# Multigenerational effects of nanoplastics on life-history traits and physiological responses in Drosophila melanogaster

Chloé M.C. Richard, Hervé Colinet, Mélanie Boël, Stéphane A.P. Derocles, Clémentine Goupil, Théo Laguilliez, David Renault

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Chloé M.C. Richard, Hervé Colinet, Mélanie Boël, Stéphane A.P. Derocles, Clémentine Goupil, et al. Multigenerational effects of nanoplastics on life-history traits and physiological responses in Drosophila melanogaster. NanoImpact, 2025, 40, pp.100597. 10.1016/j.impact.2025.100597. hal-05379280

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Research Paper

# Multigenerational effects of nanoplastics on life-history traits and physiological responses in *Drosophila melanogaster*

Chloé M.C. Richard<sup>a</sup>, Hervé Colinet<sup>a</sup>, Mélanie Boël<sup>b</sup>, Stéphane A.P. Derocles<sup>a</sup>, Clémentine Goupil<sup>a</sup>, Théo Laguilliez<sup>a</sup>, David Renault<sup>a,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

Editor: Bernd Nowack

Keywords:

Nanotoxicity

Phenotypic traits

Physiological responses

Terrestrial invertebrates

## ABSTRACT

The ubiquitous presence of nanoplastics (NP) in the environment has emerged as a global concern. Their impacts have been mostly studied in aquatic ecosystems and species, and more recently, in terrestrial species. In addition, the toxicity of NP on organisms exposed continuously to this pollutant and over multiple generations remains poorly considered. Our study focused on the effects of chronic exposure of a polydisperse mix of polystyrene NP (20, 80, and 200 nm) on the fruit fly *Drosophila melanogaster* to four NP concentrations (0, 1, 100, and 500 µg NP. g⁻¹ of food) over 10 generations. Their toxicity was assessed at the first (F1) and tenth (F10) generations by evaluating phenotypic (viability, development time, body size and body weight) and physiological responses, through enzyme activities related to oxidative stress and the expression of specific target genes (e.g. apoptosis, immunity). Our results showed a decrease in organisms’ key traits in the first and tenth generations, with a reduced viability and body size. Additionally, superoxide dismutase (SOD) activity was altered, with a decrease observed in the F1 generation, followed by an increase in F10, in flies exposed to 500 µg NP.g⁻¹. Finally, the expressions of target genes involved in apoptosis and immune responses were either up- or down-regulated, depending on the generation. These results highlighted the effects NP can have on life-history or physiological traits without higher effect across generations. In addition, the study reveals the complex response across generation and highlights the importance to study long-term exposure.

## 1. Introduction

Nanoplastics (NP) are now considered as an emergent environmental pollutant of high concern for human and biodiversity health (Chen et al., 2024a; Sana et al., 2020). NP are tiny plastic particles (<1 µm) (Gigault et al., 2018) comprising primary and secondary NP. Primary NP are intentionally synthesized for industrial purposes and applications such as in personal care and cosmetics products (Hernandez et al., 2017; Lorenz et al., 2011) while secondary NP are generated through the breakdown of larger plastic debris into smaller particles (Blancho et al., 2021). In the environment, several physico-chemical processes, including exposure to sunlight (Andrady et al., 2022), temperature, wind erosion, mechanical action from waves in the ocean, and biological fragmentation through microorganisms (Bacha et al., 2023), contribute to the release of countless numbers of plastic particles of constantly decreasing sizes. Secondary NP can also be generated in smaller environments: for instance, in a cup of tea, a single teabag can approximately

release three billion of NP (Hernandez et al., 2019), and in a washing machine, approximately 3.3 × 10¹¹ NP per gram of washed textile can be produced (Yang et al., 2021). These mechanisms result in a wide diversity of NP with different shapes, sizes and polymer natures, which are subsequently released and dispersed in the environment, thus placing biodiversity at high exposure risk (Renault et al., 2024).

NP are now observed everywhere from urban areas (Kirchsteiger et al., 2023) to agricultural soils (Wahl et al., 2021), remote areas as in the Alps (Materić et al., 2021) and even in South and North polar circles (Materić et al., 2022). In terrestrial environments, the detection of NP remains challenging due to the difficulty of distinguishing carbon-based plastic particles from organic matter (Junhao et al., 2021). Despite these technical difficulties, previous reports revealed that the most observed NP in agricultural soils were composed of polyethylene, polystyrene and polyvinyl chloride (Wahl et al., 2021).

The number of investigations on the effects of NP on terrestrial species is growing rapidly for invertebrates such as nematodes,

<footnote><sup>a</sup> University of Rennes, CNRS, Ecobio ([(Ecosystème, Biodiversité, Evolution)]) – UMR, 6553 Rennes, France</footnote>

<footnote><sup>b</sup> Univ Lyon, Université Claude Bernard Lyon 1, ENTPE, CNRS, INRA, USC 1369, UMR 5023 LEHNA, F-69518 Vaulx-en-Velin, France</footnote>

<footnote>* Corresponding author at: University of Rennes, UMR CNRS 6553 ECOBIO, 263 Avenue du Gal Lecler, CS 74205, 35042 Rennes Cedex, France.
E-mail address: david.renault@univ-rennes.fr (D. Renault).</footnote>
earthworms or insects (Richard et al., 2024). It has been demonstrated that NP-exposed earthworms *Eisenia fetida* internalized NP in their intestinal epithelium and chloragogenous tissue (Tang et al., 2023). Similarly, NP ingested by the fruit fly *Drosophila melanogaster* were observed in the midgut before being internalized by enterocytic cells and hemocytes as they reached the hemolymph (Alaraby et al., 2022). In most invertebrate-related studies, the ingestion of NP did not impair the survival, as reported in the silkworm *Bombyx mori* (Parenti et al., 2020) and *D. melanogaster* (Matthews et al., 2021; Alaraby et al., 2022). However, even if NP ingestion may appear as not lethal, it may affect various life-history traits, with studies reporting delays in development time or body mass modifications (Aloisi et al., 2024; Muhammad et al., 2024). Yet, other studies did not observe NP effects on life-history traits (Alaraby et al., 2022; Parenti et al., 2020). NP ingestion might induce physiological disorders in the form of oxidative stress. For instance, the quantity of Reactive Oxygen Species (ROS) increased in *E. fetida* exposed to various NP concentrations, whereas the activity of two antioxidant enzymes, the catalase and the superoxide dismutase, decreased with NP concentration range and exposure duration (He et al., 2023). The antioxidant activity of *D. melanogaster* exposed to NP was found to increase in the midgut (Urbisz et al., 2024). Expression of genes involved in cellular stress responses (Kültz, 2005) may also be induced following NP exposure. The expression of genes coding Heat shock protein, related to apoptosis, oxidative stress or immunity were either upregulated or downregulated in *D. melanogaster*, *E. fetida* and *B. mori* (Aloisi et al., 2024; He et al., 2023; Matthews et al., 2021; Muhammad et al., 2021).

Most investigations on invertebrates to date have focused on short-term exposure to NP, from a few hours to a single generation (He et al., 2023; Kim et al., 2019; Matthews et al., 2021; Zhu et al., 2018), whereas organisms are potentially chronically exposed to NP in the environment over multiple generations. As a result, although the knowledge gained from such works was crucial for improving our comprehension of NP toxicity, testing the effects of long-term would provide a more realistic assessment. Only recently studies have begun to examine the long-term effects of NP across generations, firstly on aquatic species as *Daphnia pulex* (Liu et al., 2020), and later on terrestrial species such as in *Caenorhabditis elegans* (Liu et al., 2021; Yu et al., 2021) and *D. melanogaster* (Huang et al., 2025; Tu et al., 2023). These two latter studies mainly considered the NP reproductive toxicity over four and five continuously exposed generations. The authors found that NP accumulated in the flies' ovaries, subsequently decreasing egg production across generations exposed to NP concentrations (Tu et al., 2023). In addition, NP damages likely bioaccumulated over generations, and became visible at F4 and F5 generations, as evidenced by a decrease of the eclosion rate and an increase in development time (Tu et al., 2023), suggesting that some exposure duration or levels of bioaccumulation has to be reached before effects could be tracked at the individual level. These earlier studies highlight the need for further research on the effects of multigenerational NP exposure to better understand the toxicity of NP in organisms, beyond five generations, and across a range of phenotypic and physiological traits.

In this study, the effects of a multigenerational polystyrene NP exposure were thus investigated in the fruit fly *D. melanogaster* by measuring key life history traits such as viability, development time, size and body mass, as well as physiological parameters (oxidative stress and expression of stress genes). Polystyrene is a thermoplastic polymer extensively produced for food packaging as containers, bottles and others (Tawfik and Huyghebaert, 1998). Because of its widespread use, polystyrene is found everywhere in the environment, including in agricultural soils (Wahl et al., 2021). The fruit flies are potentially exposed in the environment to plastic particles mostly through ingestion as they feed and develop in decaying fruits (Reaume and Sokolowski, 2006), which may contain micro- and nanoplastics (Oliveri Conti et al., 2020). In addition, *D. melanogaster* has a short life cycle and a high fertility rate, making it an excellent model for experimental tests over multiple generations. In the present study, flies were exposed

continuously over ten generations to a polydisperse suspension composed of three NP sizes, better reflecting environmental NP, which vary greatly in sizes (Wahl et al., 2021). Based on recent findings on *D. melanogaster* using fluorescent NP and transmission electron microscopy (Alaraby et al., 2022; Tu et al., 2023; Urbisz et al., 2024), we assumed that NP were ingested by the flies, as the NP were added directly in the food, with a possible accumulation in tissues. The measurements of life history traits as well as of physiological parameters were performed on the first and tenth generations. NP toxicity was expected to increase across generations due to the assumed bio-accumulation of NP and/or progressively accumulated damages. According to previous studies highlighting no drastic impact on life-history traits (Alaraby et al., 2022; Matthews et al., 2021; Tu et al., 2023), it was hypothesized that NP ingestion would not strikingly impair viability, development time, size/body mass at the first generation (F1). While, we predicted that changes in the level of oxidative stress and stress genes expression would be detectable as soon as the flies were exposed to NP (Alaraby et al., 2022; Aloisi et al., 2024). Since stress responses are energetically costly, the amount of energy used in repairing the damage caused by oxidative stress will increase at the likely expense of other energy-dependent functions (such as the reproduction or survival). As the result, after a prolonged NP-exposure over ten generations, it was expected that the life-history traits would be altered.

## 2. Material and methods

### 2.1. Rearing of *Drosophila melanogaster*

Experiments were conducted on a wild population of *D. melanogaster* established in September 2015 from the collection of 500 to 1000 flies in Planoët (Brittany, France) (Henry et al., 2020). Individuals were maintained in 100 mL glass bottles containing standard fly medium composed of agar (15 g/L), sugar (50 g/L), dry active yeast (80 g/L) and nipagin (1 g/L). For the rearing and all experiments, flies were maintained in thermoregulated incubators (model TC 255 S, Lovibond) at 25 ± 1 °C in a 12 h light/12 h dark cycle.

### 2.2. Preparation of NP-contaminated food

Polystyrene NP suspensions at 1 % solid content and particle sizes of three different sizes (20, 80 and 200 nm) were used (ThermoFisher Scientific, PS NIST, Catalog number 3020 A, 3080 A and 30200 A). According to the manufacturer, additives such as Tween-20 and Triton X-100 are present in these commercial suspensions at very low concentrations to prevent particle aggregations. Prior experiments confirmed the absence of toxicity of these two surfactants at low concentrations (Richard et al., 2025b). Nanoparticles of the three sizes were mixed in equal volume to obtain a polydisperse suspension, which is more environmentally relevant (Wahl et al., 2021). The concentration of the stock solution was 10 mg.mL⁻¹ of NP, and the size, shape and polymer nature were characterized as previously described in (Richard et al., 2025a, 2025b)). A series of 10-fold dilutions in ultrapure water were performed to obtain solutions from 1 mg.mL⁻¹ to 1 µg.mL⁻¹. Finally, stock or diluted NP-suspensions were added directly in the nutritive diet to obtain contaminated food at 0, 0.01, 0.1, 1, 10, 100 and 500 µg NP.g⁻¹ of food. As the quantification of NP in complex environmental matrices remains very challenging, this wide range of concentrations was selected to cover both low levels, which may occur in low polluted areas, and high levels, such as 500 µg of NP.g⁻¹, which may be observed in highly polluted areas (Wang et al., 2025). Vials (25 × 95 mm) were filled with 5 mL of controlled or contaminated food.

### 2.3. Rearing of the flies for the multigenerational experiment

The flies were exposed to polystyrene NP throughout their entire
development and continuously over ten generations. For the first gen-
eration (F1), a large range of NP concentrations was used, e.g. 0, 0.01,
0.1, 1, 10, 100 and 500 µg NP.g⁻¹ to examine the effects of NP ingestion
on life-history traits. For the enzymatic assays and genes expression, a
restricted number of NP concentrations was used: 0, 1, 100 and 500 µg
NP.g⁻¹. The selection of these concentrations was based on the results
obtained on life history traits. The same concentrations (0, 1, 100 and
500 µg NP.g⁻¹) were tested at generation 10 (F10) for all traits, enzy-
matic assays and genes expression, as the continuous exposure was
highly demanding. The experiments were performed on the F1 and F10
generations to assess the effects after both a relatively short generational
exposure and a prolonged generational exposure. For logistical and
resource constraints, NP effects could not be observed in each genera-
tion, which might have provided a more comprehensive understanding
of their effects. At emergence of the first generation, approximately 50
adults of each NP concentration were placed in vials (25 × 95 mm) filled
with 3 mL of contaminated food, and food vials were replaced every two
days. After 4 days, mature adults were transferred into new vials filled
with 5 mL of food to lay eggs for two hours to produce the next gener-
ation. The process was repeated until the tenth generation (F10). For
controls, the exact same procedure was applied except that food was not
contaminated with NP.

## 2.4. Life-history traits

Several experiments on life-history traits were performed on F1 and F10. We assessed egg to pupa viability, egg to adult viability, body mass and size. Since there was no constraint on the number of individuals or available time in F1, we were able to replicate the life-history trait assays. In contrast, due to limited sample availability and temporal constraints in F10, these assays were conducted only once. Prior to the F1 and F10 experiments, approximately 200 4-day-old females from respectively the rearing stock, and from the F9 flies, were placed in egg-laying cages on grape agar medium (Genesee Scientific) for 2 h to collect their eggs.

### 2.4.1. Viability and development time

To assess viability and development time, 20 eggs were deposited in each vial containing either control or contaminated food. Ten replicates (n = 10; N = 200) were performed for each concentration (i.e. 0, 0.01, 0.1, 1, 10, 100 and 500 µg NP.g⁻¹) at F1 and all these assays were replicated two times. At F10, 20 replicates (n = 20; N = 400) were performed for each concentration (0, 1, 100 and 500 µg NP.g⁻¹). The viabilities from eggs to pupae and from eggs to adults were assessed by determining respectively the proportion of pupae and adults produced per vial for 20 deposited eggs. At the onset of emergence, emerged adults were checked every 2 h during the day, between 8 a.m. and 8 p.m., and removed in order to record the time between the deposition of eggs and the emergence. After 24 h of maturation, emerged adults were stored at -20 °C for morphological measurements.

### 2.4.2. Morphological measurements

The size of the flies was assessed by measuring the tibia length as a proxy (Siomava et al., 2016). For these measures, 30 females were randomly collected (n = 30 for the F1 and n = 60 for the F10). The left hind leg of each female was dissected, and mounted between a slide and a slip cover, to measure the tibia length with a Leica device (Leica M205 C) combined with a camera (Leica DMC4500). Then, the same flies were dried at 60 °C for 72 h to assess individual dry mass with a microbalance XP2U (Mettler Toledo, precision 1 µg).

## 2.5. Enzymatic assays

The level of oxidative stress was indirectly measured by recording
the activities of two antioxidant enzymes: the catalase (CAT) and the
superoxide dismutase (SOD). The experimental flies were obtained by

depositing 70 eggs on either controlled or contaminated food (i.e. 0, 1,
100 and 500 µg NP.g⁻¹ at F1 and F10). Four-day-old matured females
were grouped by 5 in microtubes (1.5 mL) and stored at -80 °C after
snap freezing in N₂. Briefly, the flies were homogenized in a phosphate
buffer (90 mL phosphate buffer at 0.1 M, 10 mL of glycerol (CAS 56-81-
5, Sigma-Aldrich) and 0.022 g of 1,4-dithioerythritol (CAS 6892-68-8,
Sigma-Aldrich); pH = 7, w/v = 1:50) using 15 lysing silica beads. The
homogenate was centrifugated at 14,000 rpm at 4 °C for 20 min to
subsequently collect the supernatant. A spectrophotometer was used to
monitor enzyme activities. For each concentration, 8 biological repli-
cates were performed with at least 2 technical replicates.

CAT catalyzes the decomposition of hydrogen peroxide (H₂O₂) into water and dioxygen and its activity was measured as described by Aebi (1984) with minor modifications. CAT activity was assessed by the consumption of H₂O₂ at 240 nm over 3 min, with a measurement every 10 s. Microplate wells were filled with 185 µL of 0.05 M of phosphate buffer, 10 µL of sample (diluted by ¼), and 105 µL of diluted H₂O₂ (CAS 7722-84-1, Sigma-Aldrich). A blank was performed for each sample without H₂O₂ and subtracted to the values. CAT activity was calculated using Beer-Lambert equation adapted to microplates.

SOD catalyzes the dismutation of the superoxide anion in H₂O₂ and dioxygen and its activity was measured as described by Giannopolitis and Ries (1977). Briefly, SOD activity was assessed by the disappearance of the nitroblue tetrazolium (NBT). Microplate wells contained 170 µL ultrapure water, 30 µL 0.05 M phosphate buffer, 30 µL 130 mL methionine (CAS 63-68-3, Sigma-Aldrich), 30 µL 750 µM NBT (CAS 298-33-9, Sigma-Aldrich), 10 µL sample and 30 µL 20 µM riboflavin (CAS 83-88-5, Sigma-Aldrich). The microplate was placed under uniform light for 12 min to initiate the reaction. In addition, two blanks were performed without sample and without NBT and then subtracted to the sample. Finally, one unit of SOD was defined as the amount of enzyme required to inhibit the reduction of NBT by 50 %.

The enzyme activities were standardized by the protein content of
each sample measured with Bradford method (Bradford, 1976) by
measuring absorbance at 595 nm.

## 2.6. Genes expression

### 2.6.1. RNA extraction and cDNA synthesis

Ten adult flies were ground to fine powder using pestles in 1.5 mL tubes placed in liquid nitrogen. Samples were mixed with 350 µL of lysis buffer (containing 1 % β-mercaptoethanol) from NucleoSpin RNA extraction kit (Macherey-nagel, Germany). RNA extraction and purification were performed using a NucleoSpin spin column following the manufacturer's instructions. Besides the on-column DNA digestion, any potential genomic DNA was removed using an rDNase (Macherey-nagel, Germany). Total RNA was eluted in 50 µL of diethylpyrocarbonate-treated water. The quality and concentration of the obtained RNA was then measured by NanoDrop spectrophotometer ND-1000 (NanoDrop Technologies,USA). 1000 ng of total RNA was used in the reverse transcription to cDNA, using Superscript III first-strand synthesis system (Invitrogen Pty) following manufacturer's instructions. The cDNA was then diluted 3-fold in diethylpyrocarbonate-treated water and stored at -20 °C until use. For each concentration, five independent biological samples were performed (n = 5).

### 2.6.2. Housekeeper (HK) genes selection & qRT-PCR protocols

We targeted 17 target genes for qRT-PCR (Table SA) involving gen-
eral stress-response genes (Hsp23, Hsp70), antioxidant enzymes (Cat,
Sod) regulating ROS detoxification, apoptosis regulators (Dronc, Drice,
Damm, Dcp-1, Diap-1, Decay, Dredd, Dream, Reaper), which can be acti-
vated by oxidative stress or mitochondrial damage, and immune-related
genes (AttA, LysD, TotA, TotX). These pathways are functionally con-
nected: oxidative stress can induce apoptosis and modulate immune
signaling, while heat-shock as well as Tot proteins mediate stress
tolerance and cellular protection. We focused on these genes as they
provide a targeted overview of molecular responses potentially affected by NP (Alaraby et al., 2022; Aloisi et al., 2024; Matthews et al., 2021; Muhammad et al., 2021; Zhou et al., 2023). We also tested 2 housekeeping (HK) genes (RpS20, RpL32) and the best HK gene, RspS20, was selected as the most stable based on the RefFinder algorithm (Xie et al., 2012). A Roche LightCycler® 480 (Roche, Basel, Switzerland) using SybrGreen I mix (Roche) was used to perform qPCRs, following the protocol described in Wiil et al. (2023). Briefly, the PCR reaction mixture contained 10 µL in total, including 5 µL Green I Master mix, 4 µL of gene-specific primers and 1 µL of cDNA template. The thermal cycling conditions were as follows: 1 cycle of 95 °C for 10 min, 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 15 s. The expression level of the

target genes was transformed into fold change relative to the untreated control and normalized using the HK gene using the ΔΔCt technique (Pfaffl, 2001). To validate the specificity of amplification, a post-amplification melt curve analysis was performed as described in Colinet et al. (2010a). All qRT-PCR assays yielded specific products (i.e. single melting peak).

## 2.7. Statistical analyses

All statistical analyses were performed on R studio Version 4.2.1 (R Core Team Year, 2022). Linear models or generalized linear models were used with lme4 and nlme packages and by adding a random effect

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Life-history traits of flies exposed to nanoplasics for one generation (F1). Percentage of viability from A) eggs to pupae and B) eggs to adults exposed to nanoplasics (n = 20, N = 400); C) Density plot of the development time from eggs to adults for flies exposed to nanoplasics (n = 400); D) Dry body mass of flies exposed to nanoplasics (n = 60); E) Measurements of tibia length, i.e., size of flies exposed to nanoplasics (n = 60). Black dots represent each replicate. Different letters indicate significant differences among NP concentrations (Estimated marginal means test with Tukey correction, *p* < 0.05).</figcaption></figure>
for batch in the F1 generation to account for the two replicated exper-
iments assessing viability, development time, size, and body mass. For
viability (egg to pupa and egg to adult), a generalized linear model was
performed with a binomial family for F1 and F10, as these outcomes
represent binary data (survived vs. dead). For the development time, a
double approach was adopted. First, a generalized model with a gamma
family, which is appropriate for continuous, positive-valued time-to-
event data, was performed. Second, a Kolmogorov-Smirnov test was
conducted to assess temporal distribution variations between condi-
tions. Pairwise comparisons were performed for the test and adjusted
with the Bonferroni correction. For morphological measurements and
antioxidant activities, linear models were performed for each trait. For

genes expression, a linear model was performed for each gene to
compare the three NP concentrations, and t-tests were performed to
evaluate the difference with the control. After computing all the models,
estimated marginal means (EMMs) with Tukey correction were per-
formed for pairwise comparisons.

3. Results

3.1. *Life-history traits*

3.1.1. Viability from egg to pupa, egg to adult, and development time

For the first generation, the viability from egg to pupae decreased

<figure><img src="image_5.png" /><figcaption>Fig. 2. Life-history traits of flies exposed to nanoplastics for ten generations (F10). Percentage of viability from A) eggs to pupae and B) eggs to adults exposed to nanoplastics (n = 20, N = 400); C) Density plot of the development time from eggs to adults for flies exposed to nanoplastics (n = 400); D) Dry body mass of flies exposed to nanoplastics (n = 30); E) Measurements of tibia length, i.e., size of flies exposed to nanoplastics (n = 30). Black dots represent each replicate. Different letters indicate significant differences among NP concentrations (Estimated marginal means test with Tukey correction, p < 0.05).</figcaption></figure>
with increasing NP concentrations (glmer, $\chi^2 = 22.15$, Df = 6, *p* = 0.001; Fig. 1A). Pupal viability was the lowest at the highest NP concentration (500 µg NP.g⁻¹) (Fig. 1 A). The viability from eggs to adults was also affected by NP (glmer, $\chi^2 = 38.89$, Df = 6, *p* < 0.001; Fig. 1B). The adult viability was decreased at the highest NP concentration (500 µg NP.g⁻¹) with 59.50 ± 2.76 % of viability compared to the control with 69.50 ± 3.89 %, corresponding approximately to a decrease of 10 %. The development time was also affected by NP concentrations (glmer, $\chi^2 = 15.48$, Df = 6, *p* = 0.02). The emergence of flies was delayed at 500 µg NP.g⁻¹ (Fig. 1 C). Flies' emergence ranged from 231.8 ± 0.57 h to 232.9 ± 0.53 h when exposed to NP concentrations of 0 to 10 µg NP.g⁻¹. The flies emerged after 234.8 ± 0.62 h at the highest concentration. However, the temporal distribution pattern did not differ between conditions (Kolmogorov-Smirnov test, *p* > 0.01).

For the tenth generation, the viability from eggs to pupae was not altered by NP (glm, $\chi^2 = 0.15$, Df = 3, *p* = 0.99, Fig. 2A), while the viability from eggs to adults was affected (glm, $\chi^2 = 12.32$, Df = 3, *p* = 0.006). The viability was decreased at the highest NP concentration (500 µg NP.g⁻¹) with a viability of 75.75 ± 2.62 % compared to the control group with 85.28 ± 1.43 %, corresponding approximately to a decrease of 10 % (Fig. 2B). Development time was not delayed by NP (glm, $\chi^2 = 5.91$, Df = 3, *p* = 0.12, Fig. 2 C) with the control group emerging around 217 ± 0.87 h. However, the temporal distribution pattern differed between conditions with a significative difference between the control and all the NP concentrations (Kolmogorov-Smirnov test, *p* < 0.01), but also between the low concentration (1 µg NP.g⁻¹) and the highest NP concentrations (100 and 500 µg NP.g⁻¹) (Kolmogorov-Smirnov test, *p* < 0.01).

### 3.1.2. Morphological measurements

For the first generation, the dry body mass was affected by NP

concentration (lmer, $\chi^2 = 13.129$, Df = 6, *p* = 0.04): lower dry mass was measured at low and intermediate NP concentrations (0.1 and 10 µg NP.g⁻¹) (Fig. 1 D). The tibia length was also decreased (lmer, $\chi^2 = 14.891$, Df = 6, *p* = 0.02) when the NP concentration was increased (except for 100 µg NP.g⁻¹) (Fig. 1 E). For the tenth generation, the dry body mass remained similar over the range of tested NP concentrations (lm, F = 0.58, Df = 3, *p* = 0.63), whereas the tibia length was affected (lm, F = 4.14, Df = 3, *p* = 0.007) with a length reduction at 500 µg NP.g⁻¹ compared to the other NP concentrations and the control (Fig. 2 D & 2E).

## 3.2. Enzymatic assays

For the first generation, the superoxide dismutase (SOD) activity was affected by NP concentration (lm, F = 3.20, Df = 3, *p* = 0.04): its activity was decreased at the highest NP concentration (500 µg NP.g⁻¹ of food) (Fig. 3 A). For the CAT activity, a trend towards a reduction in activity at the highest NP concentrations compared to the control was observed (lm, F = 2.71, Df = 3, *p* = 0.06), but this pattern was not significant (Fig. 3 B). For the tenth generation (F10), SOD activity was also disturbed in NP-exposed flies (lm, F = 5.23, Df = 5.23, *p* = 0.005), but with an opposite pattern was recorded in comparison to the first fly generation. The SOD activity was increased at 500 µg NP.g⁻¹ (Fig. 3 C). CAT activity was not impaired in NP-exposed flies (lm, F = 2.09, Df = 3, *p* = 0.12; Fig. 3 D).

## 3.3. Gene expression

For F1, several genes (*Hsp23*, *Cat*, *Sod*, *Dronc*, *Dcp-1*, *Diap-1*) were up-regulated in flies exposed at the highest NP concentration (500 µg NP.g⁻¹) compared to control flies (Fig. 4 & Table SB). *TotA* was more expressed at higher NP concentrations (100 and 500 µg NP.g⁻¹). *LysD*

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Activity of two antioxidant enzymes: A) the superoxide dismutase (SOD) (U/mg of protein) and the B) catalase (CAT) (nmol/min/mg of protein) of flies exposed to nanoplastics for one generation (F1) and C) SOD and D) CAT activities of flies exposed to nanoplastics for ten generations (F10). Black dots represent each replicate (n = 8) composed of five flies. Different letters indicate significant differences among NP concentrations (Estimated marginal means test with Tukey correction, *p* < 0.05).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Fold change variations of gene expression relative to control flies exposed to nanoplastics for one generation (F1) (with standard errors). A star corresponds to a significative difference with the control (t-test, p < 0.05). The difference between concentrations (Estimated marginal means test with Tukey correction, p < 0.05), when significant, are presented in Table SB (Supplementary material).</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Fold change variations of gene expression relative to the control of flies exposed to nanoplastics for ten generations (F10) (with standard errors). A star corresponds to a significant difference with the control (t-test, p < 0.05). The difference between concentrations (Estimated marginal means test with Tukey correction, p < 0.05), when significant are presented in Table SC (Supplementary material).</figcaption></figure>
was upregulated at 100 µg NP.g⁻¹ with a fold change around 5. On the contrary, *AttA* was down-regulated at the highest NP concentration (Fig. 4).

For the tenth generation, three genes were up-regulated after NP exposure (*Cat*, *Drice* and *Diap-1*) for 2 or 3 NP concentrations (Fig. 5 & Table SC). For the other genes, except *Dronc* and *Dcp-1*, their expressions were down-regulated at different NP concentrations (Fig. 5 & S1). For instance, expressions of *Hsp23*, *Hsp70*, *Sod*, *Decay*, *Damm*, *Dream*, *Reaper*, *TotX*, *AttA* and *LysD* were down-regulated at the lowest NP concentration. In addition, *Reaper* and *TotX* were down-regulated at the medium NP concentration (100 µg NP.g⁻¹) whereas *Hsp23*, *Sod*, *Decay*, *Damm*, *Dream*, *Reaper*, *TotA* and *TotX* were down-regulated at the highest NP concentration (500 µg NP.g⁻¹).

## 4. Discussion

The effects of polystyrene NP were assessed in the fruit fly over ten generations by examining a range of life-history traits and physiological parameters (oxidative stress and expression of genes involved in stress response). In the first NP-exposed fly generation, almost all life-history traits were affected by NP ingestion, although that was not expected. A decrease in viability (egg to pupa and egg to adult) and a delayed emergence time were observed at the highest NP concentration (500 µg NP.g⁻¹) compared to the control and/or low NP concentrations. In most of the available literature, such deleterious effects were not observed, with former investigations showing that NP ingestion did not impair the viability nor emergence time in *D. melanogaster* (Alaraby et al., 2022; Matthews et al., 2021; Tu et al., 2023) and *B. mori* (Muhammad et al., 2021; Parenti et al., 2020). However, a recent study reported a delay in pupal and adult emergence time in *D. melanogaster* exposed to polystyrene NP from 50 to 750 µg.mL⁻¹ (Aloisi et al., 2024), and our finding thus confirm this pattern. Moreover, tibia length, a proxy for adult fly body size, was decreased at medium and high NP concentrations, thus suggesting a reduced size of the flies (Siomava et al., 2016). Consistent with this result, body mass was also decreased at moderate NP concentrations, but not at the highest NP concentration where the increased development time may have permitted higher body mass. Interestingly, in some terrestrial invertebrates exposed to polystyrene NP, the body mass and size were not impaired (Matthews et al., 2021; Parenti et al., 2020). Yet, a decrease in body mass was observed in *E. fetida* after 28 days of NP exposure (He et al., 2023) and also in *D. melanogaster* (Aloisi et al., 2024). Our result evidences that NP exposure over a single generation can induce subtle disturbances detectable at the individual level. The distinction with the existing literature on NP toxicity on terrestrial invertebrates, including *D. melanogaster*, with no effect on viability or development time after one exposure generation (Tu et al., 2023), may be due to our use of a polydisperse NP suspension, which better reflects environmental exposure (Wahl et al., 2021). As the toxicity of plastic particles can vary with the size of plastic debris (Kim et al., 2020a, 2020b; Urbisz et al., 2024), a mixture made of different plastic particle sizes may enhance their toxicity. In addition, our results highlighted some dose-dependent effects of NP with a higher toxicity at the highest concentrations, which is consistent with former works (He et al., 2023; Kim et al., 2020a, 2020b).

NP toxicity was expected to increase across generations due to the assumed bioaccumulation of NP and/or progressively higher damage. After ten generations of continuous NP exposure, significant decreases in the egg to adult viability and tibia length were detected at the highest NP concentration (500 µg NP.g⁻¹). In addition, the temporal distribution pattern differed between the control and NP concentrations, showing a shift towards a delay in emergence when the NP concentrations increased. It is also interesting to note that the development time globally decreased and the viability (for instance from eggs to adults) increased from the F1 to the F10, as highlighted by the results on the control group. To establish each new fly generation, flies that emerged after 9/10 days were collected, which is the typical emergence time at

25 °C. By doing so, the small fraction that might have emerged after 10 days was excluded, and as the procedure was repeated over ten generations, it may have amplified the differences between the F1 and F10, with flies emerging earlier and with a higher viability. However, no effects were found in the egg to pupa viability and the body mass. In another drosophila study, the continuous exposure – across five generations – resulted in a cumulative increase of damages: the viability (egg to adult) decreased by 9 % at F5, and the adult emergence was delayed by approximately 2.35 days for flies exposed to one NP concentration of 100 mg.L⁻¹. Interestingly, no effect was reported in the former fly generations from F1 to F3 (Tu et al., 2023). Even at F4 generation, NP exposure was not found to alter the reproductive system of male *D. melanogaster* flies after a continuous exposure (Huang et al., 2025). As NP were detected in female ovaries, cumulative NP effects were attributed to the progressive alteration of the reproductive system which required to reach a threshold before becoming detectable (Tu et al., 2023). In our study, it is possible that flies progressively acclimated to NP exposure, with individuals adjusting physiologically to better tolerate plastic contamination over successive generations, a long process which was not observable within the first generations as reported by Tu et al. (2023). Consistently, the brood size of the nematode *C. elegans* was more impacted by exposure during the first generations (F0-F2) than in the latter ones (Yu et al., 2021), suggesting that a potential acclimation might occur through generations. Whatever the mechanisms, our study shows that some key traits, such as viability from eggs to adults and body size, were reduced at the F10, indicating persistent consequences. However, the underlying mechanisms of such toxicity were unclear and needed further investigations at sub-individual levels.

In all aerobic organisms, under normal conditions, ROS are continuously generated as byproducts by respiration and metabolic reactions (Sies, 1991), without leading to oxidative stress thanks to the antioxidative system (Prokić et al., 2019). Environmental contaminants such as NP can disrupt this balance and lead to oxidative stress in exposed organisms, either by increasing the ROS quantity or by disturbing the activities of antioxidant enzymes (He et al., 2023; Lei et al., 2018; Zhou et al., 2023). Increased ROS production not only elevates oxidative stress but also causes physiological disorders by damaging macromolecules and cells (Hu and Palić, 2020), with potential effects at the organism level. Here, the activity of two antioxidant enzymes was assessed after NP exposure: the SOD, which catalyzes the dismutation of the superoxide anion into H₂O₂ and the CAT, which converts the H₂O₂ into oxygen. After just one generation of NP exposure, the SOD was reduced at 500 µg NP.g⁻¹, and a trend towards a reduction for CAT activity at 100 µg NP.g⁻¹ was observed, without being significative. A decrease in the activity of these two enzymes was reported in *B. mori* (Parenti et al., 2020) and *E. fetida* (He et al., 2023; Zhou et al., 2023) after NP exposure of several weeks. In fruit flies, the total antioxidant activity was reported to be altered in the midgut and reproductive organs after NP exposure on adults for 28 days (Urbisz et al., 2024). In most of the cases, whether the antioxidant system is upregulated or downregulated, the quantity of ROS was found to increase in *D. melanogaster* (Alaraby et al., 2022; Urbisz et al., 2024) or in *C. elegans* (Kim et al., 2020a, 2020b; Qiu et al., 2020) after exposure to NP over several weeks. Oxidative stress results from the imbalance between ROS production and antioxidant defense capacity, including reduced activity of antioxidant defenses against free radicals such as SOD (Poljsak et al., 2013; Prokić et al., 2019). Thus, a decrease in SOD activity may disturb the antioxidant balance, potentially leading to elevated ROS levels and oxidative in the fruit flies following NP ingestion, probably damaging macromolecules as lipids and DNA (He et al., 2023).

Surprisingly, after ten generations of exposure, CAT activity was similar to control flies whereas SOD activity increased in a dose-dependent manner, showing an opposite pattern compared to the F1. The opposite patterns of SOD activity between acutely exposed (F1) and chronically exposed (F10) flies suggest that initial oxidative stress triggers an immediate antioxidant response, while long-term exposure may
lead to different physiological responses resulting in different antioxidant regulation. These results support the idea that antioxidant defense responses are not uniformly up- or downregulated over generations. Similar generation-dependent patterns of SOD and CAT activity, alternating between stimulation and inhibition, have been reported in *C. elegans* exposed to N-butylpyridinium chloride for 14 generations (Shi et al., 2021) and to antibiotics for 9 generations (Zheng et al., 2022). Likewise, a continuous exposure over three generations led to increase and decrease of *Cat* and *Sod* levels in *Daphnia pulex* (Liu et al., 2020). In addition, the consecutive exposure of *Daphnia magna* to triclosan for three generations triggered a decrease in ROS levels at the last generation leading to reduced effects on phenotypic traits (Lee et al., 2023). Further investigations are needed to determine if these generation-dependent opposite patterns result from a tolerance acquired from acclimation, which is a reversible response to NP ingestion, or adaptation, which involves genetic changes across generations (Collier et al., 2019).

The responses in antioxidant enzymes at the F10 observed in our study could mitigate the negative effects of NP on the viability from eggs to pupae, development time and body mass observed in the first generation. However, the increase in antioxidant defenses is likely energetically costly (Pamplona and Costantini, 2011), and may partially contribute explaining the decrease in viability and body size of the flies at the F10 generation. Similar trade-off for energy investment was already observed in *C. elegans* exposed to environmental pollutant (Shi et al., 2021) or in the fish *Oryzias latipes* where the increase in SOD activity led to a decrease in fecundity (Hemmer-Brepson et al., 2013).

While we expected life-history traits to be only minimally or not affected after a single generation of NP exposure, we predicted that, at the sub-individual level, changes in oxidative stress levels and stress gene expression would be detectable as soon as the flies were exposed to NP. Indeed, NP may alter the expression level of genes associated with stress responses which encode proteins involved in global stress tolerance, oxidative stress, cell death and immunity pathways (Matthews et al., 2021; Muhammad et al., 2021; Zhou et al., 2023). After a single exposure, several genes (*Hsp23*, *Cat*, *Sod*, *Dronc*, *Dcp-1*, *Diap-1*) were upregulated at the highest NP concentration (500 µg NP.g⁻¹). Heat shock protein 23 is a key molecular chaperone involved in proteostasis and responds to a wide range of stressors, including heat, cold and hypoxia exposure (Azad et al., 2009; Carmel et al., 2011; Colinet et al., 2010b), and our results suggest that it is also responsive to NP exposure. This suggests that NP exposure may disrupt normal protein folding and/or induce protein denaturation or aggregation. Indeed, NP are prone to interact with proteins, and this interaction may change the functionally crucial secondary structure of these biomolecules, and thereby denature them (Hollóczki and Gehrke, 2019).

The over-expression of the *Cat* and *Sod* genes at 500 µg NP.g⁻¹ aligns with previous reports in *E. fetida* (He et al., 2023), but contrasts with patterns reporting down-regulation in *B. mori* (Muhammad et al., 2021) and in *D. melanogaster* after NP exposure (Alaraby et al., 2022). Matthews et al. (2021) did not observe changes in expression levels of *Sod* and *Cat* in response to NP. Despite the upregulation of *Sod* gene, the activity of its associated enzyme was decreased in F1. In addition, despite the upregulation of *Cat* gene at high NP concentration compared to the control and low concentrations, the activity of the CAT remained unchanged. It is important to note that the transcript level and the enzyme activity are not necessarily correlated in a direct or linear way. Suarez and Moyes (2012) highlighted that there is no strict stoichio-metric relationship between the transcript and the protein activity, and only 40 % of the protein content could be explained through mRNA abundances (Vogel and Marcotte, 2012). Several factors may contribute to this discrepancy: first, transcriptional and translational processes have different temporal scale for their production and degradation. mRNA has a shorter half-life compared to proteins (Suarez and Moyes, 2012). As our sampling at the F1 and F10 generations was performed at one single point, without a multiple time-scale sampling, it is difficult to

establish clearly a relationship between the transcript and the protein. Second, post-translational regulations (Limaye et al., 2003) or enzyme modification by oxidative stress can modulate the activity of the enzyme activity independently of transcription (Overgaard et al., 2014). For instance, Limaye et al. (2003) reported CAT activity on diabetic rats with a decrease in CAT transcript but an increase in the enzyme activity. Finally, in the F1 generation, the SOD activity was decreased in response to NP exposure suggesting that the upregulation of its associated gene may serve as a compensatory response to early enzyme inhibition or oxidative stress, and inversely for the F10.

The NP ingestion might also trigger cell death mechanisms in the fruit fly with perturbations of gene expression involved in the transcription of apoptosis caspases or in its inhibition (Figure SA). For instance, *Dronc* encodes an initiator caspase which triggers the activity of effector caspase encoded by *Dcp-1* (Figure SA) (Kumar and Doumanis, 2000). In our study, both *Dronc* and *Dcp-1* were expressed in response to NP at the highest concentration compared to the control and 100 µg NP. g⁻¹. Another study also reported that *Dronc* expression increased in *D. melanogaster* after NP exposure, leading to cell death in the intestine (Aloisi et al., 2024). Together, these findings highlight that NP ingestion can activate key apoptotic pathways, suggesting that it may compromise cellular integrity and contribute to tissue damage in *D. melanogaster* after only one generation of exposure.

Finally, the immunity defenses were disturbed in NP-exposed fruit flies. *TotA* expression, involved in innate immunity, was upregulated in NP conditions (Agaisse and Perrimon, 2004; Wiil et al., 2023), and the expressions of two genes involved in immune (*LysD*, Marra et al., 2021) and antibacterial responses (*Atta*, Hanson and Lemaitre, 2020) were altered after NP ingestion. The innate immune system was also altered in *C. elegans* (Li et al., 2020; Qiu et al., 2020) and in the silkworm *B. mori* (Muhammad et al., 2021) exposed to NP. These changes can further weaken the organisms against pollutants or microbial exposures. For instance, *C. elegans* was more sensitive to fungal infection after NP ingestion due to perturbations in the innate immune system (Li et al., 2020). Thus, a single exposure to NP in the fruit fly triggered physiological disorders affecting molecular, cellular and biological functions, either directly impacting the life-history traits or disrupting energy allocation.

After 10 generations of NP exposure, the expression of a higher number of stress-related genes were altered compared to the first fly generation. In fact, apart from *Dronc* and *Dredd*, the expression of all other stress genes was altered (either up- or downregulated). In addition, in the F1, the gene expressions were mostly affected at the highest concentration, whereas at the F10, disruptions were also observed at low or moderate concentrations. The expression patterns of molecular chaperones and genes coding for antioxidant enzymes were significantly altered at F10, as in the F1 generation, but with opposite patterns for *Hsp23* and *Sod*. Similar patterns were observed in *D. pulex* exposed to NP for three generations with either up- or downregulated genes such as *Hsp70*, *Sod* and *Cat* depending on the generation (Liu et al., 2020). Here again, the expression of *Sod* gene was downregulated whereas the activity of the associated enzyme was increased in the F10. Several factors, already discussed for the F1, could explain this observation such as the different temporal scales between transcripts and enzymes but also transcriptional, translational and degradation regulations that may modulate the SOD activity. In F10, the observed increase in enzymatic activity despite lower transcription could reflect a long-term adaptation involving enhanced protein stability, accumulation, or post-translational activation of SOD in response to chronic NP exposure. The increase enzyme activity may parallel the effects observed on life-history traits with a decrease in viability and body size, altogether suggesting that deleterious NP effects could be measured with different biological and physiological metrics. Gene expressions related to apoptosis were also disturbed with a decreased expression of pro-apoptosis genes (*Reaper*), and an increased expression of *Diap-1* which is an apoptosis inhibitor (Figure SA). Again, this point to disruptions in
cell deaths mechanisms after long-term exposure to NP was observed in *D. melanogaster* with ovarian apoptosis (Tu et al., 2023). Interestingly, we observed a greater number of genes with altered expression at F10 compared to F1. This raises the question of whether this reflects increased cellular damage accumulating over generations or the activation of additional stress-response and tolerance pathways that develop progressively.

The multigenerational effects observed may result from various underlying mechanisms involving direct actions or compensatory mechanisms. First, it is possible that NP accumulate in the gonads of parents, altering gamete quality (Cui et al., 2017; Gonçalves et al., 2025; Tu et al., 2023). A maternal contribution could explain the multigenerational effects: Tu et al. (2023) showed an accumulation of NP in the ovaries of *D. melanogaster*. A second possible mechanism induced by a prolonged exposure could be based on transcriptional changes linked to epigenetic regulations. These mechanisms alter gene expression without changing the DNA sequence, for example through methylation or histone modifications (Sharavanan et al., 2020). The presence of epigenetic regulations has been observed in *D. melanogaster* exposed to MP (Zhang et al., 2020) and in *C. elegans* exposed to NP (Yu et al., 2021). Finally, it is very likely that multigenerational effects result in part from the accumulation of transgenerational alterations from previous generations. From the first generation of exposure, NP induced changes in SOD activity and gene expression disturbances. In the absence of complete repair of this damage, it can be passed on to the next generation, which is also exposed to NP, reinforcing the effects or inducing other mechanisms such as adaptation/acclimation. To disentangle these possibilities, more targeted studies using cell lines to directly visualize cellular damage, as well as tissue-specific approaches and transcriptomic approaches are needed to better understand the mechanisms involved. In addition, quantifying NP in the flies at the F1 and F10 generations would provide valuable insights into their effects, although it remains very challenging to quantify NP within organisms as current methods require further development.

Finally, our findings indicated that multigenerational exposure influenced both phenotypic and physiological traits in the fruit fly. Long-term exposure experiments are essential to better reflect environmental exposure, as organisms are likely subjected to continuous NP exposure in the environment. However, our study had limitations in ecological realism which could be improved. First, the experiments were performed over a large concentration range from 0 to 500 µg.g⁻¹. These doses were relevant to detect biological effects but it raised concerns about their environmental relevance. It is still very challenging to quantify NP in terrestrial compartments mostly due to technical limitations, which prevents to access to reliable reference values for ecotoxicological studies. However, based on Wang et al. (2025), low concentrations (1 and 10 µg.g⁻¹) may correspond to low polluted areas whereas higher concentrations (100 and 500 µg.g⁻¹) may better represent high polluted concentrations. In addition, it is important to note that even if some of the tested concentrations may appeared elevated today, they could unfortunately become realistic in the future due to the ongoing fragmentation of the existing plastics and the continuous addition of new plastic waste. Then, our results may be underestimated compared to environmental conditions because NP beads were used in this study, whereas environmental NP vary in shapes, sizes and polymer nature (Blanco et al., 2021). This diversity could be more harmful to the living organisms but also enhance their affinity with other pollutants (Chen et al., 2024b; Davranche et al., 2019).

## 5. Conclusion

This study demonstrates that polystyrene NP cause significant multigenerational effects on *D. melanogaster*, impacting both life-history and physiological traits. Notably, while phenotypic traits showed no decline across generations, complex and contrasting molecular and antioxidative responses reveal the complex influence of chronic NP

exposure. The quantification of NP content in flies, which remains a significant challenge, would provide valuable insights into NP effects across generations. These findings underscore the importance of considering both short and long-term effects in ecotoxicological assessments of NP, and highlight the need for more detailed and functional investigations into the mechanisms driving these responses.

## Funding sources

The project received financial support from CNRS through the MITI interdisciplinary programs and its exploratory research program (Project PLASTIBIO). The work was also supported by ANSES and ADEME (Projects PLASTICIDE and PLASTIBIO) through the French national research program for environmental and occupational health (PNR EST).

## CRediT authorship contribution statement

**Chloé M.C. Richard:** Writing – review & editing, Writing – original draft, Visualization, Formal analysis, Data curation, Conceptualization.
**Hervé Colinet:** Writing – review & editing, Visualization, Formal analysis, Conceptualization.
**Mélanie Boël:** Writing – review & editing, Visualization, Conceptualization.
**Stéphane A.P. Derocles:** Writing – review & editing, Visualization, Formal analysis, Conceptualization.
**Clémentine Goupil:** Writing – review & editing, Visualization, Formal analysis, Data curation.
**Théo Laguilliez:** Writing – review & editing, Visualization, Formal analysis, Data curation.
**David Renault:** Writing – review & editing, Visualization, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare no conflict of interest.

## Data availability

The authors declare that the data files of the study are available on the link https://github.com/chloerichard78/Article_F1_F10.git.

## Acknowledgements

We thank Pr. Claudia Wiegand and Nolenn Kermeur for their help and advices on the protocols for enzymatic assays.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.impact.2025.100597.

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