# Influence of the reproductive cycle and sex on microplastic toxicity in mussels

Marine Uguen, Sylvie Marylène Gaudron, Lorenzo Cozzolino, Laurent Seuront

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Marine Uguen, Sylvie Marylène Gaudron, Lorenzo Cozzolino, Laurent Seuront. Influence of the reproductive cycle and sex on microplastic toxicity in mussels. Jean-Yves Reynaud; Eric Armynot du Châtelet. Short Papers from the 19th French Congress of Sedimentology, Lille, 84, Association des Sédimentologistes Français, pp.125-132, 2025, ASF Publications, 2-907205-83-8. 10.70665/UGKJ1284. hal-05023922v2

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# INFLUENCE OF THE REPRODUCTIVE CYCLE AND SEX ON MICROPLASTIC TOXICITY IN MUSSELS

Marine Uguen<fnref n="1" />, Sylvie Marylène Gaudron<fnref n="1" />,<fnref n="2" />, Lorenzo Cozzolino<fnref n="3" /> and Laurent Seuront<fnref n="1" />,<fnref n="4" />,<fnref n="5" />

Corresponding author: marine.uguen@univ-lille.fr

**Abstract:** Plastic contamination has become a major scientific and societal concern. While research on this issue has been flourishing over the last decade, several aspects remain insufficiently explored, including the chemical impact of plastic leachates (i.e., a complex mix of chemical compounds released by plastic particles). Furthermore, essential biological factors, such as the reproductive cycle and sex, are still largely overlooked, particularly in marine invertebrates, further limiting our ability to fully assess the ecotoxicological risks posed by pollutants. Here, we investigate the influence of these biological factors on organismal sensitivity to microplastic leachates (MPL) using two mussel species, *Mytilus edulis* and *Mytilus galloprovincialis*, which are important ecosystem engineers with significant economic values. The role of reproductive cycle was assessed in *M. edulis* through aggregation assays, while sex-based differences in sensitivity were assessed in *M. galloprovincialis* through byssal thread production assays. *M. edulis* showed increased sensitivity to MPL post-spawning, through a delay in their time to form their first aggregation, while female *M. galloprovincialis* showed higher sensitivity to MPL through increased byssal thread production. The observed effects may result from the high energetic cost of reproduction, coupled with potential endocrine-disrupting effects of plastic additives, highlighting the need for further studies. Our work stresses the importance of incorporating biological factors, such as the reproductive cycle and sex, into pollution risk assessments to enhance their ecological relevance.

**Keywords:** contaminant; vulnerability; bivalve; gametogenesis; gender

## 1 INTRODUCTION

Plastic pollution is a pervasive threat, affecting marine ecosystem globally, from industrialized coastlines to the most remote and pristine regions, spanning from the Arctic to the Antarctic, and from the ocean surface to its deepest trenches (Chiba et al., 2018; Sebille et al., 2020). An estimated 19 to 23 million tons of plastic waste enter aquatic systems annually, making plastics the most common form of marine litter (ca. 80 – 85%; Auta et al., 2017; Borrelle et al., 2020). Among the various forms of plastics found in the marine environment, microplastics—particles smaller than 5 mm—are by far the most abundant (Eriksen et al., 2014) and have been linked to numerous detrimental effects on marine life in experimental studies.

The effects of microplastics on marine organisms have been extensively documented; see Gall and Thompson (2015), Auta et al. (2017) and Delaeter et al. (2022) for reviews. Due to their small size, microplastics are ingested by a wide variety of marine species, from invertebrates at the base of the food web to apex predators (Wright et al., 2013; Setälä et al., 2014). Such ingestion can result in numerous physical damages, such as gastrointestinal obstruction and injuries, alteration of physiological processes, all of which can act on the overall fitness of affected organisms (Franzellitti et al., 2019; Wright et al., 2013). In addition to these well-documented physical impacts, microplastics can also induce chemical effects, that remain comparatively understudied (Delaeter et al., 2022; Seuront et al., 2022). Indeed, plastic particles are associated with a diverse array of potentially toxic chemicals, either incorporated during their manufacture (e.g., functional additives) or accumulated from their surrounding environment (e.g., pesticides, heavy metals; Verla et al., 2019; Wiesinger et al., 2021; Lynch et al., 2022). Once released into the marine environment or directly into organisms following ingestion, these chemicals, referred to as plastic leachates, can induce diverse impacts across biological levels, from subcellular to behavioral alterations (Delaeter et al., 2022).

<footnote n="1">¹ Université de Lille, CNRS, Université du Littoral Côte d'Opale, IRD, UMR 8187, LOG, Laboratoire d'Océanologie et de Géosciences, Station marine de Wimereux, F-59000 Lille, France</footnote>

<footnote n="2">² Sorbonne Université, UFR 927, F-75005 Paris, France</footnote>

<footnote n="3">³ CCMAR-Centro de Ciencias do Mar, CIMAR Laboratório Associado, Universidade do Algarve, Campus de Gambelas, Faro 8005-139, Portugal</footnote>

<footnote n="4">⁴ Department of Zoology and Entomology, Rhodes University, Grahamstown, 6140 South Africa</footnote>

<footnote n="5">⁵ Department of Marine Resources and Energy, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo, 108-8477 Japan</footnote>
While research on microplastic toxicity has become one of the most prolific scientific fields, the role of biological factors, such as sex and the reproductive cycle, in modulating an organism sensitivity to these pollutants remains underexplored. This gap is particularly pronounced in marine invertebrates, despite their ecological importance (Burger, 2007). Importantly, the sex and the reproductive cycle, can influence the bioavailability, fate and effects of contaminants in organisms (Burger, 2007; Langston, 2020; McClellan-Green et al., 2007). Reproduction is an energetically demanding process—especially for females (Hayward and Gillooly, 2011)— and may heighten vulnerability to contaminants depending on individual's sex and reproductive stage. In addition, several plastic additives, such as Bisphenol A and phthalates, are acknowledged to act as endocrine disruptors (Burgos-Aceves et al., 2021; Liu et al., 2021; Oehlmann et al., 2009), interfering with hormonal systems and causing sex- and reproductive stage- specific effects (Burger, 2007; Langston, 2020; McClellan-Green et al., 2007). Such interaction adds complexity to the toxicity of plastic pollution and underscore the necessity of studying how biological factors interact with these contaminants to affect marine species, especially marine invertebrates.

Mussels are keystone species in marine ecosystem that hold significant ecological and economic functions (Avdelas et al., 2021; Borthagaray and Carranza, 2007; Buschbaum et al., 2009). Their high filtration capacity makes them effective sentinel species for monitoring environmental pollution while also making them more susceptible to plastic contamination (Beyer et al., 2017; Uguen et al., 2025). Additionally, as broadcast spawners and gonochorics with a seasonal reproduction (Gosling, 2015), mussels provide an ideal model for investigating how biological factors, such as sex and the reproductive stage, influence their sensitivity to pollutants. In this study, we focused on two mussel species, the blue mussel (*Mytilus edulis*) and the Mediterranean mussel (*Mytilus galloprovincialis*). Specifically, we aimed to examine whether the reproductive cycle and mussel sex affect the sensitivity to microplastic leachates in *M. edulis* and *M. galloprovincialis*, respectively. We hypothesize that sensitivity to these contaminants will increase during the post-spawning period and will be higher in females, reflecting the important energetic cost of reproduction.

## 2 MATERIALS AND METHODS

### 2.1 Impact of the reproductive cycle on mussel sensitivity to microplastic leachates

To study the impact of the reproductive cycle on mussel sensitivity to plastic leachates, adult blue mussels (*Mytilus edulis*; shell length: 2.5 – 3.5 cm; *N* = 180) were collected monthly from March to December 2021 (with the exception of October and November 2021) from a rocky intertidal reef located along the French shores of the eastern English Channel (Pointe aux Oies, Wimereux, France; 50°47′08.3″N, 1°36′03.9″E). Mussels were acclimated for 24 hours in 85 L tanks filled with running aerated natural seawater representative of *in situ* conditions.

Behavioral experiments were run in 22 cm glass arenas. Mussels (*N* = 15) were placed in the arena, their narrow end facing the center of the arena in two concentric circles, at ca. 1 body length from each other. The arenas were filled with 1.5 L of control seawater (*n* = 6) or microplastic leachate seawater (MPL; *n* = 6). MPL was prepared by incubating commercially available polypropylene pellets (3.80 ± 0.21 mm; Pemmiproducts, Aachen, Germany) in natural aerated seawater (12°C; 24 h) at an experimental concentration of ca 400 pellets per liter (equivalent to 12 g or 20 mL of pellets per liter), a concentration used in previous microplastic leachate studies known to induce effects in molluscs (Seuront, 2018; Uguen et al., 2023). After the incubation, pellets were removed from the solution using a wire mesh. The additive content of polypropylene pellets was analyzed in a previous study (Uguen et al., 2023) that identified a total of 27 additives, including three brominated flame retardants, six phosphorus flame retardants, five antioxidants, and 13 plasticizers.

Mussel positions were recorded every minute for 2 h using GoPro cameras (GoPro HERO7 Black, GoPro Inc., San Mateo, California, USA). At each 1 min interval, mussels were visually classified as either solitary or aggregated, i.e. two or more mussels with their shells in direct physical contact (Seuront et al., 2021; Uguen et al., 2023). The starting time (min) defined as the time needed to form the first aggregation within an arena was then quantified (Uguen et al., 2023; Zardi et al., 2024a).

Wilcoxon-Mann-Whitney tests with continuity correction (hereafter referred to as WMW test) were performed for each month of study to assess the impact of MPL on mussel starting time behavior.

### 2.2 Impact of sex on mussel sensitivity to microplastic leachates

To study the impact of sex on the mussel sensitivity to plastic leachates, adult Mediterranean mussels (*Mytilus galloprovincialis*; shell length: 4.0 – 4.6 cm; *N* = 18) were collected in January 2023 from Praia de Faro (Faro, Southern Portugal; 37°0′33.5″N, 7°59′36.3″W). Mussels were acclimated for 24 hours in 50 L tanks filled with running aerated natural seawater representative of *in situ* conditions.

Byssal thread production experiments were run in sealed glass chambers. Mussels were individually placed in chambers filled with 200 mL of control seawater (*n* = 9) or microplastic leachate seawater (MPL; *n* = 9). MPL was prepared by incubating low density polyethylene pellets collected from the shore of Northern
France (Ambleteuse; 50°80'61.9"N, 1°60'31.34"E; 4.04 ± 0.56 mm) in natural aerated seawater (17°C; 24 h) at an experimental concentration of ca 400 pellets per liter (equivalent to 10 g or 20 mL of pellets per liter), a concentration used in previous microplastic leachate studies known to induce effects in mussels (Uguen & Cozzolino, 2024; Uguen et al., 2024). After the incubation, pellets were removed from the solution using a wire mesh. The additive content of beached French pellets was analyzed in a previous study (Uguen and Cozzolino, 2024) that identified a total of 9 additives, including two antioxidants and seven plasticizers.

After a 12-h exposure, the total byssal thread production was visually quantified for each mussel by counting the total number of byssal plaques in the chamber (Seuront et al., 2021; Zardi et al., 2024b). Mussels were then dissected, and each individual was sexed through gamete observation under a microscope (Motic BA210) following a biopsy of the mantle (Fraser et al., 2016). The control group consisted of three females and six males, while the MPL group consisted of four females and five males.

Wilcoxon-Mann-Whitney tests with continuity correction were performed for each sex to assess the impact of MPL on mussel byssal thread production.

# 3 RESULTS AND DISCUSSION

## 3.1 The reproductive cycle affects mussel sensitivity to microplastic leachates

*Mytilus edulis* starting time was significantly impacted by MPL in September, with a longer time needed by mussels to form the first aggregation (2.2 times longer compared to control; WMW test; p < 0.05; Figure 1). In contrast, no significant effect of MPL was found for any of the other months studied (from March to August and in December; WMW test; p > 0.05; Figure 1). At the study site, the reproductive cycle of *M. edulis* is characterized by a spawning period occurring from May to August, September marking the end of the spawning season with the start of a new reproductive cycle (Uguen, unpublished data). These observations imply that *M. edulis* showed an increased sensitivity to MPL during the post-spawning period.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Starting time in minutes (min) taken by *Mytilus edulis* to form the first aggregation in control seawater (blue) and in microplastic leachates (red) from commercially available polypropylene pellets for each studied month. Asterisks depict significant difference (p < 0.05; Wilcoxon-Mann-Whitney test with continuity correction) among treatments.</figcaption></figure>

The increased mussel sensitivity during the post-spawning period (i.e. September) likely reflects physiological stress associated with reproduction, a process acknowledged to be energetically costly in mussels (Seed and Suchanek, 1992; Mredul et al., 2024; Osterheld et al., 2024). Spawning can significantly deplete mussel energy reserves, leading to a marked reduction in their condition index during the post-spawning period (Seed and Suchanek, 1992; Mredul et al., 2024), with individuals losing up to 40% of their soft tissue weight (Cossa, 1989). Mussels need time to regenerate their energy reserves following spawning (Lubet, 1959; Myrand et al., 2000; Mredul et al., 2024), while repairing and reorganizing their tissues (Tremblay et al., 1998). Thus, spawning induces a considerable energetic stress, which can make them more vulnerable to other stressors, such as pollutants.
Our results are consistent with previous studies that showed an increase in susceptibility to pollutant and other environmental stressors in post-spawning mussels (e.g. Myrand et al., 2000; Lupo et al., 2021; Laubscher et al., 2023) further corroborating that the reproductive cycle significantly modulates stress responses. Notably, recently spawned mussels exhibit heightened sensitivity to microplastic exposure, as evidenced by a more pronounced reduction in clearance rate compared to their pre-spawn counterparts (Laubscher et al., 2023). The post-spawning stage may increase the mussel mortality risk, especially when subjected to additional stressors (e.g. heat stress, pathogens). This is consistent with field observations of mass mortality in mussels following the spawning period (Charles et al., 2020; Myrand et al., 2000; Ajithkumar et al., 2024). Thus, as the mussel sensitivity to pollutant is not constant throughout the year, mussels may show a "window of sensitivity", i.e. a specific period when the sensitivity of species to contaminants is amplified (Winner et al., 1990; Dissanayake et al., 2011; Finch and Stubblefield, 2016), here at the post-spawning period, highlighting the critical need to take into account the reproductive cycles in pollution assessments and other stressors in general.

## 3.2 Mussel sex affects mussel sensitivity to microplastic leachates

Byssal thread production in female *Mytilus galloprovincialis* was significantly affected by MPL exposure, with females producing ca. 2.0 times more threads when exposed to MPL compared to control (WMW test; p < 0.05; Figure 2). In contrast, no significant effect of MPL was observed in male *M. galloprovincialis* byssal thread production (WMW test; p > 0.05; Figure 2). Thus, *M. galloprovincialis* showed a sex-specific difference in their sensitivity to MPL with an increased sensitivity in females.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** *Mytilus galloprovincialis* byssal thread production in control seawater (blue) and in MPL (red) from beached French pellets for each sex. Asterisks depict significant difference (p < 0.05; Wilcoxon-Mann-Whitney test with continuity correction) among treatments.</figcaption></figure>

The higher sensitivity to MPL of female compared to male mussels can be attributed to distinct energy investment. Female allocate a substantial part of their energy reserves to reproduction compared to male (Hayward and Gillooly, 2011), particularly during gametogenesis, as they produced numerous energy-rich oocytes (Bayne, 1976; Sprung, 1983; Petes et al., 2007). In contrast to males, the high reproductive energy investment in female likely intensifies their vulnerability to environmental stressors, such as MPL exposure. However, the stress response observed here in females is an increase in the byssal thread production, which is energy costly (Roberts et al., 2021). This increase in *M. galloprovincialis* byssal thread production during MPL exposure is consistent with previous observations (Seuront et al., 2021), suggesting that this species use byssal production as a resistance strategy to cope with stressors. By allocating their remaining energy to this behavior, they may prioritize maintaining attachment in stressful environments. Although high byssal thread production could enhance short-term survival under certain stress, e.g. wave disturbance (Carrington, 2002; Carrington et al., 2009) and predation pressure (Côté, 1995; Cheung et al., 2006; Caro et al., 2008), it might also deplete energy reserves, especially in females during gametogenesis, potentially impacting population dynamics and mussel ecological roles.

This sex-dependent effect may also be due to the endocrine-disrupting properties of plastic leachates. Numerous plastic additives have been established to be endocrine-disrupting chemicals (EDCs) due to their ability to interfere with the endocrine system, leading to sex-specific effects (see Oehlmann et al., 2009; Burgos-Aceves et al., 2021 and Liu et al., 2021 for reviews). For example, certain plastic additives can
stimulate gametogenesis and induce early spawning in female *M. galloprovincialis* (Wang et al., 2023). As byssal thread production in mussels follows a seasonal cycle, increasing during gametogenesis and declining during the post-spawning period (Carrington, 2002), MPL exposure could potentially enhance female byssal thread production indirectly by promoting gametogenesis.

## 4 CONCLUSION

This study highlights the significant role of biological factors, here the reproductive cycles and sex, in shaping mussel sensitivity to microplastic leachates. Our findings show that *M. edulis* exhibited an increased sensitivity to MPL during the post-spawning period, while female *M. galloprovincialis* were more sensitive than males. These results stress the need to incorporate biological factors into pollution risk assessments, as they provide a more nuanced understanding of how pollutants, especially microplastics, affect marine organisms. Additionally, this study emphasizes the importance of investigating the bioenergetic and endocrine-disrupting effects of plastic associated chemicals, that could explain the observed responses. As plastic pollution continues to rise, integrating biological factors into pollution studies is essential for developing targeted conservation strategies. This is particularly true for marine invertebrates and engineer species like mussels, where such factors are often overlooked despite their critical roles in supporting ecosystem stability. It is finally stressed that the impact of microplastic leachates on mussel reproductive cycle and sex has been assessed independently using two distinct polymers (i.e. polypropylene and polyethylene, respectively). This warrants the need for further work to decipher the role of distinct polymers, notably the emerging bio-sourced and/or biodegradable alternatives to conventional polymer such as polypropylene and polyethylene considered in the present work.

## 5 ACKNOWLEDGMENT

We thank J. Anquetin, A. Lutrand, A. Rahoui and V. Lefebvre for their technical support. This study was funded by the European Maritime and Fisheries Fund (FEAMP), the France Filière Pêche through the research project SOLACE («Resilience of the mussel *Mytilus edulis* to anthropogenic and climatic stressors»), the Fundação para a Ciência e a Tecnologia (FCT, MEC, Portugal, grant number: UIDB/04326/2022, PhD grant with reference 2020.06671.BD), the Pierre Hubert Curien PESSOA grant 2022-2023 and a PhD fellowship from the University of Lille and the Region-Hauts-de-France. This work was also funded by the Artois-Picardie Water Agency through the research project INFORMER ("Development of indices of ecological status of intertidal rocky ecosystems in response to climatic and anthropogenic forcings"). The manuscript was reviewed by Kévin Tallec.

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