The PolluRisk platform was developed at the Interuniversity Laboratory of Atmospheric Systems (LISA UMR CNRS 7583) in collaboration with the Mondor Institute for Biomedical Research (IMRB UMR U955 INSERM) located in Créteil, France. The fundamental concept of the PolluRisk platform (Fig. 1) is to simulate urban atmospheric pollution scenarios in a controlled environment, allowing researchers to expose biological models (e.g., mice) and study the resulting health effects. This platform has been previously described in several studies (Belgacemi et al., 2023; Blayac et al., 2024; Coll et al., 2018; Georgopoulou et al., 2024; Guilloteau et al., 2022).

Central to the platform is the CESAM atmospheric simulation chamber (http://cesam.cnrs.fr, last access: 21 September 2026), a 4.2 m³ stainless steel reactor designed to reproduce multiphasic oxidation processes. A detailed CESAM description is provided by Wang et al. (2011). The chamber achieves controlled precursor concentrations, with mixing times of approximately one minute ensuring gaseous compound homogeneity. An artificial irradiation system powered by three 6.5 kW high-pressure Xenon arc lamps (IREM, EX170GM3) facilitates atmospheric photochemistry, achieving a JNO₂ value of 8.3 × 10⁻³ s⁻¹. Pyrex^® filters reduce ultraviolet radiation intensity, especially below 300 nm, reproducing tropospheric solar radiation. The chamber maintains evacuable conditions (reaching 10⁻⁷ atm) with pumping systems capable of removing adsorbed compounds. Relative humidity (RH) was maintained at 30 %–50 % on average to promote condensational growth of organic material onto pre-formed ammonium sulphate seed particles, while avoiding water condensation interference with organic aerosol formation. RH was maintained constant across both scenarios to ensure experimental comparability. We acknowledge that real biomass burning episodes typically occur at higher RH (60 %–80 %), which would enhance gas-particle partitioning (Sect. 4.3).

To study the induced health effects, the generated atmosphere is continuously transferred through a 3/8 in. copper line inside an isolator (I-Box+ Erlab, Noroit^®), via an overpressure of 7 mbar applied in the smog chamber. This overpressure ensures continuous flow to the exposure isolator while preventing atmospheric contamination. The isolator houses experimental mice in individual cages (up to 19 cages) with controlled ventilation. The exposure isolator also receives filtered laboratory air (HEPA- and activated carbon-filtered room air, removing particles > 0.3 µm and gaseous compounds, respectively) with 90 % filtered room air and 10 % simulated smog atmospheres. This results in an atmosphere dilution ratio of 1 : 10 between CESAM and the exposure isolator. In addition to the exposure isolator, another isolator receives only filtered laboratory air and serves to house the reference mice group. Using one-way valves, both isolators are connected to a manifold system. The latter is connected to analytical instruments for the analysis of gas and particulate pollutants. Measurements of particles and gases were performed via a manifold system alternating between the exposure isolator (4 h) and the reference one (1 h), across 7 consecutive days of continuous monitoring (24 h d⁻¹). Irradiation was maintained continuously throughout the 7 d experiments without day/night cycling. While this ensures stable aerosol generation for chronic biological exposure, it differs from ambient photochemical cycling between OH-dominated daytime and NO₃-dominated night-time chemistry.

All pollutant concentrations and chemical characterisation results presented in this manuscript refer to measurements performed in the exposure isolator, which constitutes the actual biological exposure environment. CESAM serves exclusively as the aerosol generation and photochemical aging system. This paper presents the results of the measurements in the exposure isolator. The resulting health impacts will be described in a subsequent paper.

Following the PolluRisk platform methodology (Coll et al., 2018), we used a VOC mixture with concentrations determined by box modelling to achieve target urban pollution levels after photochemical processing in CESAM. Among the primary pollution sources affecting moderately polluted cities, road traffic represents a major VOC contributor. In Paris, for instance, this source accounts for approximately 25 % of annual VOC emissions, with toluene and m-xylene as major traffic-related aromatics, contributing to nearly 20 % of this fraction, while long-chain alkanes (such as tridecane) result from diesel emissions (Gaimoz et al., 2011; Languille et al., 2020). Biogenic emissions such as α-pinene and isoprene can dominate during warmer periods, contributing up to 30 % of oxygenated compounds (Ait-Helal et al., 2014; Baudic et al., 2016; Gaimoz et al., 2011). To replicate these urban environments, we selected representative anthropogenic VOCs including aromatic hydrocarbons (toluene (P. Normapur^®, PROLABO, puriss grade), m-xylene (Touzart & Matignon, 99 % purity)) and an aliphatic hydrocarbon (tridecane (Sigma-Aldrich, CAS 629-50-5, 99 % purity)), alongside biogenic VOCs (α-pinene (Sigma-Aldrich, CAS 80-56-8, ≥ 97.5 % purity), methylglyoxal (Sigma-Aldrich, CAS 78-98-8)) due to their atmospheric reactivity and SOA formation potential.

Ammonium sulphate particles (Sigma-Aldrich, CAS 7783-20-2, ≥ 99.9 % purity) were generated to simulate inorganic seed aerosols and to allow the condensation of secondary organic products resulting from the oxidation of VOCs. For biomass burning simulation, commercially available pellets compliant with EN ISO 17225-2 standards (70 % beech, 30 % conifers, ash < 0.6 %, RH < 8 %) were used as the biomass source (0.5–1.0 g per combustion cycle).

This study simulated two atmospheric scenarios for biological exposure assessment. Target PM₁ and NO₂ concentrations were selected with reference to ranges reported for the Paris region (AirParif, 2019; Petit et al., 2014; von der Weiden-Reinmüller et al., 2014). These comparisons concern selected concentration indicators only. They do not imply that either scenario reproduces the full chemical composition, temporal variability, or atmospheric processing of ambient urban air.

To achieve these representative urban conditions, all particulate and gaseous precursors were continuously injected into the CESAM smog chamber, previously filled with synthetic air (nitrogen N₂ and oxygen O₂) in 80 : 20 proportions. Tanks of pressurized liquid nitrogen (Messer, ≥ 99.9 % purity, H₂O content < 5 ppm) and oxygen (Air Liquide, AlphagazTM Class 1, ≥ 99.9 % purity) were used to generate the N₂ and O₂. A 10⁻² M ammonium sulphate solution was nebulized using a commercial atomizer (TSI Model 3075), followed by silica-gel dehumidification (TSI Model 3062). The biomass burning emissions were generated using a tubular furnace (CarboliteTM CTF Wire-Wound Tube Furnace) operated at 400 °C under controlled airflow conditions (3 L min⁻¹ synthetic air). Emissions were continuously transferred to an auxiliary chamber at 1 L min⁻¹, then introduced into CESAM for atmospheric aging (Fig. 1). Combustion occurred twice daily while refilling the furnace every 10 to 12 h, ensuring steady carbonaceous particle supply. A homemade VOC mixture (methylglyoxal (13 ppm), toluene (25 ppm), m-xylene (22 ppm), tridecane (33 ppm), and α-pinene (8 ppm)) was continuously injected into the smog chamber at a constant flowrate of 0.05 L min⁻¹ following (Doussin et al., 2023) methodology for chamber protocols. After 20–30 h, nitrogen monoxide (NO, Air Liquide, 99.5 % purity) was introduced at 0.5 L min⁻¹. Nitrogen monoxide was used to establish the NO-NO₂-O₃ photostationary cycle, promoting OH radical formation through NO₂ photolysis and enhancing VOC oxidation under high NO_x conditions representative of urban photochemical regimes. Irradiation began 11 h before exposure initiation to reach a steady state and replicate tropospheric photochemistry. Irradiation was maintained continuously throughout the entire experimental period to ensure stable photochemical conditions.

This study simulated atmospheric scenarios characteristic of moderately polluted urban environments, focusing on two distinct cases representing different emission source contributions:

The standard urban scenario simulated baseline urban conditions. It included the selected anthropogenic and biogenic VOC mixture (methylglyoxal, toluene, m-xylene, tridecane, α-pinene) together with ammonium sulphate seed particles. It was designed to examine secondary organic aerosol formation from the injected VOC mixture and did not include primary carbonaceous or traffic-emitted particulate matter.

A comprehensive analytical suite enabled detailed characterization of both gas-phase and aerosol-phase components, including concentration measurements, chemical composition analysis, and molecular-level identification.

The complete experimental workflow (Fig. 2) illustrates the five main methodological steps: (1) simulation setup with VOC injection, seed aerosol generation, and optional biomass burning emissions; (2) atmospheric aging under controlled irradiation; (3) controlled exposure in isolators with alternating sampling protocols; (4) extensive sampling and analysis using online monitoring (PTR-TOF-MS, ACSM, SMPS), offline filter collection, and molecular characterization (UPLC/ESI-IMS-QTOFMS); and (5) data processing yielding chemical composition profiles, molecular markers, volatility distributions, and source-specific signatures for each exposure scenario.

In the exposure isolator, PM₁ concentrations averaged 15 ± 7 µg m⁻³ for the standard urban scenario and 63 ± 24 µg m⁻³ for the biomass burning enhanced scenario. Non-refractory PM₁ chemical composition revealed distinct exposure profiles between scenarios (Table 1). Standard urban simulation showed sulphate dominance (approximately 42 %), followed by ammonium (approximately 29 %) and organics (approximately 17 %). The biomass burning enhanced simulation showed that organics contributed approximately 40 % of PM₁ mass, driven by carbonaceous particles and their atmospheric aging under controlled irradiation. Nitrates accounted for approximately 20 %, reflecting secondary formation through NO₂ oxidation. The calculated aerosol densities were 1.77 g cm⁻³ for the standard urban scenario and 1.59 g cm⁻³ for the biomass burning enhanced scenario.

Average NO₂ concentrations reached 27 ± 10 ppb for the standard urban scenario and 39 ± 4 ppb for the biomass burning enhanced scenario. Carbon monoxide concentrations increased from 171 ± 63 ppb (standard urban) to 472 ± 92 ppb (biomass burning enhanced). Ozone concentrations averaged 4 ± 3 ppb (standard urban) and 9 ± 5 ppb (biomass burning enhanced).

PTR-TOF-MS allowed the identification and quantification of 23 distinct VOCs, including both primary emissions and secondary oxidation products. Complete compound list with m/z values, O : C ratios, volatility classifications, and calculated concentrations is provided in Table 2.

Figure 4 illustrates VOC classification by chemical families. Oxygenated compounds dominated both scenarios (74 % standard urban, 77 % biomass burning), with carboxylic acids predominant (31 % standard, 42 % biomass burning).

Building upon global composition measurements, molecular-level analysis of particle phase revealed the specific organic compounds driving compositional differences between scenarios. After thorough peak selection and analysis using Waters UNIFI software, we identified 32 distinct organic compounds. Of these, only 4 were detected in both scenarios, while the remaining 28 were exclusively identified in the biomass burning enhanced scenario. Compound identification was assigned to two confidence levels following Schymanski et al. (2014): level 1 for compounds confirmed with authentic standards and level 2 for tentative identifications based on accurate mass and fragmentation information. These compounds span multiple chemical families including nitrophenols, biogenic oxidation products, ring-opening products, anhydrosugars, and other oxidation products. The complete list, including retention times, m/z values, O : C ratios, and estimated volatility classes, is provided in Table 3.

Aromatic oxidation (toluene and m-xylene) produced both ring-retaining compounds including 2,3-dihydroxy-4-oxopentanoic acid (DHOPA), 4-nitrophenol, dimethyl-nitrophenol and 3-methyl-4-nitrocatechol, as well as ring-opening products such as succinic acid, demonstrating multiple oxidation pathways for aromatic precursors under controlled conditions. We identified biogenic oxidation products including pinic acid, cis-pinonic acid, norpinonic acid, and 3-methyl-1,2,3-butanetricarboxylic acid (MBTCA).

We detected several compounds derived from biogenic and anthropogenic precursor oxidation in both scenarios, though we were able to achieve more comprehensive detection in biomass burning enhanced conditions due to higher organic aerosol concentrations. 4,6-dinitro-o-cresol (from aromatic precursor oxidation under high NO_x), dimethyl-nitrophenol (from m-xylene oxidation), and cis-pinonic acid (from α-pinene ozonolysis and OH radical oxidation) were identified in both scenarios. The limited number of compounds detected in both scenarios may reflect analytical detection limits rather than the absence of those compounds, as standard urban organic aerosol concentrations (1.7 µg m⁻³) were substantially lower than biomass burning levels (31.1 µg m⁻³). We also identified primary biomass burning emission markers, including a levoglucosan isomer, using m/z matching without retention time confirmation. This annotation should therefore remain tentative.

Quantification was attempted for five representative SOA markers (2-nitrophenol, cis-pinonic acid, MBTCA, norpinic acid, and pinic acid) using camphor-10-sulfonic acid as an internal standard, following calibration curves established for each compound (Sect. S4, Supplement). Atmospheric concentrations were calculated by referencing the mass identified on the filter to the specific surface area analysed and the total volume of air sampled. Sampled volumes ranged between 0.51 and 0.56 m³ for the CESAM chamber and 2.76 and 2.95 m³ for the exposure isolator. Reliable quantification in the exposure isolator was limited by the 1 : 10 dilution from CESAM combined with the comparatively low organic aerosol loading achieved in this campaign, particularly for the standard urban scenario (1.7 µg m⁻³), which approached instrumental detection limits for individual compound quantification. A confirmed quantified concentration was obtained for cis-pinonic acid in CESAM under standard urban conditions (255 ± 23 ng m⁻³). Comprehensive isolator-level quantification of these markers for both scenarios was not achieved in this campaign and is identified as a priority for future campaigns with higher organic loadings or increased sampling frequency (Sect. 4.3). Qualitative detection was possible for the 32 molecular features even when compound-specific quantification was not completed. However, identifications based only on m/z remain tentative and should not be interpreted as definitive structural assignments (Table 3).

The volatility analysis identified compounds distributed across semi-volatile, low-volatility, and extremely low-volatility organic compound ranges (SVOCs, LVOCs, ELVOCs; C < 300 µg m⁻³). These estimates are structure-based and indicative rather than direct measurements of volatility or mass partitioning. The oxygen-to-carbon (O : C) ratios and estimated saturation mass concentrations (C∗) provide qualitative information on possible gas–particle partitioning behaviour, but the figure does not provide mass-weighted contributions to total organic aerosol.

The distribution of estimated volatility classes is consistent with the presence of functionalized organic compounds after photochemical aging. It does not, however, establish the extent of atmospheric aging or quantify gas-to-particle transfer. In particular, Fig. 5 represents the molecular diversity of the identified compounds across volatility classes and does not give information on the relative mass contributions of individual species to total organic aerosol.

The temporal variations observed during the 7 d exposure periods (Fig. 3) document the ability to maintain distinct scenario-specific concentration ranges over extended periods. The day-by-day stability of the aerosol size distribution (Fig. S1a–b in the Supplement) supports the establishment of a quasi-steady state, consistent with continuous precursor injection and wall passivation.

The measured PM₁ concentrations in standard urban and biomass burning enhanced scenarios are, respectively, similar to those of summer background (10–20 µg m⁻³) and winter pollution episodes (50–80 µg m⁻³) in the Paris region (Petit et al., 2014; von der Weiden-Reinmüller et al., 2014). Organic aerosol concentrations show contrasting patterns between scenarios: standard urban levels (1.7 ± 1.8 µg m⁻³) align with typical urban background measurements, while biomass burning enhanced concentrations (31.1 ± 11.3 µg m⁻³) represent wintertime pollution episodes characteristic of residential wood burning periods in the Paris region (Languille et al., 2020; Petit et al., 2014; von der Weiden-Reinmüller et al., 2014).

However, VOC concentrations in both scenarios exceed typical ambient levels observed in Paris, where individual aromatic compounds rarely exceed 5–10 ppb (Gaimoz et al., 2011). The elevated VOC concentrations in our simulations (exceeding 20–50 ppb for several compounds) were defined to ensure sufficient secondary aerosol formation within the experimental timeframe, while maintaining realistic aerosol composition and mass concentrations for biological exposure studies.

Though, NO₂ concentrations are also typical of urban levels observed in the Paris region. These fall within ranges reported for moderate urban traffic conditions (20–40 ppb) and winter episodes (30–50 ppb), respectively (AirParif, 2019; von der Weiden-Reinmüller et al., 2014). Quantitatively, the concentration of CO highlights the contribution of biomass combustion to exposure profiles to urban pollution during winter (Baudic et al., 2016). O₃ concentrations in both cases were below WHO guidelines. According to Sillman (1999), VOC-sensitive regimes are characterized by VOC/NO_x ratios < 4, while NO_x-sensitive regimes exhibit ratios > 15. In the urban photochemical regimes, high NO_x concentrations limit O₃ accumulation through ozone titration reactions (NO + O₃ → NO₂ + O₂), hence reproducing the chemical behaviour typical of urban centres where ozone levels are suppressed despite high precursor concentrations (Seinfeld and Pandis, 2012).

Gas-phase characterization revealed scenario-specific VOC signals. Carboxylic acids accounted for 31 % of the reported signal in the standard urban mixture and 42 % in the biomass-burning-enhanced mixture. Oxygenated compounds accounted for 74 % and 77 %, respectively. The dominance of oxygenated compounds reflects photochemical aging of the precursor VOCs under controlled conditions, consistent with atmospheric transformation studies (Li et al., 2019; Montero et al., 2001). These observations are consistent with the oxidation of the injected precursors under the experimental conditions. The presence of glyoxylic acid (highest ratio of oxygen-to-carbon, O : C = 1.5) for instance, is indicative of highly oxidized products and has been attributed to toluene photooxidation products under high-NO_x conditions (Jang and Kamens, 2001).

The biomass burning enhanced scenario showed higher signals for several compounds, including formic acid (m/z = 47), acetic acid (m/z = 61), ethyl vinyl ketone (EVK, m/z = 85), and furfural (m/z = 97). For biogenic VOC transformations, the presence of MVK (methyl-vinyl ketone) was also evident. The differences are in line with the source specificities shown by Languille et al. (2020) in the Paris region.

Aerosol analysis showed a significant difference in chemical composition, with an organic fraction of approximately 17 % for the standard urban scenario against 40 % for the biomass burning scenario. The higher organic fraction in the biomass burning enhanced scenario is consistent with the added carbonaceous emissions and additional oxidation products, but these contributions were not quantitatively separated. The [NO3-] / [SO42-] ratio increased from 0.36 to 0.58. This observation indicates a compositional difference between both scenarios, reflecting secondary nitrate formation associated with biomass burning emissions (Bressi et al., 2013; Crippa et al., 2013; Petit et al., 2014; von der Weiden-Reinmüller et al., 2014).

The SMPS data (Fig. S1a–b) showed no detectable new particle formation below 30 nm in the presented time series and were consistent with growth on pre-existing particles under the experimental conditions (RH ∼ 40).

Molecular-level characterization identified a total of 32 distinct organic compounds, with source-specific molecular markers such as a levoglucosan isomer (primary biomass burning tracer), nitrophenolic compounds (3-methyl-4-nitrocatechol, nitroguaiacol) formed by oxidation of aromatic precursors, and biogenic oxidation products (pinic acid, norpinonic acid, MBTCA) of α-pinene. Volatility distribution analysis identified compounds distributed across the SVOC, LVOC, and ELVOC ranges (C∗ < 300 µg m⁻³), indicating a significant contribution of functionalized and atmospherically aged compounds. The presence of higher molecular weight compounds (up to m/z 220) suggested potential oligomerization mechanisms responsible for the low-volatility fraction (Kalberer et al., 2004). The highly oxidized product 5-nitrosalicylic acid (O : C ratio = 0.71) indicates ELVOC formation under stable conditions, thus demonstrating the ability of this platform to mimic complex atmospheric aging processes relevant for exposure characterization. These stable, controlled conditions allow for characterization of the aging process from highly volatile precursor molecules to low-volatility products. This process is practically impossible to trace in the ambient atmosphere due to mixed source contributions and oxidation levels.

In both scenarios, SOA formation was the product of VOC oxidation and in the enhanced scenario, primary biomass burning emissions and their atmospheric processing had additional contributions. This dual formation pathway explains the enhanced organic mass fractions and distinct volatility distributions observed between scenarios. These oxidation processes demonstrate the capacity of the platform to reproduce specific atmospheric transformations under controlled conditions. The detection of both ring-retaining and ring-opening products confirms the existence of multiple simultaneous oxidation mechanisms, providing molecular-level validation of urban atmospheric chemistry models.

These volatility distributions provide critical data for atmospheric model parameterization. Current secondary organic aerosol formation models are dependent on limited field observations wherein several sources interact continuously, hence isolating individual transformation processes becomes difficult. The molecular speciation and volatility distributions determined under controlled conditions allow a systematic evaluation of atmospheric chemistry models and an improvement of mechanistic representations. Thus, source-specific transformation pathways known to be difficult to isolate in ambient measurements become available for evaluation through the use of atmospheric simulation chambers. This leads to stronger predictive capacities of regional air quality models.

Detection of primary emission markers (levoglucosan), and secondary products (pinic acid, norpinonic acid, MBTCA) (Christoffersen et al., 1998; Jang, 1999; Mutzel et al., 2016; Yang et al., 2016), confirms the ability of the platform to reproduce direct emissions as well as atmospheric transformations (Ho et al., 2007; Kawamura and Kaplan, 1987; Yang et al., 2016). Nitrophenolic compounds, identified as abundant species in biomass burning aerosols (Lin et al., 2016), are specific molecular markers for residential heating emissions. They have not only been detected in biomass burning aerosols from biogenic sources but also in suburban and urban regions during wintertime, because of the widespread domestic wood burning for heating. Detection of methyl-nitrocatechols and methyl-nitrophenols, as products of m-cresol photooxidation from wood combustion, provides molecular-level tracers for source apportionment in exposure studies. Several compounds that originated from biogenic and anthropogenic precursor oxidation were detected in both scenarios, although more comprehensive detections were achieved with the biomass burning enhanced conditions due to higher concentrations of organic aerosol. We identified compounds in both scenarios including 4,6-dinitro-o-cresol (a product of aromatic precursor oxidation under high NO_x), dimethyl-nitrophenol (a product of m-xylene oxidation), and cis-pinonic acid (a product of α-pinene ozonolysis and OH radical oxidation).

Hildebrandt Ruiz et al. (2015) emphasized the influence of photochemical aging on organic aerosols, highlighting the progressive oxidation of organic compounds which resulted in highly oxidized low-volatility aerosol species. Paciga et al. (2016) evaluated the dominance of organic nitrogen compounds in secondary organic aerosols, providing additional insights into the distribution of semi-volatile, low-volatility, and extremely low-volatility organic aerosol compounds. The pyrolysis of lignin during biomass burning produces substituted phenols (i.e., alkylphenols, methoxyphenols), which undergo further oxidations with atmospheric radicals, primarily hydroxyl radicals (OH), or through NO₂ addition reactions, leading to the formation of nitrophenols (Haque et al., 2019; Simoneit et al., 1999; Yang et al., 2016). Beyond their utility as source tracers, these compounds have pro-oxidant and cytotoxic properties demonstrated in previous laboratory studies (Keith and Telliard, 1979; Khan et al., 2022). This detailed chemical characterization facilitates mechanistic studies linking specific molecular components to biological responses under reproducible exposure conditions.

Wall effects are an inherent consideration in extended chamber experiments. Particle wall losses have not been specifically investigated during these experiments but were characterized in previous work: polydisperse ammonium sulfate particles (submicronic) and dusts (supermicronic) were injected into CESAM, and the number size distribution was measured as a function of time to derive first-order decay rates for each size bin. These experimental loss rates were well reproduced using the parametrization of Lai and Nazaroff (2000). The aerosol lifetime in CESAM was determined to range from 10 h to 4 d depending on particle size distribution, which is therefore quite long, although not negligible for experiments lasting several days as in this study. These losses would need to be considered when performing numerical simulations, which was not the case here.

The loss of semi-volatile species has also been investigated in previous studies and found to be highly dependent on the wall state (Suarez-Bertoa et al., 2012). A passivation of the walls was observed when repeating the same experiment over several days, leading to a decreasing wall loss rate for the semi-volatile compounds injected. The impact of heterogeneous reactions occurring on the walls on gas-phase composition has also been investigated in CESAM; for example, the reduction of NO₂ into HONO has been observed, and identified heterogeneous wall reactions are described in an “auxiliary mechanism” (Wang et al., 2011).

Taken together, these considerations confirm that particles and semi-volatile compounds may be lost on the walls, affecting the composition of the mixture. However, this study does not aim to investigate detailed chemical processes but to generate mixtures representative of urban atmospheres. What matters here is the composition of the mixture actually reaching the exposure isolator. Considering that experiments last for several days, we expect that chamber walls progressively passivate, leading to an equilibrium between emissions, losses, and partitioning between gas-particles and walls. This is supported by the stability of the dilution ratio between CESAM and the exposure isolator throughout both experimental periods, consistent with the nominal 1 : 10 design specification, as well as by the day-by-day stability of the aerosol size distribution (Fig. S1a–b, Supplement). Such heterogeneous wall reactions have not been observed to lead to new compounds affecting SOA composition.

Concerning the photolysis of products, considering that the actinic flux in CESAM closely approximates the solar spectrum, photochemistry is expected to be representative of that occurring in the real atmosphere (Wang et al., 2011).

Some aspects of the platform present additional limitations. Ozone concentrations remained low due to rapid NO-O₃ reactions and chamber surface interactions. The molecular characterization was based on offline filter analysis rather than time-resolved measurements of transformation products. The targeted molecular markers were not quantified comprehensively in the exposure isolator. In addition, the experiment did not determine an integrated OH exposure or an equivalent atmospheric age, and it did not resolve transient intermediates or radical chemistry products.

Irradiation was maintained continuously throughout the 7 d experiments without day/night cycling. While this ensures stable aerosol generation for chronic biological exposure, it differs from ambient photochemical cycle between OH-dominated daytime and NO₃-dominated night-time chemistry. Relative humidity and temperature were held constant across both scenarios (RH 30 %–50 %, T 22–25 °C) to ensure experimental comparability. Real biomass burning episodes typically occur at higher RH (60 %–80 %) and lower temperatures, which would enhance gas-particle partitioning.

The standard urban scenario included primary ammonium sulphate seed particles but no primary carbonaceous or traffic-emitted particulate matter. Its organic fraction was intended to result mainly from oxidation of the injected VOC mixture, although the primary and secondary organic fractions were not quantified. Preliminary tests using a MiniCast combustion source to introduce primary carbonaceous particles in this scenario raised concerns regarding animal exposure to residual propane. Filtration and source characterization are therefore technical priorities for future campaigns. In the biomass burning enhanced scenario, the dual primary/secondary origin of organic aerosol was assessed through molecular markers rather than formal source apportionment. Positive matrix factorization (PMF) analysis of the ACSM organic mass spectra was not performed in this study. Finally, polycyclic aromatic hydrocarbons (PAHs), a recognized class of biomass burning emission products, could not be characterized with the current UPLC/ESI-IMS-QTOFMS setup, as electrospray ionization is poorly suited to these non-polar compounds. As noted in Sect. 3.2, comprehensive isolator-level quantification of the targeted SOA markers was not achieved in this campaign. This prevented a systematic comparison of quantified marker concentrations against ambient measurements, which would otherwise strengthen the assessment of atmospheric representativeness at the molecular level. Increasing the number of filters collected per scenario, or using more sensitive pre-concentration and quantification methods, would enable this comparison in future campaigns.

Nonetheless, promising developments can be achieved in the future. Ozone generation thus represents a priority to better reflect urban photochemical conditions. Extending the exposure durations while maintaining atmospheric stability presents technical challenges which will require systematic investigation. Mechanistic understanding would be enhanced through the integration of advanced analytical techniques. Chemical ionization mass spectrometry (CIMS) would allow real-time detection of highly reactive species, while derivatization methods would better capture reactive intermediates. Platform expansion should involve a variety of scenarios, from low-polluted to high-polluted urban environments, enabling standardized exposure assessments across pollution levels. The inclusion of additional source-specific scenarios (e.g., industrial, agricultural, marine) would help ensure full exposure characterization across different urban settings. Finally, the integration of real-time molecular analyses would offer a complementary temporal resolution for the transformation processes, linking controlled mechanistic laboratory studies with field observations.

Direct OH measurement in CESAM, while outside the scope of this study, represents a valuable future direction that could enable finer comparison between the simulated and ambient atmospheres. Recent developments in nitrate-ion CIMS instrumentation (Alage et al., 2024) represent a promising potential option for future integration of real-time monitoring with this platform. Gas chromatography-mass spectrometry (GC-MS) would complement the current approach for the characterization of PAHs. Elemental and metal characterization by ICP-MS or real-time instrumentation (an Xact instrument has since been used in more recent PolluRisk campaigns) would provide additional insight into the oxidative potential of biomass burning aerosols. Formal PMF analysis of ACSM organic mass spectra would allow quantitative apportionment of primary and secondary organic aerosol fractions.

Future work could also explore exposing mice to variable or cyclic atmospheres to approach more realistic exposure patterns than the stable conditions used in this study.