# No evidence for behavioral or physiological effects of nanoplastics ingestion in the fruit fly Drosophila melanogaster

Chloé M.C. Richard, David Renault, Clarisse Cadot, Hervé Colinet, Stéphane

A.P. Derocles, Silvia Argelia Peraza Ku, Karine Salin, Mélanie Boël

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Chloé M.C. Richard, David Renault, Clarisse Cadot, Hervé Colinet, Stéphane A.P. Derocles, et al. No evidence for behavioral or physiological effects of nanoplastics ingestion in the fruit fly Drosophila melanogaster. Ecotoxicology and Environmental Safety, 2025, 303, pp.118811. 10.1016/j.ecoenv.2025.118811. hal-05219689

HAL Id: hal-05219689

https://hal.univ-brest.fr/hal-05219689v1

Submitted on 25 Aug 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# No evidence for behavioral or physiological effects of nanoplasics ingestion in the fruit fly *Drosophila melanogaster*

Chloé M.C. Richard<sup>a</sup>, David Renault<sup>a</sup>, Clarisse Cadot<sup>a</sup>, Hervé Colinet<sup>a</sup>, Stéphane A.P. Derocles<sup>a</sup>, Silvia Argelia Peraza Ku<sup>b</sup>, Karine Salin<sup>c</sup>, Mélanie Boël<sup>d,*</sup>

## ARTICLE INFO

Edited by Bing Yan

**Keywords:**

Metabolic rate

Locomotion

Life history traits

Fitness

Nanoplastics

## ABSTRACT

Plastic pollution increases worldwide, releasing small plastic particles into the environment, such as nanoplasics (NP) that affect living organisms. NP can penetrate biological membranes and interact with cellular components such as mitochondria. However, the effects of NP on terrestrial insects, the most diverse and abundant taxa, remains poorly studied compared to aquatic species. Our study assesses the effect of nanoplasics on the emergence rate, mitochondrial activity, metabolism, body mass, and locomotor activity in *Drosophila melanogaster* (Meigen, 1830, Diptera: Drosophilidae), a terrestrial insect. Three experimental groups were compared: a control group (C) with flies fed food without NP, a low contamination group (LOW) with flies fed food containing 1 µg/g of NP of different sizes (20, 80 and 200 nm), and a high contamination group (HIGH) with flies fed food containing 500 µg/g of NP of the same classes of sizes. Low and High groups were exposed to polystyrene NP for their entire development cycle. NP ingestion did not affect the number of pupae and adults, emergence rate, mitochondrial activity, metabolism and body mass. The locomotor activity of the flies was also unaffected by ingestion of NP. These results, compared with those reported in the literature, showed that the effect of nano-plastics on living organisms might depend on the species and polymer nature. The absence of detectable effects on flies is positive for the ecology of the species, constantly exposed to plastic pollution. Further studies in this area are still needed to fully determine the effects of NP on *Drosophila* biology and behavior.

## 1. Introduction

The worldwide use of plastics has significantly increased in the past decades (Geyer, 2020) and reached 400.3 million metric tons in 2022 (Plastics Europe, 2023). The sharp increase in plastic production over the years, combined with inadequate plastic waste management strategies (Geyer et al., 2017), result in a massive release of macroplastics in the environment (Ryberg et al., 2019; Li et al., 2021), in turn polluting all types of habitats (Stegmann et al., 2022). Once in the environment, plastics are fragmented and degraded into smaller particles through abiotic (UV, temperature, wave, etc.) and biotic (bacteria, fungi, insect, etc.) factors (Andrady, 2011; Bacha et al., 2023). Depending on their size, these smaller particles are described as microplastics (MP; 1 µm - 5 mm) or nanoplasics (NP; < 1000 nm) (da Costa et al., 2016; Gigault et al., 2018). The atmospheric and water diffusion of these smaller-sized

plastic particles is further facilitated, and the presence of micro- and nano-plastics (MNP) has now been reported in some of the most remote areas of the globe, such as Antarctica (Piehl et al., 2018; Elisa Bergami et al., 2020; Wang et al., 2020).

Micro- and nano-plastics (MNP) can be inhaled (Lu et al., 2021) or ingested by living organisms (Huerta Lwanga et al., 2016), leading to harmful effects. MNP ingestion is generally non-lethal but may cause alterations in the behavior, physiology, growth or reproduction of organisms (Haegerbaeumer et al., 2019). Impacts of MNP on aquatic species have been widely studied (Avio et al., 2015; Z. Li et al., 2020), while their impacts on terrestrial invertebrates remain largely unexplored (Richard et al., 2024). This is surprising as significant amounts of MP have been detected from soil samples (Zhu et al., 2019), from 40 mg/kg in China (W. Li et al., 2020) to 67,500 mg/kg in Australia (Fuller and Gautam, 2016), thus placing terrestrial invertebrates at high

<footnote><sup>a</sup> University of Rennes, CNRS, ECOBIO [(Ecosystèmes, biodiversité, évolution)] - UMR 6553, Rennes 35000, France</footnote>

<footnote><sup>b</sup> University of Rennes, CNRS, ISCR – UMR 6226, Rennes F-35000, France</footnote>

<footnote><sup>c</sup> University of Brest, CNRS, IRD, Ifremer, LEMAR (Laboratory of Environmental Marine Sciences), Plouzane F-29280, France</footnote>

<footnote><sup>d</sup> Université Claude Bernard Lyon 1, LEHNA UMR 5023, CNRS, ENTPE, Vaulx-en-Velin, France</footnote>

<footnote>* Correspondence to: Université Claude Bernard Lyon 1, ENTPE, CNRS, INRA, USC 1369, UMR 5023 LEHNA, Vaulx-en-Velin F-69518, France.
E-mail address: Melanie.BOELVOISIN@entpe.fr (M. Boël).</footnote>
MNP exposure risks (Huerta Lwanga et al., 2016; De Felice et al., 2021; El Kholy and Al Naggar, 2023). For instance, Jiang et al. (2020) observed an accumulation of polystyrene MP in the intestine of the worm *Eisenia fetida* (Savigny, 1826, Crassiclitellata: Lumbricidae), which caused alterations to epithelial cells, oxidative stress and DNA damage. In *Caenorhabditis elegans* (Maupas, 1899, Rhabditida: Rhabditidae), ingestion of MP caused oxidative stress and neurotoxic effects, which lead to behavioral alterations (Lei et al., 2018).

NP are expected to induce higher deleterious effects on living organisms than MP as smallest plastic debris, which can also adsorb other environmental pollutants or pathogens to their surface, can be more easily transported across biological barriers (Shen et al., 2019) and diffuse among body compartments. However, we are still far from having a good understanding of NP impacts on terrestrial invertebrates, although their presence in soil is clearly demonstrated (Wahl et al., 2021), with polystyrene being the most frequently found NP plastic particles in soils (Zhou et al., 2018; Wahl et al., 2021). The lower number of studies concerning the effects of NP ingestion on terrestrial species compared to aquatic species could be partly explained by the challenge and difficulty to characterize and quantify NP from soils (Junhao et al., 2021).

Among studies exploring NP toxicity on terrestrial invertebrates, less than 30% have focused on insects (Richard et al., 2024), even though these organisms belong to the most diverse and abundant taxa of the animal kingdom (Scudder, 2017). When available, the information is often limited to a few model species, such as *Bombyx mori* (Linnaeus, 1758, Lepidoptera: Bombycidae) and *Drosophila melanogaster* (Meigen, 1830, Diptera: Drosophilidae). After ingestion and observation of polystyrene NP in the digestive tract of *D. melanogaster* (Matthews et al., 2021), NP can cross biological membranes, as already observed in flies' ovaries and even reaching the hemolymph (Alaraby et al., 2022; Urbisz et al., 2024), and in *B. mori* (Parenti et al., 2020). There is little evidence that NP ingestion impacts the survival of insects. The adult viability and larval length, for instance, were not impaired in *D. melanogaster* after the ingestion of polystyrene NP with sizes from 20 to 600 nm (Matthews et al., 2021; Alaraby et al., 2022, 2023). Polystyrene NP ingestion did not alter the larval mortality nor the duration of the larval cycle in *B. mori* (Parenti et al., 2020, Muhammad et al., 2021, 2024). Even if the viability or mortality of insects is not affected by NP exposure, sublethal toxicity on other traits may be observed. For instance, Alaraby et al. (2022), (2023) highlighted that polystyrene NP ingested by *D. melanogaster* led to genotoxic effects. The accumulation of NP can also induce oxidative stress (Alaraby et al., 2022; Urbisz et al., 2024) and alter locomotion in *D. melanogaster* (Matthews et al., 2021). In *B. mori*, polystyrene NP caused an increase in abnormal locomotor activity that could have harmful ecological consequences for organisms (Parenti et al., 2020). In addition, NP ingestion in larvae of silkworm led to metabolism and microbiota perturbations potentially contributing to increase – yet not always systematic - larval body mass (Parenti et al., 2020; Muhammad et al., 2021, 2024). Zhu et al. (2018) conversely measured a reduction of body mass in *Enchytraeus crypticus* when high concentrations of polystyrene NP were ingested. These opposite results could be explained by the fact that growth, and consequently the body mass of organisms, depends on metabolic activity of individual and energy allocation for different biological functions, but also on other factors such as microbiota disturbance or feeding activity (Matthews et al., 2021).

At the cellular level, mitochondria, often referred to as the “powerhouses of the cell”, are responsible for energy (ATP) production through oxidative phosphorylation (Rolfe and Brown, 1997). In addition to being the main source of energy, mitochondria are also important sites of reactive oxygen species generation (Murphy, 2009). Any modulation in the activity or function of mitochondria could thus promote adaptive capacities of stress-exposed insects. It is now widely admitted that mitochondria are critical organelles involved in the performance and life-history traits of organisms (Salin et al., 2015; Hood et al., 2018),

including growth in *D. melanogaster* (Jacobs et al., 2020). Studying the impacts of NP on mitochondrial activity could therefore provide valuable information on NP-induced physiological alterations at the cellular level in terrestrial insects. Thus, any modulation of their activity or function can have adverse effects on individual performance. So far, it has been demonstrated that NP can enter cells such as enterocytes which surround mitochondria (Alaraby et al., 2022, 2023) and alter morphology of mitochondria (Urbisz et al., 2024). However, to our knowledge, no information is available on the effects of NP ingestion on mitochondrial activity and metabolism in insects. Thus, this study is the first to address this gap.

The present study aimed to assess the effects of polystyrene NP ingestion on emergence rate, mitochondrial activity and metabolism for the first time, body mass and locomotor activity of the fruit fly *D. melanogaster*. This species is naturally exposed to MNP ingestion when growing on contaminated fruits for their entire developmental cycle, but also when feeding on fruits at adult stage in various environments (Reaume and Sokolowski, 2006; Oliveri Conti et al., 2020; Sun et al., 2020, 2021; Zhou et al., 2021). In addition, this insect is a versatile model whose developmental cycle is well known and can be controlled, thus allowing the detection of subtle biological effects. Considering that NP in soils greatly vary in size (Wahl et al., 2021), a polydisperse suspension, composed of three different sizes of NP, was used for the first time in our study to better reflect environmental conditions. Based on recent observations using fluorescent NP or transmission electron microscopy (Alaraby et al., 2022; Urbisz et al., 2024), we assumed that polystyrene NP accumulated in fly tissues after ingestion, as NP were directly added in the food. We hypothesized that the exposure to NP would not seriously impair the pupa or adult viability as well as the emergence rate. However, we predicted sublethal toxicity resulting in a rise of mitochondrial activity and metabolic rate to cope with the stress and damage potentially caused by NP ingestion. We also expected that these changes in mitochondrial activity and metabolism would have a negative impact on the growth and locomotion of the insect. Indeed, NP could lead to a reallocation of energy in support of damage repair, resulting in slower growth and lower body mass, as well as in lower locomotion activity.

## 2. Materials and methods

### 2.1. Biological sample

Experiments were conducted on a laboratory population of *Drosophila melanogaster* derived from wild individuals collected in September 2015 in Brittany (France). Flies were maintained in 100 mL bottles containing a standard fly medium adapted from Toivonen et al. (2007) (15 g L⁻¹ agar, 50 g L⁻¹ sucrose, 80 g L⁻¹ inactive brewer's yeast and 10 mL L⁻¹ Nipagin at a 10 % concentration, at 25 °C, 60 % of humidity with a 12 h light:12 h dark cycle.

### 2.2. NP characteristics

Spherical polystyrene (PS) particles of different sizes (20, 80 and 200 nm) stored in an aqueous suspension (10 mg mL⁻¹, based on the manufacturer's information), were purchased from ThermoFischer Scientific (respectively 3020 A, 3080 A and 3200 A, Nanospheres, ThermoFischer Scientific, USA). The three particle sizes were mixed in equal volume to obtain a unique suspension of NP of different sizes at 10 mg mL⁻¹. Before being used for the experiments, the suspension mix was characterized with different methods:

(i) Scanning electron microscope (SEM) was used to visualize the shape and size of the mixed NP suspensions (Jeol JSM 7100 F EDS EBSD, Oxford).

(ii) Nanoparticle tracking analysis (NTA) was performed to complete information on the sizes of NP in the mixture by using a NanoSight LM10 device system (Malvern Panalytical) equipped with a 40 mW laser
working at 405 nm. The measurements were performed on NANOSIGHT NTA 3.1 Analytical Software Suite. NP were measured for 30 s at three different locations of the suspension.

(iii) Pyrolysis (PY-3030, Frontier Lab) coupled with a gas chromatography and a mass spectrometry detector (Py-GCMS) (5977B, Agilent Technologies) was performed to confirm the nature of the polymer. The pyrolysis was performed at 600 °C, with a 60m-column (Rtx-5, Restek) and a split of 20:1. The NP suspension was diluted twice by 1:10 with ultrapure water before being added to artificial food (around 40–50 °C) used for rearing the flies as described by Tu et al. (2023).

## 2.3. NP contamination and emergence rate

Three experimental groups were designed: the control group (C, nanoplastic-free food), the low contamination group (LOW, 1 µg of NP g⁻¹ food) and the high contamination group (HIGH, 500 µg of NP g⁻¹ food). As the quantification of NP in the environment is not yet possible, we hypothesized that the LOW group would represent a chronic NP exposure, while the HIGH group would be an acute NP exposure reflecting elevated NP concentrations as potentially observed in highly polluted areas. Fifteen mature and mated females, not exposed to NP and aged of 5 days, were placed in vials containing 5 mL of food with (LOW and HIGH groups) or without (C) NP to lay eggs for 2 h before being removed. The eggs laid were then allowed to develop under the above-described rearing conditions until the adult stage, meaning that individuals were exposed to a fixed concentration of NP (except control flies) during their entire development cycle.

The number of pupae produced as well as the number of emerging adults were counted in each vial (i.e. replicate) to indirectly assess the larval viability (i.e. number of pupae) and emergence (n = 10 vials per condition). We prepared extra vials to produce adults in the same way. Once reaching the adult stage, individuals were transferred to bottles of 200 mL containing NP-contaminated (LOW and HIGH) or non-contaminated (C) food, so that adults aged between 2 and 5 days could be used for the experiments described below. For all the experimental procedures that follow, only females from the first generation of unexposed adults, were used.

## 2.4. Mitochondrial activity

For each experimental condition, two females aged of 2 days were anaesthetized by cold and placed in a petri dish at 4 °C. Heads and abdomens of the flies were removed to keep only the thoraces containing the flight muscles, and thus large amounts of mitochondria (Levenbook, 1953). Each sample was made of two thoraces of females exposed to the same level of plastic exposure which provided a muscle tissue mass of around 0.89 ± 0.23 mg for each measurement session, as previously recommended (Jørgensen et al., 2020). Then, permeabilization of thoraces was carried out as previously described by Simard et al. (2018) and Jørgensen et al. (2020). Briefly, thoraces were transferred in a 100 mm petri dish containing 1 mL of ice-cold BIOPS solution (2.77 mM Ca₂EGTA, 7.23 mM K₂EGTA, 5.77 mM Na₂ATP, 6.56 mM MgCl₂, 20 mM Taurine, 15 mM Na₂Phosphocreatine, 20 mM Imidazole, 0.5 mM Dithiothreitol, 50 mM K-MES, pH 7.1) and mechanically permeabilized with forceps by repeatedly tearing apart the tissue to obtain a loosely connected network. Then, two thoraces were placed in 1 mL of BIOPS complemented with 12.5 µL of saponin (5 mg mL⁻¹) and incubated with mild agitation for 20 min to be chemically permeabilized. Thoraces were rinsed to remove the saponine: 2 times in 1 mL of BIOPS and once in 1 mL of respiration medium (120 mM KCl, 5 mM KH₂PO₄, 3 mM Hepes, 1 mM EGTA, 1 mM MgCl₂ (hexahydrate) and 0.2 % bovine serum albumin (BSA) (w/v), pH 7.2), with gentle agitation for 5 min each time. Finally, the thoraces were weighted and immediately transferred into an Oxygraph-O₂K chamber (Oroboros Instruments, Innsbruck, Austria) containing 2 mL of respiration medium supplemented with pyruvate (10 mM) and malate (2.5 mM). The Oxygraph-O₂K chamber was closed

and the signal was allowed to stabilize in order to measure the LEAK respiration at the level of complex I (CI-LEAK). No inhibitor of ATP synthase activity was used, so it is important to note that endogenous ADP could still be present and activate residual ATP production at this stage. Then, injection of ADP (5 mM) stimulated the mitochondrial respiration as the electron transport from complex I was coupled to the phosphorylation of ADP into ATP (CI-OXPHOS). The respiratory control ratio (RCR) was calculated by the ratio CI-OXPHOS/CI-LEAK and was used to assess the mitochondrial quality and coupling. Functional integrity of mitochondria was verified by adding cytochrome c (10 µM). Two substrates were sequentially added: succinate which provides electrons to the mitochondrial complex II (5 mM, CII-OXPHOS) and glycerol-3-phosphate (15 mM) that stimulates the transport of electron via the mitochondrial glycerol-3-phosphate dehydrogenase (mG3PDH-OXPHOS). In most cases, the addition of succinate did not stimulate mitochondrial respiration beyond the CI-OXPHOS flux. Consequently, this mitochondrial state will not be addressed for the remainder of the study. Antimycin (2.5 µM) was injected to inhibit the complex III of the mitochondrial respiratory chain and to assess the residual oxygen consumption. This residual oxygen consumption was used to correct the previous mitochondrial respiration measurement. Finally, ascorbate (2 mM) and N,N,N',N'-Tetramethyl-p-phenylenediamine (0.5 mM) were added to measure the maximum capacity of complex IV (CIV). This respiration was corrected by the respiration in presence of sodium azide (20 mM) that inhibited the complex IV for considering auto-oxidation of TMPD. The contribution of glycerol-3-phosphate was calculated: (mG3PDH-OXPHOS - CII-OXPHOS)/CII-OXPHOS (Cormier et al., 2019). Mitochondrial respiration in presence of different substrates was performed for each condition and expressed in pmol O₂·s⁻¹·mg of tissue⁻¹ (n = 8 samples of two thoraces for LOW and HIGH conditions and n = 9 samples for control).

## 2.5. Metabolic rate

Metabolic rate of female *D. melanogaster* was assessed by respirometry following the same method as previously described by Colinet and Kustre (2024). The individual CO₂ production of each fly was measured using a multichannel (16-chamber) flow-through MAVEN respirometry system (Sable Systems International, Las Vegas, USA) at 20 °C ± 0.5. The system comprised a differential CO₂ analyser (Li-Cor7000) and a 16-channel flow management and signal processing system. The MAVEN was calibrated before experiments using pure nitrogen and CO₂ at 10 ppm. To prevent flies from drying out during measurements, the pumped air passed through a humidifier (Nafion bottle, Sable Systems International) before entering the chamber. The flow rate in each chamber was set at 20 mL·min⁻¹. Flies were placed without anesthesia in individual chambers, each consisting of a transparent glass tube (46 mm long and 6 mm in diameter). The MAVEN system was configured to measure CO₂ emission in each chamber for 120 s (Dwell Time) with baseline measurements (for 120 s) taken every 8 chambers (Interleave ratio). For each chamber, 11 cycles of measurements were taken and the mean of these 11 values was calculated per individual. After the test, each fly was weighed using a microbalance (precision 1 µg) (Mettler Toledo XP2U, Columbus, USA). All data transformations and extractions were conducted in ExpeData Software (Sable Systems International, Las Vegas, USA), utilizing macros provided by Sable Systems for the transformation of raw data. After extracting the data for each chamber, the mass-corrected metabolic rate was calculated as the ratio of CO₂ produced (mL/min) to body weight (mg) of the fly. Thirty-two flies were assessed per experimental condition (n = 32).

## 2.6. Body mass measurements

The flies were dried in an oven for three days at 60 °C to determine dry body mass. Dry body mass was assessed by weighting each fly used
in the metabolic rate experiment with a microbalance (precision 1 µg) (Mettler Toledo XP2U, Columbus, USA) (n = 32 flies per experimental condition).

## 2.7. Locomotor activity

Trikinetics LAM10H (Inc., Waltham, MA, USA) was used to record the locomotor activity of the flies (n = 32 per condition), as described by Chiu et al. (2010) with modifications. This system depicted the activity of the fly by detecting each passage through infrared beams for 32 individuals in parallel, randomly placed in the machine, and then directly recorded on a software. To avoid dehydration and starvation, a moistened cotton and NP-free food were placed at both ends of the closed cylindric tube containing a fly. Activity measurements were carried out at 25 °C, 60% of humidity and a 12 h light: 12 h dark cycle. The number of passages was recorded every 5 min for 4 consecutive days with the software DAMSystel3 (Trikinetics, Inc USA). In addition, daily activity was estimated using the area under the curve of number of passages over 24 h. It was expressed in arbitrary units (a.u.) and corresponds to the total locomotor activity (number of passages) of flies over 24 h. For locomotor activity analysis, only the values of days 2 (daytime and nighttime) and 4 (daytime and nighttime) were analyzed, as the flies were acclimatized for 24 h (first day) and some flies laid eggs at day 3, requiring the cleaning of the tubes and the resetting of the system.

## 2.8. Statistical analyses

All results were statistically analyzed with R software (R Core Team, 2023), with a risk factor of 0.05. The effects of NP concentration were assessed on the number of pupae, the number of adults, and the emergence rate by linear models. A nlme model ('nlme' package) (Pinheiro et al., 2024) was used to evaluate the mitochondrial respiration to NP concentrations with the substrate as random effect, after a 10-log transformation. Then, linear models were performed to assess the respiratory control ratio (RCR), the contribution of glycerol-3-phosphate and the maximal activity of complex IV (CIV) to NP concentrations.

The effect of NP on the metabolic rate was also assessed after a 10-log transformation with a linear model. The effects of NP on body mass were assessed by a linear model. Finally, the effects of NP ingestion on the number of passages during the daytime or the nighttime and the daily activity of individuals (estimated by the aera under the curve) were analyzed, at day 2 and 4, after a square-root transformation, by a nlme model (Pinheiro et al., 2024) with the individual as a random effect.

# 3. Results

## 3.1. NP characterization

The SEM images confirmed the spherical shape of the NP (Fig. 1A). The sizes of the beads (20, 80 and 200 nm) were also verified and were consistent with the range indicated by the manufacturer. The resolution of the device made it difficult to observe the beads of 20 nm, and they could be distinguished at the background of the images. NP of the different sizes were observed as individual beads or in aggregation. The NTA spectrum showed that particles mixed in the suspension were around 151 +/- 2.0 nm, as illustrated in Fig. 1B, with a mean diameter of particles and aggregate particles ranging from 41.5 to 577.5 nm. Similarly to the SEM images, the smallest particles (20 nm) were not detected individually with NTA analysis due to the device's resolution. The m/z 104 revealed the presence of styrene pyrolyzates in the pyrogram (Fig. 1C), in accordance with the polystyrene nature of the beads. The mass spectrum was very similar to the reference spectrum from the NIST library.

## 3.2. Emergence rate

NP ingestion did not affect the number of pupae and adults produced per 15 females (Figs. 2A and 2B), with an average of 149 ± 45 pupae (lm; F(2)= 1.53, p = 0.24; Table SA) and 134 ± 39 adults (lm; F(2)= 1.46, p = 0.25; Table SA). Consequently, the emergence rate did not differ among experimental conditions (Fig. 2C), and was on average

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Characterization of NP suspension made from a mix of three NP sizes (20, 80 and 200 nm). A) Scanning Electron Microscopy images with observation of the beads of 80 and 200 nm, the 20 nm-beads are less visible, and can be distinguished in the background. B) Nanoparticle Tracking Analysis spectrum of the NP suspension C) NP suspension pyrogram for 104 m/z.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Effect of nanoplastic ingestion on the number of pupae or adults, and on the emergence rate of the fruit fly Drosophila melanogaster. Life history traits were measured for flies from each condition (n = 10 per condition). C = Control, LOW = 1 µg of nanoplastic g⁻¹ food, and HIGH = 500 µg of nanoplastic g⁻¹ food. A) Number of pupae per condition. B) Number of emerged adults. C) Emergence rate calculated by the ratio between the number of emerged adults and the number of pupae.</figcaption></figure>

$$0.84 \pm 0.06 (\text{lm}; F_{(2)}= 0.01, p = 1.0; \text{Table SA}).$$

### 3.3. Mitochondrial activity

Mitochondrial respiration varied significantly after substrate addition (Ime; $F(3)= 929.59, p < 0.001$; Table 1; Table SA), and this was indicative of the good quality of the mitochondrial preparation. For all substrates, NP ingestion had no effect on the mitochondrial respiration rates such as CI-LEAK, CI-OXPHOS and mG3PDH-OXPHOS (Ime; $F(2)= 0.25, p = 0.88$; Table 1; Table SA), nor on the respiratory control ratio (Im; $F(2)= 0.10, p = 0.90$; Table 1; Table SA). Finally, NP contamination did not alter the G3P contribution nor the maximal activity of complex IV (CIV, Table 1) (Im; $F(2)= 1.81, p = 0.19$ for G3P contribution and Im; $F(2)= 1.07, p = 0.36$ for CIV; Table SA).

### 3.4. Metabolic rate

The metabolic rate of adult *D. melanogaster* did not differ among experimental conditions with an average of 0.13 ± 0.03 µL CO2.min⁻¹. mg⁻¹ (Im; $F(2)= 2.10, p = 0.13$, Fig. 3; Table SA).

<caption>**Table 1**</caption>

Mitochondrial parameters for flight muscles from flies exposed or not to nanoplastics (mean ± standard error). Mitochondrial functioning was investigated in flight muscles from thoraces of *D. melanogaster* for each experimental condition (n = 8 for LOW and HIGH and n = 9 for C). C=control, LOW = 1 µg of nanoplastic g⁻¹ food, and HIGH = 500 µg of nanoplastic g⁻¹ food.

<table><thead><tr><th colspan="4">Experimental conditions</th></tr><tr><th></th><th>C</th><th>LOW</th><th>HIGH</th></tr></thead><tbody><tr><td colspan="4"><strong>Mitochondrial respiration for different substrates (pmol O<sub>2</sub>.s<sup>-1</sup>.mg of tissue<sup>-1</sup>)</strong></td></tr><tr><td>CI-LEAK</td><td>12.00 ± 3.75</td><td>13.76 ± 7.40</td><td>14.53 ± 6.79</td></tr><tr><td>CI-OXPHOS</td><td>18.80 ± 7.05</td><td>21.33 ± 8.10</td><td>22.19 ± 11.55</td></tr><tr><td>mG3PDH-OXPHOS</td><td>86.54 ± 25.56</td><td>100.69 ± 35.69</td><td>119.24 ± 50.14</td></tr><tr><td colspan="4"><strong>Respiratory control ratio</strong></td></tr><tr><td>RCR</td><td>1.62 ± 0.67</td><td>1.80 ± 0.97</td><td>1.70 ± 0.78</td></tr><tr><td colspan="4"><strong>Maximum capacity of complex IV (pmol O<sub>2</sub>.s<sup>-1</sup>.mg of tissue<sup>-1</sup>)</strong></td></tr><tr><td>CIV</td><td>265.86 ± 84.02</td><td>296.11 ± 74.36</td><td>326.34 ± 96.42</td></tr><tr><td colspan="4"><strong>Other mitochondrial parameters</strong></td></tr><tr><td>G3P contribution</td><td>6.45 ± 3.00</td><td>6.12 ± 3.25</td><td>8.70 ± 2.62</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>Fig. 3. Impact of nanoplastic ingestion on the metabolic rate of individuals in Drosophila melanogaster. Metabolic rate was estimated by the CO₂ released by flies for each condition (n = 32 per condition). C=control, LOW = 1 µg of nanoplastic g⁻¹ food, and HIGH = 500 µg of nanoplastic g⁻¹ food.</figcaption></figure>

### 3.5. Body mass

The dry mass of adult *D. melanogaster* did not differ among the experimental conditions (F(2)= 0.23, *p* = 0.79; Table SA), with masses of: 0.88 ± 0.19, 0.91 ± 0.13 and 0.88 ± 0.32 mg for control, LOW and HIGH NP conditions, respectively.

### 3.6. Locomotor activity

Independently of the period of the day (nighttime and daytime), the same amounts of infrared beams were crossed by adult flies for the three tested conditions (lme; F(2)= 0.24, *p* = 0.89 for day 2 (24 h) and lme; F(2)= 1.19, *p* = 0.55 for day 4 (24 h); Fig. 4A; Table SA). Flies were 4.9–6.3 times more active during daytime compared to nighttime (lme; F(2)= 380.67, *p* < 0.001 for day 2 and lme; F(2) = 341.0, *p* < 0.001 for day 4; Fig. 3A; Table SA). Daily activity (AUC over 24 h) was not affected by an ingestion of NP at day 2 (lm; F(2)= 0.45, *p* = 0.64) and day 4 (lm; F(2)= 0.81, *p* = 0.45; Fig. 4B; Table SA & SB).

## 4. Discussion

This study assessed the effects of a polydisperse suspension of NP, an environmentally relevant plastic, on the emergence rate, mitochondrial activity, metabolism, body mass, and locomotion of *D. melanogaster*. The NP characterization confirmed the spherical shape, the nature of the polymer (polystyrene) and the presence of three sizes of particles in the suspension (20, 80 and 200 nm). The NTA analysis detected NP particles whose sizes approximately ranged from 40 to 575 nm. Thus, the smallest particles (20 nm) were not detected individually due to the device's resolution, and only became detectable when they aggregated. The tendency of particles to aggregate can explain the measured size which exceeded individual sizes of NP (Pradel et al., 2023). Thus, flies were prone to ingest NP particles of 20, 80 and 200 nm in addition to NP aggregation varying between 40 and 575 nm.

As expected, polystyrene NP ingestion had no toxic effect on the developmental cycle of the flies. NP ingestion did not affect the number of pupae and adults and consequently did not alter the emergence rate. This finding is consistent with former studies showing that polystyrene NP ingestion did not alter the development of *B. mori* (Parenti et al., 2020, Muhammad et al., 2021) nor the development and the mortality of *D. melanogaster* (Alaraby et al., 2022, 2023). The results obtained in

the present study support the findings of Matthews et al. (2021), as similar conclusions were obtained despite that our highest NP concentration was five times higher than the one used by these authors (Matthews et al., 2021).

Ingestion of polystyrene NP did not impair the mitochondrial activity of skeletal muscles of the flies, nor the metabolic rate. As NP can cross biological membranes, organelles such as mitochondria could be surrounded by NP (Alaraby et al., 2022, 2023). Urbisz et al. (2024) highlighted that after NP ingestion, the morphology of gut mitochondria changed and became filamentous, finding their way into microvilli of epithelium. In addition, other studies demonstrated toxicological effects of NP on mitochondrial morphology, function and dynamics (Lee et al., 2022; Dal Yöntem, Aydogan Ahbab, 2024). In the present study, NP ingestion seemed to have no significant impact on mitochondrial functioning and thus, on the metabolic rate of organism. These results were consistent with a study showing that metabolic rate was not impacted by NP after 15 days of ingestion in *Clamys farreri* (Sun et al., 2024) or that mitochondrial function was not altered by NP in heart and gonads of zebrafish (Pitt et al., 2018). The contrasting results concerning mitochondrial function could be explained by the fact that the effects of NP are tissue dependent. Indeed, Félix et al., (2023) demonstrated that ingestion of polystyrene NP impacts mitochondrial function in the liver but not in the brain.

Mitochondria convert oxygen and nutrients into energy in the form of ATP through oxidative phosphorylation (Rolfe and Brown, 1997). The efficiency of this energy production process is influenced by various factors such as temperature (Havird et al., 2020; Lubawy et al., 2022) and can be altered by environmental pollutants. In zebrafish, for example, mitochondrial functioning was impaired in larvae exposed to polystyrene NP, as evidenced by the decrease in ATP production and mitochondrial coupling efficiency (Trevisan et al., 2019). Consequently, although mitochondrial oxygen consumption in *D. melanogaster* was unaffected by NP ingestion, other mitochondrial parameters could have been altered, particularly the mitochondrial ATP synthesis. Respiratory control ratio (RCR) was used to assess mitochondrial efficiency, but was not affected by NP ingestion, suggesting no alteration of the ATP production. Nevertheless, RCR does not really assess the ATP synthesis in living organisms, and measurements of the amount of ATP generated per molecule of oxygen would be more appropriate (Salin et al., 2018), especially to study the effect of pollutants on mitochondrial efficiency. Therefore, quantification of mitochondrial ATP production combined with oxygen consumption should be considered in future studies as this information would provide a more complete understanding of the impact of NP ingestion on mitochondrial activity in flies. For example, it was observed that modulation of mitochondrial efficiency had an effect on the growth of organisms (Salin et al., 2019; Jacobs et al., 2020).

Mitochondria generate also reactive oxygen species, molecules that can lead to cellular damages on lipids, proteins, and DNA, oxidative stress, and to apoptosis of the cell (Murphy, 2009; Bhatti et al., 2017). Several studies showed that NP ingestion led to oxidative stress and DNA damages. Martin-Folgar et al. (2024) observed that NP ingestion caused genotoxic effects in *Chironomus riparius*. ROS production and DNA damages have been enhanced after NP ingestion in some tissues in *D. melanogaster* (Alaraby et al., 2023; Urbisz et al., 2024). In these situations, organisms must devote more energy to maintenance costs to repair the damage. The reallocation of energy in favor of repairing damages caused by pollutants could then lead to an alteration in other biological functions of organisms, such as growth or locomotion (Qiu et al., 2020). However, no effect of NP on the dry mass of the flies was detected, regardless of the concentration used, suggesting that our NP mix had no impact on the growth rate. Even if a decrease in body mass was formerly demonstrated in the earthworm *E. crypticus* fed with polystyrene NP with sizes ranging from 0.05 to 0.1 µm (Zhu et al., 2018), or increased body mass measured in *B. mori* (Muhammad et al., 2024), our findings were consistent with other available studies. For instance, Muhammad et al. (2021) did not observe changes in body mass for the
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Number of passages made by flies at day 2 (24 h) and 4 (24 h) depending of the period of the day. A) Number of passages made by individuals were recorded at day 2 (left side) and at day 4 (right side) during the daytime (top part) or the nighttime (bottom part) for each condition (n = 32 per condition). *** Day vs night for the both days 2 and 4 (p < 0.001). B) Daily activity (Aera Under Curve, AUC) for 24 h of individuals estimated for each condition (n = 32 per condition). C=control, LOW = 1 µg of nanoplastic g⁻¹ food, and HIGH = 500 µg of nanoplastic g⁻¹ food.</figcaption></figure>

silkworm after a polystyrene NP ingestion for 72 h.

Concerning the locomotor activity, it was observed that adults of *D. melanogaster* were more active during day compared to night, in accordance with diurnal nature of the species (Pegoraro et al., 2020). No increase of the locomotor activity was observed in flies developed on polystyrene NP, which contrasted with the results of Matthews et al. (2021). This study demonstrated an increased locomotion in males exposed at 50 ppm of polystyrene NP of 20 nm and 1 µm. Some other studies also highlighted changes in mobility, as for instance in larvae of the silkworm whose activity was increased when exposed to polystyrene

NP (Parenti et al., 2020), or hyperactivity in *Caenorhabditis elegans* exposed to polystyrene NP (Lei et al., 2018). Our results suggested that exposure to polystyrene NP did not lead to a marked increase in locomotor activity in *D. melanogaster*. However, the absence of locomotor alteration observed in the present study could also be attributed to several factors, such as the specific concentration of NP used, the duration of exposure, or potential differences in how *D. melanogaster* responded to NP compared to other species.

Altogether, our investigations revealed that fruit flies were not affected by the polystyrene NP. Based on previous studies using similar
exposure protocols on *D. melanogaster*, we assumed that NP were ingested by the flies (Matthews et al., 2024) and accumulated in tissues and organs at larval and adult stages (Alaraby et al., 2022). Some authors hypothesized that adult mouthparts, which are sucking and sponging, were not suitable for nanoparticles ingestion (Alaraby et al., 2024). However, others reported that adults can ingest and accumulate NP using similar exposure protocol (Tu et al., 2023). As the accumulation of polystyrene NP in flies' tissues and organs was not confirmed in this study, further investigations are needed to understand their distribution within flies. For instance, fluorescent or metal-doped NP could be effective candidates to visualize and quantify NP distribution in specific organs.

Instead of diffusing and accumulating in the insect's body, it is possible that ingested NP were excreted by the flies. Consistently, in the scallop *Pecten maximus*, the majority of NP were depurated after 3 days with a complete depuration of small NP (24 nm) after 14 days (Al-Sid-Cheikh et al., 2018). The depuration may occur through the excretion of MNP in feces/pseudofeces, as highlighted in fish exposed to MNP (Yang et al., 2023), or by regurgitation as it was suggested on the diving beetle *Cybister japonicus* (Kim et al., 2018). Further studies are thus needed to investigate this hypothesis and to understand the potential role of depuration processes which have so far only been investigated in marine or freshwater organisms.

Finally, our results indicated that commercial polystyrene NP had no significant deleterious effects on the biological and physiological traits we measured. These results also confirmed that the effects of plastic pollutants are species-specific, and may greatly differ according to the nature, size, and concentration of the studied polymers. This nevertheless raises a concern that potential effects will be very difficult to detect under real ecological conditions, making it challenging to demonstrate their long-term impacts through experimentation. Although our results did not show a direct effect of NP ingestion in *D. melanogaster*, it is important to keep in mind that there could still be subtle or sublethal effects that we did not detect in our study. These effects might be especially significant if NP exposure occurs over a longer period, or when animals have limited food access, as it occurs in the environment. Consistently, Tu et al. (2023) demonstrated that alterations in reproduction of *D. melanogaster* appeared only from the 4th generation. These results highlighted the importance of assessing the multi-generational effects of pollution and open up possibilities for future research assessing long-term or repeated exposure of NP. Furthermore, the lack of toxicity observed in this study is undoubtedly underestimated compared to environmental conditions. Environmental NP are composed by several polymers and have different shapes (Blancho et al., 2021) with possible sharper sides that can more damage tissues and cells. He et al. (2023), for instance, demonstrated recently that the ingestion of randomly-shaped NP, which are more environmentally relevant, by the earthworm *E. fetida* were more toxic than commercial NP.

## 5. Conclusion

This work provided insights on the effects of polystyrene NP used as a mix of different sizes which was environmentally relevant, and provided, for the first time, data on the effects of polystyrene NP on mitochondria activity in terrestrial insect (*D. melanogaster*). In our experimental context, the NP ingestion showed no sign of toxicity in flies: the emergence rate, mitochondrial activity, metabolic rate, body mass, and locomotor activity were unaffected. However, even if no effect was observed, it is possible that NP altered other physiological parameters at infra-individual levels. In addition, it is possible that NP ingestion did not affect these traits after a single generation, however, potential effects may emerge after several generations or through trophic chains due to bioaccumulation or biomagnification. Consequently, NP alterations on mitochondrial efficiency, oxidative stress, or immune responses and across several generations or trophic chains remain to be elucidated.

## CRediT authorship contribution statement

**Chloé M. C. Richard:** Writing – review & editing, Writing – original draft, Data curation, Conceptualization. **David Renault:** Writing – review & editing, Funding acquisition, Conceptualization. **Clarisse Cadot:** Visualization, Formal analysis, Data curation. **Hervé Colinet:** Writing – review & editing, Conceptualization. **Stéphane A. P. Derocles:** Writing – review & editing, Visualization, Formal analysis, Conceptualization. **Silvia Argelia Peraza Ku:** Writing – review & editing, Data curation. **Karine Salin:** Writing – review & editing, Conceptualization. **Mélanie Boël:** Writing – review & editing, Writing – original draft, Visualization, Formal analysis, Data curation, Conceptualization.

## 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).

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: David Renault reports financial support was provided by ANSES. David Renault reports financial support was provided by The French Agency for Ecological Transition. David Renault reports financial support was provided by National Centre for Scientific Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgements

We thank the scanning electron microscopy center (CMEBA) at Rennes and Frangis GoutteFangeas for his help and acquisition of SEM images. We also thank the team Nanoscale (Geosciences, Rennes) for the acquisition of Py-GCMS spectrum.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2025.118811.

## Data Availability

The data are shared in a repository available on the link https://github.com/chloerichard78/Data_nanoplastics_emergence_metabolic.git.

## References

* Alaraby, M., Abass, D., Domenech, J., Hernández, A., Marcos, R., 2022. Hazard assessment of ingested polystyrene nanoplastics in *drosophila* larvae. Environ. Sci. Nano 9, 1845–1857. https://doi.org/10.1039/D1EN01199E.

* Alaraby, M., Villacorta, A., Abass, D., Hernández, A., Marcos, R., 2023. The hazardous impact of true-to-life PET nanoplastics in drosophila. Sci. Total Environ. 863, 160954. https://doi.org/10.1016/j.scitotenv.2022.160954.

* Alaraby, M., Abass, D., Gutiérrez, J., Velázquez, A., Hernández, A., Marcos, R., 2024. Reproductive toxicity of nanomaterials using silver nanoparticles and drosophila as models. Molecules 29 (23), 5802. https://doi.org/10.3390/molecules29235802.

* Al-Sid-Cheikh, M., Rowland, S.J., Stevenson, K., Rouleau, C., Henry, T.B., Thompson, R. C., 2018. Uptake, Whole-Body distribution, and depuration of nanoplastics by the scallop *pecten maximus* at environmentally realistic concentrations. Environ. Sci. Technol. 52, 14480–14486. https://doi.org/10.1021/acs.est.8b05266.

* Andrady, A.L., 2011. Microplastics in the marine environment. Mar. Pollut. Bull. 62, 1596–1605. https://doi.org/10.1016/j.marpolbul.2011.05.030.
Avio, C.G., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., d'Errico, G., Pauletto, M., Bargelloni, L., Regoli, F., 2015. Pollutants bioavailability and toxicological risk from microplastics to marine mussels. Environ. Pollut. 198, 211–222. https://doi.org/10.1016/j.envpol.2014.12.021.

Bacha, A.-U.-R., Nabi, I., Zaheer, M., Jin, W., Yang, L., 2023. Biodegradation of macro- and micro-plastics in environment: a review on mechanism, toxicity, and future perspectives. Sci. Total Environ. 858, 160108. https://doi.org/10.1016/j.scitotenv.2022.160108.

Bergami, E., Rota, E., Caruso, T., Birarda, G., Vaccari, L., Corsi, I., 2020. Plastics everywhere: first evidence of polystyrene fragments inside the common antarctic colembolan cryptopygus antarcticus. Biol. Lett. 16, 20200093. https://doi.org/10.1098/rslb.2020.0093.

Bhatti, J.S., Bhatti, G.K., Reddy, P.H., 2017. Mitochondrial dysfunction and oxidative stress in metabolic disorders — a step towards mitochondria based therapeutic strategies. Biochim. Et. Biophys. Acta (BBA) Mol. Basis Dis. 1863, 1066–1077. https://doi.org/10.1016/j.bbadis.2016.11.010.

Blanco, F., Davranche, M., Fumagalli, F., Ceccone, G., Gigault, J., 2021. A reliable procedure to obtain environmentally relevant nanoplastic proxies. Environ. Sci. Nano 8 (11), 3211–3219. https://doi.org/10.1039/D1EN00395J.

Chiu, J.C., Low, K.H., Pike, D.H., Yildirim, E., Edery, I., 2010. Assaying locomotor activity to study circadian rhythms and sleep parameters in drosophila. JoVE 2157. https://doi.org/10.3791/2157.

Colinet, H., Kustre, A., 2024. The apparent seasonal biphenism in DROSPHILA SUZUKII stems in reality from continuous reaction norms. Pest Manag. Sci., ps.8452 https://doi.org/10.1002/ps.8452.

Cormier, R.P.J., Champigny, C.M., Simard, C.J., St-Coeur, P.-D., Pichaud, N., 2019. Dynamic mitochondrial responses to a high-fat diet in drosophila melanogaster. Sci. Rep. 9, 4531. https://doi.org/10.1038/s41598-018-36060-5.

da Costa, J.P., Santos, P.S.M., Duarte, A.C., Rocha-Santos, T., 2016. Nano)plastics in the environment - sources, fates and effects. Sci. Total Environ. 566–567, 15–26. https://doi.org/10.1016/j.scitotenv.2016.05.041.

Dal Yöntem, F., Aydogan Ahbab, M., 2024. Mitochondria as a target of micro- and nanoplastic toxicity. Camb. Prisms Plast. 2, e6. https://doi.org/10.1017/plc.2024.6.

De Felice, B., Ambrosini, R., Bacchetta, R., Ortenzi, M.A., Parolin, M., 2021. Dietary exposure to polyethylene terephthalate microplastics (PET-MPs) induces faster growth but not oxidative stress in the giant snail achatina reticulata. Chemosphere 270, 129430. https://doi.org/10.1016/j.chemosphere.2020.129430.

El Kholy, S., Al Naggar, Y., 2023. Exposure to polystyrene microplastic beads causes sex-specific toxic effects in the model insect drosophila melanogaster. Sci. Rep. 13, 204. https://doi.org/10.1038/s41598-022-27284-7.

Félix, L., Carreira, P., Peixoto, F., 2023. Effects of chronic exposure of naturally weathered microplastics on oxidative stress level, behaviour, and mitochondrial function of adult zebrafish (Danio rerio). Chemosphere 310, 136895. https://doi.org/10.1016/j.chemosphere.2022.136895.

Fuller, S., Gautam, A., 2016. A procedure for measuring microplastics using pressurized fluid extraction. Environ. Sci. Technol. 50, 5774–5780. https://doi.org/10.1021/acs.est.6b00816.

Geyer, R., 2020. Production, use, and fate of synthetic polymers. Plastic Waste and Recycling. Elsevier, pp. 13–32. https://doi.org/10.1016/B978-0-12-817880-5.00002-5.

Geyer, R., Jambeck, J.R., Law, K.L., 2017. Production, use, and fate of all plastics ever made. Sci. Adv. 3, e1700782. https://doi.org/10.1126/sciadv.1700782.

Gigault, J., Halle, A.T., Baudrimont, M., Pascal, P.-Y., Gauffre, F., Phi, T.-L., El Hadri, H., Grassl, B., Reynaud, S., 2018. Current opinion: what is a nanoplastic? Environ. Pollut. 235, 1030–1034. https://doi.org/10.1016/j.envpol.2018.01.024.

Haegerbaumer, A., Mueller, M.-T., Fueser, H., Traunspurger, W., 2019. Impacts of Micro- and Nano-Sized plastic particles on benthic invertebrates: a literature review and gap analysis. Front. Environ. Sci. 7, 17. https://doi.org/10.3389/fenvs.2019.00017.

Havird, J.C., Shah, A.A., Chicco, A.J., 2020. Powerhouses in the cold: mitochondrial function during thermal acclimation in montane mayflies. Philos. Trans. R. Soc. B 375, 20190181. https://doi.org/10.1098/rstb.2019.0181.

He, F., Shi, H., Liu, R., Tian, G., Qi, Y., Wang, T., 2023. Randomly-shaped nanoplastics induced stronger biogeochemistry targeted to earthworm eisenia fetida species: differential effects and the underlying mechanisms of realistic and commercial polystyrene nanoplastics. Sci. Total Environ. 877, 162854. https://doi.org/10.1016/j.scitotenv.2023.162854.

Hood, W.R., Austad, S.N., Bize, P., Jimenez, A.G., Montooth, K.L., Schulte, P.M., Scott, G. R., Sokolova, I., Treberg, J.R., Salin, K., 2018. The mitochondrial contribution to animal performance, adaptation, and Life-History variation. Integr. Comp. Biol. 58, 480–485. https://doi.org/10.1093/icb/icy089.

Huerta Lwanga, E., Gertsen, H., Gooren, H., Peters, P., Salánki, T., van der Ploeg, M., Besseling, E., Koelmans, A.A., Geissen, V., 2016. Microplastics in the terrestrial ecosystem: implications for lumbricus terrestris (Oligochaeta, Lumbricidae). Environ. Sci. Technol. 50, 2685–2691. https://doi.org/10.1021/acs.est.5b05478.

Jacobs, H.T., George, J., Kemppainen, E., 2020. Regulation of growth in drosophila melanogaster: the roles of mitochondrial metabolism. J. Biochem. 167, 267–277. https://doi.org/10.1093/jb/mvaa002.

Jiang, X., Chang, Y., Zhang, T., Qiao, Y., Klobučar, G., Li, M., 2020. Toxicological effects of polystyrene microplastics on earthworm (Eisenia fetida). Environ. Pollut. 259, 113896. https://doi.org/10.1016/j.envpol.2019.113896.

Jørgensen, L., Overgaard, J., Hunter-Manseau, F., Pichaud, N., 2020. Dramatic changes in mitochondrial substrate use at critically high temperatures: a comparative study using Drosophila. https://doi.org/10.1101/2020.11.19.389924.

Junhao, C., Xining, Z., Xiaodong, G., Li, Z., Qi, H., Siddique, K.H.M., 2021. Extraction and identification methods of microplastics and nanoplastics in agricultural soil: a

review. J. Environ. Manag. 294, 112997. https://doi.org/10.1016/j.jenvman.2021.112997.

Kim, S.W., Kim, D., Chae, Y., An, Y.-J., 2018. Dietary uptake, biodistribution, and depuration of microplastics in the freshwater diving beetle cyberstone japonicus: effects on predacious behavior. Environ. Pollut. 242, 839–844. https://doi.org/10.1016/j.envpol.2018.07.071.

Lee, S.E., Yi, Y., Moon, S., Yoon, H., Park, Y.S., 2022. Impact of micro- and nanoplastics on mitochondria. Metabolites 12, 897. https://doi.org/10.3390/metabo12100897.

Lei, L., Liu, M., Song, Y., Lu, S., Hu, J., Cao, C., Xie, B., Shi, H., He, D., 2018. Polystyrene (nano)microplastics cause size-dependent neurotoxicity, oxidative damage and other adverse effects in caenorhabditis elegans. Environ. Sci. Nano 5, 2009–2020. https://doi.org/10.1016/j.c8en00412A.

Levenbook, L., 1953. The mitochondria of insect flight muscle. J. Histochem. Cytochem. 1, 242–247. https://doi.org/10.1177/1.4.242.

Li, P., Wang, X., Su, M., Zou, X., Duan, L., Zhang, H., 2021. Characteristics of plastic pollution in the environment: a review. Bull. Environ. Contam. Toxicol. 107, 577–584. https://doi.org/10.1007/s00128-020-02820-1.

Li, W., Wufuer, R., Duo, J., Wang, S., Luo, Y., Zhang, D., Pan, X., 2020. Microplastics in agricultural soils: extraction and characterization after different periods of polythene film mulching in an arid region. Sci. Total Environ. 749, 141420. https://doi.org/10.1016/j.scitotenv.2020.141420.

Li, Z., Feng, C., Wu, Y., Guo, X., 2020. Impacts of nanoplastics on bivalve: fluorescence tracing of organ accumulation, oxidative stress and damage. J. Hazard. Mater. 392, 122418. https://doi.org/10.1016/j.jhazmat.2020.122418.

Lu, K., Lai, K.P., Stoeger, T., Ji, S., Lin, Z., Lin, X., Tef, F., Fang, J.K.-H., Lo, M., Gao, L., Qiu, C., Chen, S., Chen, G., Li, L., Wang, L., 2021. Detrimental effects of microplastic exposure on normal and asthmatic pulmonary physiology. J. Hazard. Mater. 416, 126069. https://doi.org/10.1016/j.jhazmat.2021.126069.

Lubawy, J., Chowański, S., Adamski, Z., Słocińska, M., 2022. Mitochondria as a target and central hub of energy division during cold stress in insects. Front. Zool. 19 (1). https://doi.org/10.1186/s12983-021-00448-3.

Martin-Folgar, R., Sabroso, C., Cañas-Portilla, A.I., Torres-Ruiz, M., González-Caballero, M.C., Dorado, H., Velasco, I., Morales, M., 2024. DNA damage and molecular level effects induced by polystyrene (PS) nanoplastics (NPs) after chironomus riparius (Diptera) larvae. Chemosphere 346, 140552. https://doi.org/10.1016/j.chemosphere.2023.140552.

Matthews, S., Genbo Xu, E., Dumont, E.R., Meola, V., Pikuda, O., S., Cheong, R., Guo, M., Tahara, R., E., Larsson, H.C., Tufenkji, N., 2021. Polystyrene micro- and nanoplastics affect locomotion and daily activity of drosophila melanogaster. Environ. Sci. Nano 8, 110–121. https://doi.org/10.1039/D0EN00942C.

Muhammad, A., Zhou, X., He, J., Zhang, N., Shen, X., Sun, C., Yan, B., Shao, Y., 2021. Toxic effects of acute exposure to polystyrene microplastics and nanoplastics on the model insect, silkworm bombyx mori. Environ. Pollut. 285, 117255. https://doi.org/10.1016/j.envpol.2021.117255.

Muhammad, A., Zhang, N., He, J., Shen, X., Zhu, X., Xiao, J., Qian, Z., Sun, C., Shao, Y., 2024. Multiomics analysis reveals the molecular basis for increased body weight in silkworms (Bombyx mori) exposed to environmental concentrations of polystyrene micro- and nanoplastics. J. Adv. Res. 57, 43–57. https://doi.org/10.1016/j.jare.2023.09.010.

Murphy, M.P., 2009. How mitochondria produce reactive oxygen species. Biochem J. 417, 1–13. https://doi.org/10.1042/BJ20081386.

Oliveri Conti, G., Ferrante, M., Banni, M., Favara, C., Nicolosi, I., Cristaldi, A., Fiore, M., Zuccarello, P., 2020. Micro- and nano-plastics in edible fruit and vegetables. The first diet risks assessment for the general population. Environ. Res. 187, 109677. https://doi.org/10.1016/j.envres.2020.109677.

Parenti, C.C., Binelli, A., Caccia, S., Della Torre, C., Magni, S., Pirovano, G., Casartelli, M., 2020. Ingestion and effects of polystyrene micro particles in the silkworm bombyx mori. Chemosphere 257, 127203. https://doi.org/10.1016/j.chemosphere.2020.127203.

Pegoraro, M., Flavell, L.M.M., Menegazzi, P., Colombi, P., Dao, P., Helfrich-Förster, C., Tauber, E., 2020. The genetic basis of diurnal preference in drosophila melanogaster. BMC Genom. 21, 596. https://doi.org/10.1186/s12864-020-07020-z.

Piehl, S., Leibner, A., Löder, M.G.J., Dris, R., Bogner, C., Laforsch, C., 2018. Identification and quantification of macro- and microplastics on an agricultural farmland. Sci. Rep. 8, 17950. https://doi.org/10.1038/s41598-018-36172-y.

Pinheiro J., Bates D., R Core Team (2024). nlme: Linear and Nonlinear Mixed Effects Models. R package version 3.1-166, (https://CRAN.R-project.org/package=nlme).

Pitt, J.A., Trevisan, R., Massarsky, A., Kojal, J.S., Levin, E.D., Di Giulio, R.T., 2018. Maternal transfer of nanoplastics to offspring in zebrafish (Danio rerio): a case study with nanopolystyrene. Sci. Total Environ. 643, 324–334. https://doi.org/10.1016/j.scitotenv.2018.06.186.

Plastics Europe. 2023. Plastics - the fast Facts 2023. (https://plasticseurope.org/knowledgehub/plastics-the-fast-facts-2023/).

Pradel, A., Catrouillet, C., Gigault, J., 2023. The environmental fate of nanoplastics: what we know and what we need to know about aggregation. Nanolmpact 29, 100453. https://doi.org/10.1016/j.impact.2023.100453.

Qiu, Y., Liu, Y., Li, Y., Li, G., Wang, D., 2020. Effect of chronic exposure to nanopolystyrene on nematode caenorhabditis elegans. Chemosphere 256, 121712. https://doi.org/10.1016/j.chemosphere.2020.121712.

Reaume, C.J., Sokolowski, M.B., 2006. The nature of drosophila melanogaster. Curr. Biol. 16, R623–R628. https://doi.org/10.1016/j.cub.2006.07.042.

Richard, C.M.C., Dejoie, E., Wiegand, C., Gouesbet, G., Colinet, H., Balzani, P., Siaussat, D., Renault, D., 2024. Plastic pollution in terrestrial ecosystems: current knowledge on impacts of micro and nano fragments on invertebrates. J. Hazard. Mater. 477, 135299. https://doi.org/10.1016/j.jhazmat.2024.135299.
Rolfe, D.F., Brown, G.C., 1997. Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Physiol. Rev. 77, 731–758. https://doi.org/10.1152/physrev.1997.77.3.731.

Ryberg, M.W., Hauschild, M.Z., Wang, F., Averous-Monnery, S., Laurent, A., 2019. Global environmental losses of plastics across their value chains. Resour. Conserv. Recycl. 151, 104459. https://doi.org/10.1016/j.resconrec.2019.104459.

Salin, K., Auer, S.K., Rey, B., Selman, C., Metcalfe, N.B., 2015. Variation in the link between oxygen consumption and ATP production, and its relevance for animal performance. Proc. R. Soc. B Biol. Sci. 282, 20151028. https://doi.org/10.1098/rspb.2015.1028.

Salin, K., Villasevil, E.M., Anderson, G.J., Selman, C., Chinopoulos, C., Metcalfe, N.B., 2018. The RCR and ATP/O indices can give contradictory messages about mitochondrial efficiency. Integr. Comp. Biol. 58, 486–494. https://doi.org/10.1093/icb/icy085.

Salin, K., Villasevil, E.M., Anderson, G.J., Lamarre, S.G., Melanson, C.A., McCarthy, I., Selman, C., Metcalfe, N.B., 2019. Differences in mitochondrial efficiency explain individual variation in growth performance. Proc. R. Soc. B 286, 20191466. https://doi.org/10.1098/rspb.2019.1466.

Scudder, G.G.E., 2017. The importance of insects. In: Foottit, R.G., Adler, P.H. (Eds.), Insect Biodiversity. Wiley, pp. 9–43. https://doi.org/10.1002/978118945568.ch2.

Shen, M., Zhang, Y., Zhu, Y., Song, B., Zeng, G., Hu, D., Wen, X., Ren, X., 2019. Recent advances in toxicological research of nanoplastics in the environment: a review. Environ. Pollut. 252, 511–521. https://doi.org/10.1016/j.envpol.2019.05.102.

Simard, C.J., Pelletier, G., Boudreau, L.H., Hebert-Chatelain, E., Pichaud, N., 2018. Measurement of mitochondrial oxygen consumption in permeabilized fibers of drosophila using minimal amounts of tissue. J. Vis. Exp. 57376. https://doi.org/10.3791/57376.

Stegmann, P., Daioglou, V., Londo, M., Van Vuuren, D.P., Junginger, M., 2022. Plastic futures and their CO2 emissions. Nature 612, 272–276. https://doi.org/10.1038/s41586-022-05422-5.

Sun, H., Lei, C., Xu, J., Li, R., 2021. Foliar uptake and leaf-to-root translocation of nanoplastics with different coating charge in maize plants. J. Hazard. Mater. 416, 125854. https://doi.org/10.1016/j.jhazmat.2021.125854.

Sun, X.-D., Yuan, X.-Z., Jia, Y., Feng, L.-J., Zhu, F.-P., Dong, S.-S., Liu, J., Kong, X., Tian, H., Duan, J.-L., Ding, Z., Wang, S.-G., Xing, B., 2020. Differentially charged nanoplastics demonstrate distinct accumulation in arabidopsis thaliana. Nat. Nanotechnol. 15, 755–760. https://doi.org/10.1038/s41565-020-0707-4.

Sun, Y., Zhao, X., Sui, Q., Sun, X., Zhu, L., Booth, A.M., Chen, B., Qu, K., Xia, B., 2024. Polystyrene nanoplastics affected the nutritional quality of chlamys farreri through disturbing the function of gills and physiological metabolism: comparison with microplastics. Sci. Total Environ. 910, 168457. https://doi.org/10.1016/j.scitotenv.2023.168457.

Toivonen, J.M., Walker, G.A., Martinez-Diaz, P., Bjedov, I., Driege, Y., Jacobs, H.T., Gems, D., Partridge, L., 2007. No influence of indy on lifespan in drosophila after correction for genetic and cytoplasmic background effects. PLoS Genet. 3, e95. https://doi.org/10.1371/journal.pgen.0030095.

Trevisan, R., Voy, C., Chen, S., Di Giulio, R.T., 2019. Nanoplastics decrease the toxicity of a complex PAH mixture but impair mitochondrial energy production in developing zebrafish. Environ. Sci. Technol. 53, 8405–8415. https://doi.org/10.1021/acs.est.9b02003.

Tu, Q., Deng, J., Di, M., Lin, X., Chen, Z., Li, B., Tian, L., Zhang, Y., 2023. Reproductive toxicity of polystyrene nanoplastics in drosophila melanogaster under multi-generational exposure. Chemosphere 330, 138724. https://doi.org/10.1016/j.chemosphere.2023.138724.

Urbisz, A.Z., Małota, K., Chajec, Ł., Sawadro, M.K., 2024. Size-dependent and sex-specific negative effects of micro- and nano-sized polystyrene particles in the terrestrial invertebrate model drosophila melanogaster. Micron 176, 103560. https://doi.org/10.1016/j.micron.2023.103560.

Wahl, A., Le Juge, C., Davranche, M., El Hadri, H., Grassl, B., Reynaud, S., Gigault, J., 2021. Nanoplastic occurrence in a soil amended with plastic debris. Chemosphere 262, 127784. https://doi.org/10.1016/j.chemosphere.2020.127784.

Wang, S., Chen, H., Zhou, X., Tian, Y., Lin, C., Wang, W., Zhou, K., Zhang, Y., Lin, H., 2020. Microplastic abundance, distribution and composition in the mid-west Pacific Ocean. Environ. Pollut. 264, 114125. https://doi.org/10.1016/j.envpol.2020.114125.

Yang, L., Tang, B.Z., Wang, W.-X., 2023. Near-Infrared-II *in vivo* visualization and quantitative tracking of Micro/Nanoplastics in fish. ACS Nano 17, 19410–19420. https://doi.org/10.1021/acsnano.3c07571.

Zhou, C.-Q., Lu, C.-H., Mai, L., Bao, L.-J., Liu, L.-Y., Zeng, E.Y., 2021. Response of rice (Oryza sativa L.) roots to nanoplastic treatment at seedling stage. J. Hazard. Mater. 401, 123412. https://doi.org/10.1016/j.jhazmat.2020.123412.

Zhou, Q., Zhang, H., Fu, C., Zhou, Y., Dai, Z., Li, Y., Tu, C., Luo, Y., 2018. The distribution and morphology of microplastics in coastal soils adjacent to the bohai sea and the yellow sea. Geoderma 322, 201–208. https://doi.org/10.1016/j.geoderma.2018.02.015.

Zhu, B.-K., Fang, Y.-M., Zhu, D., Christie, P., Ke, X., Zhu, Y.-G., 2018. Exposure to nanoplastics disturbs the gut microbiome in the soil oligochaete enchytraeus crypticus. Environ. Pollut. 239, 408–415. https://doi.org/10.1016/j.envpol.2018.04.017.

Zhu, F., Zhu, C., Wang, C., Gu, C., 2019. Occurrence and ecological impacts of microplastics in soil systems: a review. Bull. Environ. Contam. Toxicol. 102, 741–749. https://doi.org/10.1007/s00128-019-02623-z.