Mohammad Wazne
email: mohammad.wazne@univ-lyon1.fr

Florian Mermillod-Blondin

Manon Vallier

Frédéric Hervant

Adeline Dumet

Holly A Nel

Anna Kukkola

Stefan Krause

Laurent Simon

Microplastics in freshwater sediments impact the role of a main bioturbator in ecosystem functioning

Keywords: Microcosms, tubificid worms, exposure, sediment-water interface, biogeochemical fluxes

While microplastic transport, fate and effects have been a focus of studies globally, the consequences of their presence on ecosystem functioning have not received the same attention.

With increasing evidence of the accumulation of microplastics at sediment-water interfaces there is a need to assess their impacts on ecosystem engineers, also known as bioturbators, which have direct and indirect effects on ecosystem health. This study investigated the impact of microplastics on the bioturbator Tubifex tubifex alongside any effects on the biogeochemical processes at the sediment-water interface. Bioturbators were exposed to four sediment microplastic concentrations: 0, 700, 7000 and 70000 particles kg -1 sediment dry weight. Though no mortality was present, a significant response to oxidative stress was detected in tubificid worms after exposure to medium microplastic concentration (7000 particles kg -1 sediment dry weight). This was accompanied by a reduction in worm bioturbation activities assessed by their ability to rework sediment and to stimulate exchange water fluxes at the sediment-water interface. Consequently, the contributions of tubificid worms on organic matter mineralization and nutrient fluxes were significantly reduced in the presence of microplastics. This study demonstrated that environmentally realistic microplastic concentrations had impact on biogeochemical processes at the sediment-water interface by reducing the bioturbation activities of tubificid worms.

INTRODUCTION

Despite the global attempts to manage plastic waste, humanity is still far from confronting the plastic crisis. [START_REF] Shen | Micro)Plastic Crisis: Un-Ignorable Contribution to Global Greenhouse Gas Emissions and Climate Change[END_REF][START_REF] Shen | Are Biodegradable Plastics a Promising Solution to Solve the Global Plastic Pollution?[END_REF] Even with efforts to reduce plastic pollution, the amount of mismanaged plastic waste expected to be released in aquatic ecosystems by 2030 may reach 53 million metric tons (Mt), twice the amount estimated for 2016. [START_REF] Borrelle | Predicted Growth in Plastic Waste Exceeds Efforts to Mitigate Plastic Pollution[END_REF] Microplastics (plastic particles ≤ 5 mm in size) [START_REF] Arthur | Proceedings of the International Research Workshop on the Occurrence, Effects, and Fate of Microplastic Marine Debris[END_REF] are part of this plastic waste, and are transported to the ocean via coastal runoff and river discharge. [START_REF] Kooi | Modeling the Fate and Transport of Plastic Debris in Freshwaters: Review and Guidance[END_REF][START_REF] Malli | Transport Mechanisms and Fate of Microplastics in Estuarine Compartments: A Review[END_REF] Once in the river environment, depending on their density, [START_REF] Drummond | Microplastic Accumulation in Riverbed Sediment via Hyporheic Exchange from Headwaters to Mainstems[END_REF] microplastics can float in the water column, or sink into sediment beds [START_REF] Kaiser | Effects of Biofouling on the Sinking Behavior of Microplastics[END_REF][START_REF] Waldschläger | The Way of Microplastic through the Environment -Application of the Source-Pathway-Receptor Model (Review)[END_REF][START_REF] Sandgaard | Sediment Matters as a Route of Microplastic Exposure: A Call for More Research on the Benthic Compartment[END_REF] where they can be temporary trapped. [START_REF] Horton | Microplastics: An Introduction to Environmental Transport Processes[END_REF] Microplastic concentrations estimated in European riverbed sediments range from 18 up to 75000 particles kg - 1 dry weight, [START_REF] Hurley | Microplastic Contamination of River Beds Significantly Reduced by Catchment-Wide Flooding[END_REF][START_REF] Rodrigues | Spatial and Temporal Distribution of Microplastics in Water and Sediments of a Freshwater System (Antuã River, Portugal)[END_REF] with the majority of particles detected being polyethylene, polypropylene, and polystyrene polymer types [START_REF] Klein | Occurrence and Spatial Distribution of Microplastics in River Shore Sediments of the Rhine-Main Area in Germany[END_REF] in a wide range of sizes and shapes (fragments, fibers, and spherical forms). [START_REF] Hurley | Microplastic Contamination of River Beds Significantly Reduced by Catchment-Wide Flooding[END_REF][START_REF] Klein | Occurrence and Spatial Distribution of Microplastics in River Shore Sediments of the Rhine-Main Area in Germany[END_REF][START_REF] Horton | Large Microplastic Particles in Sediments of Tributaries of the River Thames, UK -Abundance, Sources and Methods for Effective Quantification[END_REF] The sediment-water interface is a hotspot of biological activity in streams and rivers, which play a fundamental role in ecosystem functioning through biogeochemical processes involved in carbon (C) and nitrogen (N) cycles. [START_REF] Bardini | Nutrient Cycling in Bedform Induced Hyporheic Zones[END_REF][START_REF] Krause | Organizational Principles of Hyporheic Exchange Flow and Biogeochemical Cycling in River Networks Across Scales[END_REF][START_REF] Krause | Ecohydrological Interfaces as Hot Spots of Ecosystem Processes[END_REF] Although these biogeochemical processes are driven mainly by microbial activities, [START_REF] Krause | Organizational Principles of Hyporheic Exchange Flow and Biogeochemical Cycling in River Networks Across Scales[END_REF][START_REF] Arora-Williams | Dynamics of Microbial Populations Mediating Biogeochemical Cycling in a Freshwater Lake[END_REF][START_REF] Krause | Inter-Disciplinary Perspectives on Processes in the Hyporheic Zone[END_REF] benthic invertebrates acting as bioturbators can significantly influence these microbial processes [START_REF] Kristensen | What Is Bioturbation? The Need for a Precise Definition for Fauna in Aquatic Sciences[END_REF] by sediment reworking, biogenic structure building (e.g., burrows), and bioirrigation (i.e., the action of benthic organisms flushing their burrows with overlying water). These processes can modify pore water chemistry, which will in turn have consequences on microbial communities and associated biogeochemical processes. [START_REF] Cariou | Effects of Bioturbation by Tubificid Worms on Biogeochemical Processes, Bacterial Community Structure and Diversity in Heterotrophic Wetland Sediments[END_REF][START_REF] Svensson | Effects of Bioturbation by Tube-dwelling Chironomid Larvae on Oxygen Uptake and Denitrification in Eutrophic Lake Sediments[END_REF][START_REF] Svensson | Nitrification and Denitrification in a Eutrophic Lake Sediment Bioturbated by Oligochaetes[END_REF] Chironomid larvae (Diptera, Chironomidae) and tubificid worms (Oligochaeta, Tubificidae) have been identified as important bioturbators in freshwater ecosystems. [START_REF] Baranov | Effects of Bioirrigation of Non-Biting Midges (Diptera: Chironomidae) on Lake Sediment Respiration[END_REF][START_REF] Gautreau | The Influence of Bioturbation and Water Column Oxygenation on Nutrient Recycling in Reservoir Sediments[END_REF][START_REF] Pigneret | Urban Pollution of Sediments: Impact on the Physiology and Burrowing Activity of Tubificid Worms and Consequences on Biogeochemical Processes[END_REF] Despite this recognized importance of bioturbators on organic matter processing and nutrient recycling at sediment-water interfaces of freshwater ecosystems, the consequences of microplastics exposure of sediments on these bioturbation-driven processes remain unexplored.

Bioturbators like tubificid worms are deposit feeders that can be exposed to microplastics trapped in river bed sediments. [START_REF] Hurley | Ingestion of Microplastics by Freshwater Tubifex Worms[END_REF][START_REF] Kukkola | Nano and Microplastic Interactions with Freshwater Biota -Current Knowledge, Challenges and Future Solutions[END_REF][START_REF] Nel | Sinks and Sources: Assessing Microplastic Abundance in River Sediment and Deposit Feeders in an Austral Temperate Urban River System[END_REF][START_REF] Scherer | Feeding Type and Development Drive the Ingestion of Microplastics by Freshwater Invertebrates[END_REF][START_REF] Van Cauwenberghe | Microplastics Are Taken up by Mussels (Mytilus Edulis) and Lugworms (Arenicola Marina) Living in Natural Habitats[END_REF][START_REF] Frank | Ability of Benthic Oligochaetes to Bury Microplastics in Aquatic Bottom Sediments[END_REF] Yet, it is still unclear how this exposure may impact their physiology, and activities in sediments. Although there has been little research on this topic, a reduction in the survival rates of deposit feeding organisms like Chironomus tepperi [START_REF] Ziajahromi | Environmentally Relevant Concentrations of Polyethylene Microplastics Negatively Impact the Survival, Growth and Emergence of Sediment-Dwelling Invertebrates[END_REF] and Caenorhabditis elegans [START_REF] Lei | Microplastic Particles Cause Intestinal Damage and Other Adverse Effects in Zebrafish Danio Rerio and Nematode Caenorhabditis Elegans[END_REF] exposed to microplastics has been previously reported. In contrast, other exposure studies did not find any adverse effects on growth and survival. [START_REF] Redondo-Hasselerharm | Microplastic Effect Thresholds for Freshwater Benthic Macroinvertebrates[END_REF][START_REF] Scopetani | Microplastics Exposure Causes Negligible Effects on the Oxidative Response Enzymes Glutathione Reductase and Peroxidase in the Oligochaete Tubifex Tubifex[END_REF][START_REF] Urban-Malinga | Microplastics Cause Neurotoxicity and Decline of Enzymatic Activities in Important Bioturbator Hediste Diversicolor[END_REF] In aquatic benthic organisms, it has been shown that microplastics could significantly decrease energy reserves, [START_REF] Bour | Environmentally Relevant Microplastic Exposure Affects Sediment-Dwelling Bivalves[END_REF][START_REF] Wright | Microplastic Ingestion Decreases Energy Reserves in Marine Worms[END_REF] increase lipid peroxidation, [START_REF] Oliveira | Effects of Microplastics and Mercury in the Freshwater Bivalve Corbicula Fluminea (Müller, 1774): Filtration Rate, Biochemical Biomarkers and Mercury Bioconcentration[END_REF] trigger oxidative enzymes, [START_REF] Cole | Microplastics, Microfibres and Nanoplastics Cause Variable Sub-Lethal Responses in Mussels (Mytilus Spp.)[END_REF] and generate neurotoxicity. [START_REF] Urban-Malinga | Microplastics Cause Neurotoxicity and Decline of Enzymatic Activities in Important Bioturbator Hediste Diversicolor[END_REF] However, it has also been reported that exposing deposit feeders to microplastics did not affect their energy storage [START_REF] Van Cauwenberghe | Microplastics Are Taken up by Mussels (Mytilus Edulis) and Lugworms (Arenicola Marina) Living in Natural Habitats[END_REF] nor cause any oxidative damage. [START_REF] Scopetani | Microplastics Exposure Causes Negligible Effects on the Oxidative Response Enzymes Glutathione Reductase and Peroxidase in the Oligochaete Tubifex Tubifex[END_REF] The feeding activity of benthic macro-organisms could also be reduced by the presence of microplastics in sediments, [START_REF] Foley | A Meta-Analysis of the Effects of Exposure to Microplastics on Fish and Aquatic Invertebrates[END_REF] although this is also not universally accepted and may vary with respect to microplastic concentration, size, shape, polymer type, exposure time and species sensitivity itself. [START_REF] Van Cauwenberghe | Microplastics Are Taken up by Mussels (Mytilus Edulis) and Lugworms (Arenicola Marina) Living in Natural Habitats[END_REF][START_REF] Bour | Environmentally Relevant Microplastic Exposure Affects Sediment-Dwelling Bivalves[END_REF] The present study aims to fill this gap by investigating the impacts microplastic exposure may have on the tubificid worm Tubifex tubifex and on the ecosystem services they offer, by assessing changes to survival, physiological state and bioturbation activity. [START_REF] Kang | Enhancement of the Performance of Constructed Wetlands for Wastewater Treatment in Winter: The Effect of Tubifex Tubifex[END_REF][START_REF] Mermillod-Blondin | Influence of Tubificid Worms on Sediment Structure, Benthic Biofilm and Fauna in Wetlands: A Field Enclosure Experiment[END_REF][START_REF] Yang | Enhanced Organic Matter Decomposition in Sediment by Tubifex Tubifex and Its Pathway[END_REF] Laboratory experiments were conducted in microcosms at four sediment microplastic concentrations. Health (survival, energetic reserves and oxidative stress) of tubificid worms and their potential consequences on bioturbation activities (sediment reworking and bioirrigation rate) and ecological processes (nutrient and CO2 fluxes) at the sediment-water interface were assessed.

MATERIALS AND METHODS

Collection and characterization of sediments used

Sediments were collected using a metal shovel and metal buckets from the streambed of the Lone des Pêcheurs, a dead arm of the Rhône river in the South-east of France (45°48′41.1"N, 5°6′1.6″E).

Collected sediments were sieved (< 3.6 mm) to exclude any large debris and homogenized in a large glass container using a metal scoop. Sediments were then stored at -20°C to kill any macroorganisms present. In addition, sediment samples were analyzed to determine their particle size distribution, total organic carbon (TOC) and total nitrogen (TN) content. Particle size distribution was obtained using laser granulometer (Mastersizer, 2000, Malvern Instrument) as reported by Gette-Bouvarot et al. (2014). [START_REF] Gette-Bouvarot | Coupling Hydraulic and Biological Measurements Highlights the Key Influence of Algal Biofilm on Infiltration Basin Performance[END_REF] Three measurements (i.e. three replicates) were carried out for the sediment after ultrasound treatment (50-60 Hz, 1 min) to eliminate non-stable particle aggregates. [START_REF] Gette-Bouvarot | Coupling Hydraulic and Biological Measurements Highlights the Key Influence of Algal Biofilm on Infiltration Basin Performance[END_REF] TOC and TN contents were determined by high-temperature in situ combustion on pre-acidified (i.e., HCL 2 mol/L) dry samples (60°C, 48hr) for three replicates of 15 mg each, using an elemental analyzer (FlashEA, Thermo Electron Corporation). The microplastic content of the sediment was assessed following an approach modified from those of Frei et al. (2019), [START_REF] Frei | Occurence of Microplastics in the Hyporheic Zone of Rivers[END_REF] .

Briefly, after drying at 50 °C for 24 hours, a sediment sample of 50 g was suspended in zinc chloride (ZnCl2) solution (density 1.7 g cm -3 ) for 24 hours to collect microplastic particles and organic components which were later degraded with 30% H2O2 in the presence of 0.05 M Fe 2+ (i.e., aqueous). The remaining material was then stained with Nile Red solvent, [START_REF] Nel | Detection Limits Are Central to Improve Reporting Standards When Using Nile Red for Microplastic Quantification[END_REF][START_REF] Maes | A Rapid-Screening Approach to Detect and Quantify Microplastics Based on Fluorescent Tagging with Nile Red[END_REF] filtered, and microplastics > 20 µm sizes were quantified under Nikon SMZ1270 fluorescent stereo microscope.

(Table 1).

Table 1 Physical characteristics of the sediment (mean ± SD, n = 3).

Microplastics preparation

A heterogeneous mixture of two types of microplastic fragments (polystyrene (PS) and polyamide (PA)) and one type of microplastic fibers (PA) was used in this study. The PS particles were madein-house by cutting cuvettes (VWR) into small pieces and then ground in a Retsch ball mill MM 400 at 30 Hz for 30 s with liquid nitrogen. The resulting powder was sieved to exclude polystyrene fragments > 1000 µm using a stainless steel sieve mesh. To obtain multiple shapes of PA particles, fibers (L = 500 µm, W = 15 µm) were obtained from Flock Depot (Stuttgart, Germany) and pellets were sourced from Resinex Ltd (High Wycombe, United Kingdom). The pellets were subsequently frozen at -80°C for 72hr and ground using a Fritsch Pulverisette 0 ball mill with liquid nitrogen for 20 minutes. The resulting PA powder was size fractionated by stainless steel sieves to exclude PA fragments > 1000 µm. Prior to their use in the controlled experiments, the particle size distribution of PS and PA particles (i.e., both fragments and fibers) was measured by laser granulometry with the same method and equipment than those used previously to measure sediment grain size distribution (Supplementary Figure 1). The size and morphological features of microplastic particles were investigated with a fluorescent stereo microscope (Nikon SMZ1270) without fluorescence under the brightfield mode. Overall, the microplastic particles used in the experiment presented a wide variety of shapes and sizes varying from 15 to 1500 µm

Measurements

Grain size distribution (%) 64 µm < Sand < 2 mm 35.9 ± 5.8 2 µm < Silt < 64 µm 60.2 ± 2.9 Clay < 2 µm 3.9 ± 0.3 Organic matter composition (% sediment dry mass) Total organic carbon 2.4 ± 0.10 Total nitrogen 0.2 ± 0.02 (Supplementary Figure 2), including particles that could be easily ingested by tubificid worms (< 63 µm) [START_REF] Hurley | Ingestion of Microplastics by Freshwater Tubifex Worms[END_REF] .

Bioturbation model organisms

The tubificid worm, Tubifex tubifex was chosen to investigate the impacts of microplastics on freshwater bioturbators as this species has a key role in the biogeochemistry and ecology of freshwater benthic habitats, [START_REF] Mermillod-Blondin | Influence of Tubificid Worms on Sediment Structure, Benthic Biofilm and Fauna in Wetlands: A Field Enclosure Experiment[END_REF] and due to its wide use in measuring the ecotoxicity of sediments. [START_REF] Hurley | Ingestion of Microplastics by Freshwater Tubifex Worms[END_REF][START_REF] Paris-Palacios | Toxic Effects and Bioaccumulation of the Herbicide Isoproturon in Tubifex Tubifex (Oligocheate, Tubificidae): A Study of Significance of Autotomy and Its Utility as a Biomarker[END_REF] Tubificid worms were bought from a commercial breeder (GREBIL & Fils, Arry, France) and acclimatized to experimental conditions (i.e., temperature of 20°C, sediments collected from Lone des Pêcheurs, aerated synthetic water, and under a 16:8 h light: dark cycle) for two weeks prior to the start of the experiment.

Experimental design and preparation

A complete randomized block design was used to test the impacts of the addition of four microplastic concentrations (0 particles/kg sediment dry weight (DW) (control); 700 particles/kg sediment DW (low); 7000 particles/kg sediment DW (medium); 70000 particles/kg sediment DW (high)) on microcosms with or without tubificid worms. By measuring the interaction between microplastic treatments and worm presence, this experimental design aimed to evaluate whether the presence of microplastics impacted the role of tubificid worms in sediments compared with the direct impact of microplastics on microorganisms (treatments without worms). The four microplastic treatments were chosen based on reports of actual environmental realistic concentrations reported from Europe to China. [START_REF] Hurley | Microplastic Contamination of River Beds Significantly Reduced by Catchment-Wide Flooding[END_REF][START_REF] Klein | Occurrence and Spatial Distribution of Microplastics in River Shore Sediments of the Rhine-Main Area in Germany[END_REF][START_REF] Huang | The Occurrence and Abundance of Microplastics in Surface Water and Sediment of the West River Downstream, in the South of China[END_REF] These investigations indicated the presence of hundreds to thousands of plastic particles per kilogram of dry sediment, with the greatest concentration of microplastics discovered in the riverbeds of the Irwell River, with up to 75,000 particles per kilogram of dry sediment. [START_REF] Hurley | Microplastic Contamination of River Beds Significantly Reduced by Catchment-Wide Flooding[END_REF] A total of eight treatments (4 microplastic concentrations * 2 worm conditions) was tested with five replicates per treatment (Ntotal = 40). Each replicate was made up of a 2 L glass bottle (internal diameter: 13 cm, height: 25 cm) filled with 8 cm of sediment (i.e., 0.738 kg DW) (Supplementary Figure 3) previously prepared by mixing microplastic particles with sediments to obtain the four desired concentrations. The mixing process took place one week before filling the 2 L glass bottles by using one glass container (L = 50 cm, W = 20 cm, H = 25 cm) per microplastic treatments (4 glass containers were used for the initial preparation of the 4 tested microplastic concentrations, Supplementary Table 1). After a week of homogeneous mixing of sediment with the desired concentration of microplastics (for each treatment, microplastics and sediments were thoroughly mixed in a glass container for 30 min every day during 1 week using a stainless steel spoon), sediments were transferred to 2 L glass bottles (i.e. microcosms), with 10 glass bottles per microplastic treatment. By homogeneously mixing sediment and microplastics, we used a simplified distribution of microplastics in sediments compared with heterogeneous distributions of microplastics in freshwater environments, [START_REF] Drummond | Microplastic Accumulation in Riverbed Sediment via Hyporheic Exchange from Headwaters to Mainstems[END_REF][START_REF] Frei | Occurence of Microplastics in the Hyporheic Zone of Rivers[END_REF] but it was the best way to control the exposure of tubificid worms to microplastic particles. After sediment addition, 10 cm of overlying synthetic freshwater (96 mg L -1 NaHCO3, 39.4 mg L -1 CaSO4.2H2O, 60 mg L -1 MgSO4.7H2O, 4 mg L -1 KCl, and 6.4 mg L -1 (CH3CO2)2Ca.H2O; pH = 7.5) [START_REF] Weber | Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms[END_REF] was added to each microcosm. Synthetic water was gently added to each microcosm to avoid any sediment resuspension. Microcosms were aerated to maintain a dissolved oxygen (DO) concentration between 7.5 and 8.5 mg L -1 in the water column throughout the full duration of the experiment. The experiment was conducted at a constant room temperature of 20°C under a 16:8 h light: dark cycle. After microcosm preparation, 180 individuals of tubificid worms having lengths ranging between 1 and 3 cm were introduced into each microcosm dedicated to treatments with worms (half of the microcosms), representing a density of 13,274 individuals per m 2 commonly observed in the braided arms of the Rhône river. [START_REF] Martinet | Descripteur Des Flux Organiques Liés Aux Sédiments: Exemples Dans Diverses Annexes Fluviales Du Rhône[END_REF] Then, the experiment started for 77 days, an experimental duration which was long enough to observe long-term impacts on tubificid worms without having a deletion in sedimentary organic matter that could impair food availability for tubificid worms.

During the last four weeks of the experiment, the bioturbation activities (surface sediment reworking and bioirrigation rate) of worms and biogeochemical processes (CO2, CH4, and nutrient fluxes) were assessed to evaluate the influences of microplastic contaminated sediments on individuals and associated ecosystem processes. At the end of the experiment (i.e., day 77), microcosms were dismantled and sediments were sieved on a 250 µm mesh sieve to recover tubificid worms. After collection, tubificid worms were counted to determine their survival rate in each microplastic treatment. Surviving worms were collected with each worm being examined to see whether it moves or not. Then worms from each microcosm were separated in two groups: a first group of around 130 individuals was used to evaluate the physiological state of worms and a second group of around 50 individuals was used to determine the quantity of microplastics ingested by worms. In microcosms where less than 180 individuals were alive, individuals were divided into two groups while maintaining the same ratio of division.

To avoid contamination with microplastics from laboratory equipment, we used glass material for handling of particles, manipulating tubificid worms and for exposure experiments. Materials were covered to reduce airborne contamination.

Exposure impact analysis

Physiological analyses

The first group of collected worms (130 individuals per microcosm) were kept for 48hr in glass beakers filled with 0.7 µm filtered synthetic water for depuration. Afterwards, tubificid worms were freeze-dried and weighed collectively. These freeze-dried 130 individuals were then divided randomly into four small subgroups, each subgroup being used to perform a given physiological analysis (2 analyses associated with energy body stores and 2 analyses associated with oxidative stress). Energy body stores were determined in the whole animal by extracting and measuring the amount of triglycerides (Sigma Aldrich, T2449 and F6428) and glycogen (Sigma Aldrich, G3293)

as described in Hervant et al. (1999) and Salin et al. (2010). [START_REF] Hervant | Comparative Study on the Metabolic Responses of Subterranean and Surface-Dwelling Amphipods to Long-Term Starvation and Subsequent Refeeding[END_REF][START_REF] Salin | Cave Colonization without Fasting Capacities: An Example with the Fish Astyanax Fasciatus Mexicanus[END_REF] Two markers of oxidative stress were measured: malondialdehyde (MDA) content which is a biomarker of lipid peroxidation (LPO) and the antioxidant enzyme superoxide dismutase (SOD) activity which is induced by the formation of reactive oxygen species (ROS). The thiobarbituric acid (TBARS) assay (Cayman chemicals, 10009050) was used to determine the MDA content [START_REF] Lawniczak | Preventive Antioxidant Responses to Extreme Oxygen Level Fluctuation in a Subterranean Crustacean[END_REF] and the SOD activity (Cayman chemicals, 706002) was estimated according to Flohe (1984) and Lawniczak et al. (2013). [START_REF] Lawniczak | Preventive Antioxidant Responses to Extreme Oxygen Level Fluctuation in a Subterranean Crustacean[END_REF]58 MDA contents were expressed in nanomoles (nmol) of MDA mg -1 dry mass, whereas SOD catalytic activities were expressed as U g -1 dry mass, where one unit was defined as the quantity of enzyme that reduced 50% of oxidized cytochrome c (which is oxidized under superoxide production).

Microplastics ingestion analysis

The second group of collected worms (50 individuals combined per microcosm) were cleaned from external debris using vacuum filtered ultrapure (VFUP) water (i.e., filtered through Whatman GF/F filters (0.7 µm)) without any depuration phase. Then, microplastics were extracted from tubificid worms according to Hurley et al. ( 2017) with some modifications. [START_REF] Hurley | Ingestion of Microplastics by Freshwater Tubifex Worms[END_REF] Fresh tubificid worms were weighed and placed into glass tubes previously washed three times with VFUP water.

Worms were digested with 30% H2O2 in the presence of 0.05 M Fe 2+ (i.e., aqueous) catalyst solution at an ambient room temperature for 24hr. After digestion, samples were washed with VFUP water then filtered through Whatman GF/F filters (0.7 µm). The number of microplastics were determined using a Nikon SMZ1270 fluorescent stereo microscope after staining with Nile Red of 5 µm mL -1 as final concentration. [START_REF] Nel | Detection Limits Are Central to Improve Reporting Standards When Using Nile Red for Microplastic Quantification[END_REF][START_REF] Maes | A Rapid-Screening Approach to Detect and Quantify Microplastics Based on Fluorescent Tagging with Nile Red[END_REF] The stereo microscope was fitted with Intensilight C-HGFIL Lamp (130w) with a 0.75 X objective lens. All measurements were done at an overall magnification of 15 X. Three procedural blanks were prepared simultaneously. These blanks were assumed to indicate background microplastic contamination, and as such, blank values were subtracted from the final results.

Sediment reworking analysis

As microplastic contamination has been shown to reduce the egestion of fecal pellets by the marine worm A. marina, [START_REF] Green | Effects of Conventional and Biodegradable Microplastics on a Marine Ecosystem Engineer (Arenicola Marina) and Sediment Nutrient Cycling[END_REF] we also expected that microplastic exposure could reduce the production of fecal pellets at the sediment surface by tubificid worms. To assess this potential effect, the impact of microplastic exposure was measured on the surface sediment reworking (SSR) process induced by tubificid worms during 5 days in the last week of the experiment (from day 70 to day 74, Supplementary Figure 4) using the method of De Nadaï-Monoury et al. (2013). [START_REF] Nadaï-Monoury | A Cost-Effective Method to Quantify Biological Surface Sediment Reworking[END_REF] For each microcosm, a 2 mm thick layer of a particulate tracer (white sand of Fontainebleau) was uniformly distributed at the surface of the sediment seven days before the end of the experiment.

Translocation of the tracer due to the fecal deposition by tubificid worms was monitored by taking pictures every 12 hours for five days. Pictures were analyzed using ImageJ software. [START_REF] Schneider | NIH Image to ImageJ: 25 Years of Image Analysis[END_REF] Automatic pixel counts were performed based on colors (sandy tracer was white whereas fecal pellets appeared in dark, Supplementary Figure 5) to estimate the surface covered by tracer. The remaining area covered by fecal pellets was calculated as the difference between total surface area of microcosms minus surface occupied by the tracer. SSR rate was calculated and expressed in % of area recovered by fecal pellets per hour.

Bioirrigation activity

A dissolved conservative tracer (i.e., KCl) was added to the water column (as the methodology used by Mermillod-Blondin et al. ( 2004) [START_REF] Mermillod-Blondin | Influence of Bioturbation by Three Benthic Infaunal Species on Microbial Communities and Biogeochemical Processes in Marine Sediment[END_REF] with Br -as conservative tracer) to quantify water fluxes from the water column to sediment. This measurement was done for 1 day, two weeks before the end of the experiment (from day 63 to day 64, Supplementary Figure 4), by monitoring the concentration shift over time of the dissolved tracer. More precisely, for each microcosm, water was replaced with synthetic freshwater enriched with KCl to obtain a concentration of 37 mg L -1 of Cl -, a concentration which was not a stress for Tubifex tubifex, [START_REF] Soucek | Influence of Water Hardness and Sulfate on the Acute Toxicity of Chloride to Sensitive Freshwater Invertebrates[END_REF] and commonly measured in freshwater environments. [START_REF] Voisin | Aquifer Recharge with Stormwater Runoff in Urban Areas: Influence of Vadose Zone Thickness on Nutrient and Bacterial Transfers from the Surface of Infiltration Basins to Groundwater[END_REF] Using acid-washed 100 ml syringes, water samples were collected immediately after water column replacement and 1 day after for each microcosm. Water samples were filtered through Whatman GF/F filters (0.7 µm) and Cl -concentration were analyzed using a sequential analyzer based on colorimetric methods (Smartchem 200, AMS Alliance, France). The decrease in Cl -concentrations between the two days was used to evaluate the water fluxes at the sediment-water interface in each microcosm. Based on changes in Cl -concentrations and the water volume in microcosms, these fluxes were expressed in L of water exchanged per day and m -2 .

Biogeochemical analysis

The influence of microplastic contamination on biogeochemical processes (organic matter mineralization and nutrient cycling) was estimated by measuring the CO2 and CH4 fluxes and the fluxes of nutrients (N-NH4 + , N-NO3 -, N-NO2 -and P-PO4 3-) at the sediment-water interface for each microcosm. [START_REF] Pigneret | Urban Pollution of Sediments: Impact on the Physiology and Burrowing Activity of Tubificid Worms and Consequences on Biogeochemical Processes[END_REF][START_REF] Mermillod-Blondin | Do Tubificid Worms Influence the Fate of Organic Matter and Pollutants in Stormwater Sediments?[END_REF] CO2 and CH4 fluxes were measured for 5 days according to Pigneret et al. (2016), [START_REF] Pigneret | Urban Pollution of Sediments: Impact on the Physiology and Burrowing Activity of Tubificid Worms and Consequences on Biogeochemical Processes[END_REF] three weeks before the end of the experiment (from day 56 to day 60, Supplementary Figure 4). Each microcosm was tightly sealed with a lid connected to a pump and a greenhouse gas analyzer (G2201i PICARRO). CO2 and CH4 fluxes were measured by monitoring the increase in concentrations every 2 hr during 10 hr at 20 °C for each microcosm. Then, CO2 and CH4 fluxes were expressed in mmol per hour and m 2 of sediment-water interface.

For nutrient fluxes, 2/3 of the water column was replaced in each microcosm four weeks before the end of the experiment (from day 49 to day 53, Supplementary Figure 4). For five days, water samples (100 ml) were collected in the water column of each microcosm at daily intervals. Water samples were taken using acid-washed 100 ml syringes, filtered through Whatman GF/F filters (0.7 µm), and analyzed for nutrient concentrations within 24hr using a sequential analyzer based on colorimetric methods (Smartchem 200, AMS Alliance, France). The fluxes of N-NH4 + , N-NO3 - , N-NO2 -and P-PO4 3-at the sediment-water interface were calculated from the linear changes over time of the concentration of each nutrient in the water column. Then, nutrient fluxes were expressed in mg per day and m 2 of sediment-water interface. Positive fluxes indicated an efflux of nutrients from sediments to the water column whereas negative fluxes indicated an influx of nutrients from the water column to sediments.

Statistical Analyses

For the three physiological variables measured on tubificid worms (glycogen content, triglyceride content, SOD activity), the influence of microplastic treatments (control, low, medium, and high) was tested using one-way analyses of variance (1-way ANOVAs) with microplastic concentrations as the fixed factor. A one-way ANOVA was also used to test the influence of microplastic treatments on the surface sediment reworking rate due to tubificid worms. For chloride, nutrient fluxes, CO2 and CH4 fluxes, the effects of microplastic treatments, tubificid worm treatment and their interactions were tested using two-way analyses of variance (2-ways ANOVA) with microplastic concentrations and tubificid worms as fixed factors. When 2-ways ANOVA revealed significant differences among treatments (i.e., p < 0.05), Tukey's post hoc tests were performed to evaluate which treatments significantly differed. For all variables, the normality and the homoscedasticity of the residues were verified using the Shapiro-Wilk's test and the Bartlett's test, respectively. As for survival rates and MDA contents the requirements for normality and homoscedasticity were not met, non-parametric Kruskal-Wallis test were performed. All statistical analyses were done with the RStudio software. 66

RESULTS AND DISCUSSION

Survival and physiology of Tubifex tubifex

After 77 days of exposure, the survival rates of tubificid worms were higher than 75% in all microplastic treatments (Figure 1A). Survival rate was not significantly affected by microplastic concentrations (Kruskal-Wallis test, H (3) = 3.8, p = 0.28) although it tended to decrease for individuals exposed to the medium microplastic concentration in comparison with the control treatment. Interestingly, this trend was associated with a significant increase in SOD activity in tubificid worms exposed to the medium microplastic concentrations (Figure 1B, 1-way ANOVA, p < 0.05) with a mean value of 12.8 ± 6.2 U g -1 dry mass which was 16-fold higher than in the control treatment (0.8 ± 1.9 U g -1 dry mass). These differences in SOD activity among microplastic treatments were not associated with significant changes in MDA concentrations among tubificid worms exposed to different microplastic treatments (Figure 1C, Kruskal-Wallis test, H (3) = 2.49, p = 0.48). Observed increase in SOD activity showed that tubificid worms exposed to microplastics produced antioxidant defense enzymes to catalyze the dismutation of superoxide into molecular oxygen and hydrogen peroxide. [START_REF] Pigneret | Urban Pollution of Sediments: Impact on the Physiology and Burrowing Activity of Tubificid Worms and Consequences on Biogeochemical Processes[END_REF][START_REF] Piddington | Zn Superoxide Dismutase of Mycobacterium Tuberculosis Contributes to Survival in Activated Macrophages That Are Generating an Oxidative Burst[END_REF] Thus, the physiology of tubificid worms was influenced by reactive oxygen species (ROS) generated by microplastic exposure, especially for the medium microplastic concentration. In general, the presence of ROS causes lipid peroxidation (LPO) damage that leads to an increase in MDA which is a by-product of LPO. [START_REF] Yang | The Combined Toxicity Influence of Microplastics and Nonylphenol on Microalgae Chlorella Pyrenoidosa[END_REF] Nevertheless, the lack of significant increase in MDA concentrations in tubificid worms despite an increased production of SOD enzymes indicated that tubificid worms succeeded in neutralizing both ROS production and oxidative damage to maintain their homeostasis (a mechanism described by Trestrail et al.

(2020)). [START_REF] Trestrail | Invertebrate Responses to Microplastic Ingestion: Reviewing the Role of the Antioxidant System[END_REF] This mechanism has never been detected in invertebrates but has been already observed in the freshwater fish Oreochromis niloticus by Ding et al. (2018). [START_REF] Ding | Accumulation, Tissue Distribution, and Biochemical Effects of Polystyrene Microplastics in the Freshwater Fish Red Tilapia (Oreochromis Niloticus)[END_REF] These authors found that the presence of polystyrene microplastics significantly increased SOD activity in the fish without affecting its MDA content. [START_REF] Ding | Accumulation, Tissue Distribution, and Biochemical Effects of Polystyrene Microplastics in the Freshwater Fish Red Tilapia (Oreochromis Niloticus)[END_REF] Thus, in the present experiment, it is likely that oxidative stress provoked by microplastic exposure was efficiently managed by antioxidative defenses of tubificid worms, preventing cellular damages caused by high lipid peroxidation levels. [START_REF] Trestrail | Invertebrate Responses to Microplastic Ingestion: Reviewing the Role of the Antioxidant System[END_REF] In addition, the presence of microplastics at different concentrations did not significantly affect energy reserves in tubificid worms (Figure 1D, E, 1-way ANOVAs, p = 0.2 for glycogen and p = 0.1 for triglyceride concentration). These findings are in agreement with Van Cauwenberghe et al.

(2015) who observed that polystyrene microspheres had no significant impact on the energy reserves of the lugworm Arenicola marina (deposit feeder) or the blue mussel Mytilus edulis (filter feeder). [START_REF] Van Cauwenberghe | Microplastics Are Taken up by Mussels (Mytilus Edulis) and Lugworms (Arenicola Marina) Living in Natural Habitats[END_REF] Theoretically, the antioxidative defenses activated in tubificid worms when exposed to microplastics should have generated a significant consumption of energy reserves, leading to lower stocks of glycogen and triglycerides in worms exposed to medium microplastic concentrations. [START_REF] Lu | Uptake and Accumulation of Polystyrene Microplastics in Zebrafish (Danio Rerio) and Toxic Effects in Liver[END_REF][START_REF] Xia | Polyvinyl Chloride Microplastics Induce Growth Inhibition and Oxidative Stress in Cyprinus Carpio Var. Larvae[END_REF] However, the present study indicated that tubificid worms did not rely on their energy stores to manage oxidative stress induced by microplastics exposure.

Ingestion of microplastics

Although the microplastic mixture contained particles that could be easily ingested by tubificid worms (< 63 µm) [START_REF] Hurley | Ingestion of Microplastics by Freshwater Tubifex Worms[END_REF] and despite the presence of high microplastic concentrations in sediments (up to 70000 particles/kg DW), no significant numbers of microplastic particles (neither fragments nor fibres) were detected by fluorescence microscopy in the 50 organisms digested with Fenton reagent at the end of the experiment (Supplementary Table 2). This lack of ingested microplastics could partly explain the lack of observed effects on energy reserves in tubificid worms. For example, Wright et al. ( 2013) reported that longer gut residence time of ingested microplastics was positively correlated with the consumption of energy reserves in marine worms (Arenicola marina). [START_REF] Wright | Microplastic Ingestion Decreases Energy Reserves in Marine Worms[END_REF] As we did not detect microplastics particles in tubificid worms tissues, tubificid worms were more likely able to exclude the microplastics when feeding on sediments, thereby avoiding a large oxidative stress and the high energy expenditure associated with defense against oxidative damages. This interpretation is supported by Rodriguez et al. (2001) who showed that tubificid worms could selectively feed on fine organic rich particles in sediment. [START_REF] Rodriguez | Selective Feeding by the Aquatic Oligochaete Tubifex Tubifex (Tubificidae, Clitellata)[END_REF] Selective feeding excluding microplastic particles has been also reported by other aquatic deposit feeders as polychaetes of the genus Marenzelleria 74 Therefore, the observed elevation in SOD activity in tubificid worms exposed to microplastics could be the result of external physical damage induced by plastic particles rather than due to ingestion. [START_REF] Yardy | What the Fluff Is This? -Gammarus Pulex Prefer Food Sources without Plastic Microfibers[END_REF] It is also worth noting that the physiological impacts induced by microplastic exposure on biota have been reported to be species-specific. [START_REF] Bour | Environmentally Relevant Microplastic Exposure Affects Sediment-Dwelling Bivalves[END_REF] In the present study, it can be concluded that the benthic species T. tubifex was relatively tolerant to the microplastic exposure applied, explaining the low impact on MDA and worm survival. Such conclusion was consistent with other field studies that have reported that T. tubifex is one of the most pollution-resistant species living in freshwater environments. [START_REF] Chapman | Effects of Species Interactions on the Survival and Respiration of Limnodrilus Hoffmeisteri and Tubifex Tubifex (Oligochaeta, Tubificidae) Exposed to Various Pollutants and Environmental Factors[END_REF][START_REF] Milbrink | Biological Characterization of Sediments by Standardized Tubificid Bioassays[END_REF]

Bioturbation activities of Tubifex tubifex

While surface sediment reworking (SSR) was absent in the treatments without tubificid worms, SSR rapidly occurred after the addition of the white Fontainebleau sand (non-fluorescent tracer)

in microcosms with Tubifex tubifex (Figure 2A). The mean surface reworked by tubificid worms exceeded 50% of microcosm surface for all treatments after 12hr (Figure 2B). The surface of sediment reworked increased strongly within the first 12hr to reach values higher than 50% (Figure 2A). As most of the sediment surface was already reworked within these first 12hr, SSR rates decreased after this time (Figure 2B). For this reason, only the areas occupied by fecal pellets within the first 12hr were used to compare treatments (Figure 2C). Although statistical analyses were only marginally significant (1-way ANOVA, 0.05 < p < 0.1), SSR due to fecal pellet deposition by worms tended to decrease with the increasing concentration of microplastics added to sediments. After 12hr of tubificid worm bioturbation, the SSR was 60.9 ± 5% in the treatment without microplastic (control) and was reduced by around 12% in the treatment with the highest microplastic concentration.

Tubificid worms significantly increased the water flux between water and sediments in the microcosms (2-way ANOVA, worm effect, p < 0.05) regardless of the microplastic treatment (2way ANOVA, interaction between worm treatments and microplastic treatments, p = 0.8). For all microplastic treatments, the exchange of Cl -measured at the sediment-water interface was around two-fold higher in treatments with tubificid worms than in treatments without worms (Figure 2D).

Nevertheless, it is worth noting that no significant difference in water fluxes was detected between treatments with and without worms at medium microplastic concentrations (Tukey's test, p = 0.1) whereas significant differences between treatments with and without worms were measured for all other microplastic concentration treatments (Tukey's tests, p < 0.05).

SSR measurements indicate that the presence of microplastics tended to decrease the feeding activity and the deposition of fecal pellets by tubificid worms at the sediment surface. In contrast, the bio-irrigation process generated by tubificid worms and assessed by water flux measurements was not strongly affected by microplastic treatments. Thus, microplastic contamination did not affect similarly bioirrigation and feeding activities of tubificid worm, highlighting the complex influences of microplastic contaminations on bioturbators. Remarkably, the different impacts of microplastics on bioirrigation and sediment reworking processes could have different effects on ecosystem functioning because these two bioturbation processes are known to differently stimulate nutrient fluxes and mineralization processes in sediments.

Biogeochemical Fluxes

No significant CH4 fluxes were measured from all microcosms, whereas significant CO2 fluxes were calculated from linear increases of CO2 concentration over time (Supplementary Figure 6A, and 6B). Overall, tubificid worms significantly increased CO2 fluxes (Figure 3A, 2-way ANOVA, worm effect, p < 0.05) depending on microplastic concentrations in sediments (2-way ANOVA, statistical interaction between worm treatments and microplastic treatments, p < 0.05). CO2 fluxes were around 2.5-fold higher with worms (0.47 mmol h -1 m -2 ± 0.2) than without worms (0. Nutrient flux measurements did not report any linear increase in N-NH4 + and P-PO4 3- concentrations with incubation time, indicating no significant fluxes of these two nutrients from sediments to water column (Supplementary Figure 6C, and 6D). The absence of N-NH4 + and P-PO4 3-fluxes associated with the lack of measured CH4 flux (associated with methanogenesis processes) indicate that no strong anoxic conditions occurred in the sediment used for the experiment. More precisely, aerobic conditions in sediments probably inhibited the fluxes of PO4 3- released to the water column by facilitating the adsorption of P with Fe(OH)3 and Mn(OH)2. [START_REF] Gautreau | The Influence of Bioturbation and Water Column Oxygenation on Nutrient Recycling in Reservoir Sediments[END_REF] Such aerobic conditions could also have minimized the release rates of NH4 + from sediments by facilitating a rapid nitrification of NH4 + produced by organic matter mineralization into N-NO3 - . [START_REF] Hedin | Thermodynamic Constraints on Nitrogen Transformations and Other Biogeochemical Processes at Soil-Stream Interfaces[END_REF] In line with this facilitated nitrification process, N-NOx (N-NO3 -+ N-NO2 -) concentrations linearly and significantly increased during the incubation, indicating a significant release rate of NO3 -from sediments to the water column (Supplementary Figure 6E, and6F). Overall, tubificid worms significantly increased the NOx fluxes from sediments to the water column in microcosms (Figure 3B, 2-way ANOVA, worm effect, p < 0.05). However, this stimulation of NOx fluxes due to the presence of worms was significantly reduced in presence of microplastics at low, medium and high concentrations (Figure 3B, 2-way ANOVA, interaction "worm*MPs" effect, p < 0.05, Tukey's HSD post-hoc tests, p < 0.05). As observed for CO2 fluxes, NOx fluxes were comparable among the four microplastic treatments without tubificid worms (Figure 3B, Tukey's HSD, p > 0.05), suggesting no direct influence of microplastics on microbial processes involved in N cycling.

These results clearly highlighted that microplastic exposure significantly reduced the contribution of tubificid worms on organic matter processing and nutrient cycling at the sediment-water interface. To our knowledge, this is the first evidence of an adverse impact of microplastics not only on organisms, but also on their contributions to ecosystem functioning.

Main conclusions and perspectives

The present study highlighted a significant impact of microplastic exposure on bioturbation-driven processes (organic matter mineralization and nutrient fluxes). These effects were probably associated with a reduction of bioturbation activity of tubificid worms monitored by fecal pellet production at the sediment-water interface. However, the obtained results were only marginally significant because more than 50% of the sediment surface area was already covered by fecal pellets at the first time of analysis (12hr). Under these conditions, it was difficult to observe significant results among treatments. Thus, for future experiments, monitoring fecal pellet production at higher frequencies (every 3hr for example) would permit more pertinent calculations of fecal pellet deposition rates. Furthermore, the use of luminophore tracer distribution in the sediment column at the end of the experiment [START_REF] Gerino | Comparison of Different Tracers and Methods Used to Quantify Bioturbation During a Spring Bloom: 234-Thorium, Luminophores and Chlorophylla[END_REF][START_REF] Ciutat | Effects of Bioturbation on Cadmium Transfer and Distribution into Freshwater Sediments[END_REF][START_REF] Lagauzère | Effects of Uranium-Contaminated Sediments on the Bioturbation Activity of Chironomus Riparius Larvae (Insecta, Diptera) and Tubifex Tubifex Worms (Annelida, Tubificidae)[END_REF] or techniques such as computed tomography (CT) scanning 85 could be promising approach to evaluate more precisely the impacts of microplastic exposure on bioturbation process.

Interestingly, we also found that the effects of microplastics on the stimulation of biogeochemical processes by worms were not positively correlated with microplastic concentrations. First, the positive effects of tubificid worms on CO2 fluxes were totally inhibited at the lowest concentration of microplastics in sediments. Similarly, the role of tubificid worms in N-NOx fluxes from sediment to water column was also impacted in the presence of low microplastic concentrations.

These results indicate that concentrations exceeding 700 particles/kg sediment DW and observed in a wide range of freshwater environments [START_REF] Hurley | Microplastic Contamination of River Beds Significantly Reduced by Catchment-Wide Flooding[END_REF][START_REF] Klein | Occurrence and Spatial Distribution of Microplastics in River Shore Sediments of the Rhine-Main Area in Germany[END_REF][START_REF] Frei | Occurence of Microplastics in the Hyporheic Zone of Rivers[END_REF][START_REF] Lin | Occurrence and Distribution of Microplastics in an Urban River: A Case Study in the Pearl River along Guangzhou City, China[END_REF] may damage the role played by bioturbators on ecosystem functioning. Second, medium microplastic concentrations (7000 particles/kg sediment DW) induced stronger adverse impacts on tubificid worm physiology (higher SOD activity) and activities (worm effect on CO2 fluxes) than the highest concentration of microplastics in sediments (70000 particles/kg sediment DW). This surprising result could be due to the ability of tubificid worms to better detect and then to avoid microplastic particles at their highest concentration (may be because they are less embedded with sediment particles than for lower concentrations). This mechanism of microplastic avoidance has been already reported in other invertebrate species which were able detect the physical presence of plastic particles and/or chemical cues associated with microplastics. [START_REF] Yardy | What the Fluff Is This? -Gammarus Pulex Prefer Food Sources without Plastic Microfibers[END_REF][START_REF] Carrasco | The Influence of Microplastics Pollution on the Feeding Behavior of a Prominent Sandy Beach Amphipod, Orchestoidea Tuberculata (Nicolet, 1849)[END_REF] Under these conditions, high concentrations of microplastic particles might have modified the burrowing behavior of tubificid worms (as suggested by change in fecal pellet production) and the gallery network produced by worms in sediments. For example, it has been largely observed that contaminants could influence the structure of biogenic structures produced by bioturbators in sediments [START_REF] Pigneret | Urban Pollution of Sediments: Impact on the Physiology and Burrowing Activity of Tubificid Worms and Consequences on Biogeochemical Processes[END_REF][START_REF] Lagauzère | Effects of Uranium-Contaminated Sediments on the Bioturbation Activity of Chironomus Riparius Larvae (Insecta, Diptera) and Tubifex Tubifex Worms (Annelida, Tubificidae)[END_REF] and soils. [START_REF] Capowiez | Sublethal Effects of Imidacloprid on the Burrowing Behaviour of Two Earthworm Species: Modifications of the 3D Burrow Systems in Artificial Cores and Consequences on Gas Diffusion in Soil[END_REF] Therefore, change in gallery network would have modulated the solute fluxes at the sediment-water interface by modifying the exchange area between sediments and surface water, [START_REF] Pischedda | Imaging Oxygen Distribution in Marine Sediments. The Importance of Bioturbation and Sediment Heterogeneity[END_REF] impacting the biogeochemical processes occurring in sediments. [START_REF] Aller | The Effect of Biogenic Irrigation Intensity and Solute Exchange on Diagenetic Reaction Rates in Marine Sediments[END_REF] Further experiments based on precise quantifications of tubificid worm activities in sediments (creation of biogenic structure, sediment reworking using luminophores) are needed to decrypt these complex interactions among microplastic contaminations, tubificid worm bioturbation and biogeochemical processes at the sediment-water interface of freshwater ecosystems.

Figure 1 .

 1 Figure 1. Survival rates (A), superoxide dismutase activity (B), malondialdehyde concentration

Figure 2 .

 2 Figure 2. Sediment surface images taken from four replicates of each microplastics treated

  19 mmolh -1 m -2 ± 0.1) in the control treatment (Figure3A, Tukey's HSD, p < 0.05). In contrast, CO2 fluxes were similar between control microcosms and microcosms with worms in the presence of low and medium microplastic concentrations (Figure3A, Tukey's HSD, p > 0.5). Surprisingly, this inhibition of the effects of tubificid worms on CO2 fluxes was not observed in the treatment with the highest microplastic concentration where tubificid worms stimulated by two-fold the CO2 fluxes emitted from microcosms (Figure3A, Tukey's HSD, p < 0.05). It is also worth noting that microplastic contamination did not have a direct effect on microbial production of CO2 as CO2 fluxes were comparable among the four microplastic treatments without tubificid worms (Figure3A, Tukey's HSD, p > 0.05).

Figure 3 .

 3 Figure 3. CO2 (A) and N-NOx (N-NO3 -+ N-NO2 -) fluxes (B) measured in the water columns. For

  [START_REF] Gautreau | The Influence of Bioturbation and Water Column Oxygenation on Nutrient Recycling in Reservoir Sediments[END_REF][START_REF] Braeckman | Role of Macrofauna Functional Traits and Density in Biogeochemical Fluxes and Bioturbation[END_REF][START_REF] Anschutz | Effects of Tubificid Worm Bioturbation on Freshwater Sediment Biogeochemistry[END_REF][START_REF] Stief | Stimulation of Microbial Nitrogen Cycling in Aquatic Ecosystems by Benthic Macrofauna: Mechanisms and Environmental Implications[END_REF]

Microplastic concentration

ACKNOWLEDGEMENTS

We thank Félix Vallier, Colin Issartel, Lara Konecny-Dupré, and ACSED technical staff (Univ. Lyon 1, CNRS, UMR 5023 LEHNA, France) for all their technical assistance in the laboratory. We also thank Brice Mourier and André-Marie Dendievel (Univ. Lyon 1 and ENTPE, UMR CNRS 5023 LEHNA, France) for their advice and help, especially during the use of laser granulometer (Mastersizer, 2000, Malvern Instrument). We highly appreciate all the valuable discussions and support received from Florian Malard, Yohan Lebon and Edwige Gautreau. This work has been funded by the IDEXLYON of Université de Lyon (UdL) within the program "Investissements d'Avenir" (ANR-16-IDEX-0005), and was performed within the frameworks of the EUR H2O'Lyon (ANR-17-EURE-0018) and the Rhone Basin Long Term Environment Research (LTSER ZABR). We also wish to thank the Sedaqua+ platform of the Fédération de Recherche BioEEnViS (FR3728) and the program "Ec'Eau Campus" funded by a CPER (Contrat de Plan Etat -Région Auvergne Rhône Alpes 2015-2021) for the use of a thermoregulated lab and specific equipment (microcosms, gas analyzer) needed for our experiments. Constructive comments by 6 anonymous referees on a previous version of the manuscript were very useful to improve its quality.

SUPPORTING INFORMATION

Additional information is provided on microplastic characterization, microcosm setup, workflow for parameter analysis, sediment surface images and ImageJ analysis, gas and nutrient fluxes, the number of microplastics added to the sediment, and the number of microplastics extracted from worms.