Minguillón et al. 2018). Cr and Cu were probably emitted by wear and tear on railway equipment (e.g., pantograph, catenary, power wire) (Kang et al. 2008; Kam et al. 2013; Martins et al. 2016a). Ba can come from train brakes, but also from ballast or earth's crust (Sitzmann et al. 1999; Salma et al. 2007; Querol et al. 2012).

In the underground railway station, the characterization shows that during normal traffic, all samples collected contained the same elements and at similar concentrations, proving that sources of aerosols (for

elemental composition) were constant from day to day and hour to hour. The total concentration of elements in the sample collected during the passage of the work trains was half that observed under normal traffic conditions. However, all these samples induced similar effects *in vitro*. These results therefore highlight that there may be a threshold effect, i.e., a concentration above which *in vitro* effects are no longer linear.

On the contrary, for the AA and DTT redox activity assays, we saw variability in results between sampling days and between the types of trains that passed through the station. This indicates that these acellular tests may be more sensitive than the *in vitro* tests under these conditions. In the literature, AA and DTT have been shown to be reactive with metals, which explains why the sample with the lowest concentration induced less reactivity (Crobeddu et al. 2017; Calas et al. 2019; Perrone et al. 2019; Pietrogrande et al. 2019; Barraza et al. 2020).

In the passenger car, a 10-minutes sampling period was sufficient to observe differences in the induced effects of samples collected from the same train with only 20 min differences between sample collections. This result implies that the levels of particulate pollution vary depending on the stations. In addition, it means that the time spent in each station (less than 1 min per station) is enough to allow air infiltration into the train and modify the concentration at a place where a passenger can travel (the cyclone was placed on a seat). This phenomenon was also observed in a study on Polycyclic Aromatic Hydrocarbons (PAHs) where concentrations were found to be higher in underground stations than in overground stations (Jiang et al. 2017). As there is no ventilation in the passenger car, we can assume that aerosols are not immediately evacuated from the train, which may contribute to higher toxicological potential when crossing underground stations.

To interpret the toxicological results, a more in-depth chemical analysis is needed to characterize the species responsible for the observed induced effects. For example, in the underground railway station that we studied (see Section 3.2.1), AA reactivity was higher at the 16:05 sampling time, but this sample collected the same concentration as the 07:32 sample, whereas the AA reactivity was one of the lowest. This means that the total concentration of the elements is not sufficient to interpret the results. Some aerosols not analyzed here and identified in railway environments could contribute to the induced effects, as Poly Aromatic Hydrocarbons (PAH), such as nitro- and
<figure><img src="image_23.png" /></figure>

oxy-PAH (Song et al. 2020), Elemental and Organic Carbons (EC/OC) (Fromme et al. 1998; Martins et al. 2016b) or soluble transition metals and carbon (organic and elemental) (Kam et al. 2011; Anses 2022). To maximize the volume of samples, and therefore the number of analyses, one solution would be to accumulate several samples collected every 10 min (Rufino de Sousa et al. 2020). A less time-consuming alternative would be to run two cyclones in parallel, one dedicated to toxicological analysis, the other to chemical analysis.

To compare all the environments with each other with the protocols developed here, and ultimately classify environments according to aerosols' induced effects, the same parameters (cyclone, sampling duration, flow rate, medium collection, liquid volume) will have to be applied for future collections. This would open the door to the identification of "hot spots" in railway environments in terms of aerosol toxicity, which could help advise passengers on the preferable paths to take and indicate which places need to be cleaned up first.

## 5. Conclusion

In this study, four collection media for cyclone sampling were assessed to reduce or eliminate sonication, solvent usage and spinning down for recovering aerosol particles from the cone of collection and later conducting toxicological experiments. Two media, a Dry collection and a liquid cyclone filled with Water and surfactant, were selected as alternatives to filter sampling. The Dry collection has the advantage of requiring few preparation steps for in vitro studies, since culture medium must simply be added to the cone and vortex mixed before in vitro assays. Nevertheless, the use of sonication at weak energy remains needed to recover the sample fully. The Water-surfactant method requires sample dilution before running toxicological studies, but enables selecting suitable liquids depending on the test method, i.e., culture medium for in vitro tests on cells, Gamble for DTT/AA activities or water for chemical analyses. The use of sonication is optional, as aerosols are directly suspended in liquid and not attached to the cone walls, but remains recommended if samples undergo freezing/thawing steps prior to testing. To eliminate this step, we recommend a more in-depth study to determine the impact of freezing on the size of aerosols. Hence, the liquid collection represents a more promising method than the Dry collection to perform in vitro

tests but requires further studies to evaluate completely its impact on the properties of the collected aerosols. Further research is also needed to precisely study cyclone sampling efficiency and link toxicological results to aerosol size distribution or composition. This research could be carried out using computational fluid dynamics combined with tests bench.

Finally, both cyclones are portable and take up little space. The deployment of the liquid cyclone in a moving train showed that 10 min sampling at a flow rate of 300 L.min⁻¹ is sufficient to discriminate environments (overground and underground stations) according to aerosols induced effects. These techniques are thus ideal for studying the aerosols in restricted or limited areas in space and time, e.g., in vehicles, offices, workshops, in the city near construction sites, etc. The rapid deployment of cyclones could provide new information and knowledge on the toxicity of aerosols in diverse environments and help implement new legislation on air quality in these environments.

## Acknowledgments

This work was conducted as part of the TOXinTRANSPORT project (Queron et al. 2022) and a thesis (Delater 2023). The authors thank all the project partners, especially the SNCF Voyageurs (Agence d'Essai Ferroviaire) for providing railway environments for the measurements and Farid Ait Ben Ahmad and Yohann Baillon for their analyses. We also thank the French National Research Agency in the framework of the Investissements d'Avenir Program (ANR-15-IDEX-02) and ANR GetOP Stand-OP (ANR-19-CE34-0002-01). We are also grateful to Bertin Technologies for the loan of two cyclones. Finally, we would like to thank Thomas Loret and Patrice Coll for proofreading this article.

## Authors contributions

CRediT: **A. Delater**: Writing -original draft, Formal analysis, Data curation, Investigation, Methodology, Visualization; **M. Plumail**: Investigation, Data curation, Visualization; **G. Uzu**: Writing -original draft, Investigation, Methodology, Writing -review & editing; **L. Meunier**: Investigation; **S. Fable**: Investigation; **M. Floreani**: Investigation; **C. Gamez**: Investigation; **T. Mhadhbi**: Investigation; **J. Queron**: Conceptualization, Project administration; **O. Le Bihan**: Conceptualization, Funding acquisition, Project administration, Writing -review & editing; **I. Coll**: Conceptualization, Supervision, Validation, Writing -review & editing; **B. Berthelot**: Conceptualization, Supervision, Validation, Writing -review & editing; **G. Lacroix**: Writing -original draft, Conceptualization, Supervision, Methodology, Validation, Visualization, Writing -review & editing.
<figure><img src="image_24.png" /></figure>

## Disclosure statement

The cyclones were loaned by Bertin Technologies SAS (excluding consumables) for the duration of the field campaigns. 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 article.

## Funding

This work was supported by INERIS [P190 program]; and Agence de l'Environnement et de la Maîtrise de l'Energie [call for projects IMPACTS, 2017].

## ORCID

Ambre Delater http://orcid.org/0000-0002-3350-8949

Gaëlle Uzu http://orcid.org/0000-0002-7720-0233

Olivier Le Bihan http://orcid.org/0000-0002-4775-960X

Isabelle Coll http://orcid.org/0000-0002-4586-529X

Ghislaine Lacroix http://orcid.org/0000-0002-3260-8526

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