# Heterogeneous mixture of microplastics: impact on the freshwater ecosystem engineer (Tubifex tubifex) and its role in biogeochemical processes

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Wazne Mohammad¹*, Mermillod-Blondin Florian¹, Vallier Manon¹, Hervant Frédéric¹, Nel Holly², Kukkola Anna², Krause Stefan¹,²,#, Simon Laurent¹,#

1 Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, UMR 5023 LEHNA, F-69622, Villeurbanne, France
2 School of Geography, Earth and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

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Mohammad.wazne@univ-lyon1.fr

## Introduction

Deposit-feeding bioturbators are key actors in sediment functioning of aquatic ecosystems. Bioturbators, such as *Tubifex tubifex*, contribute to organic matter degradation and nutrient recycling in freshwater sediments due to their ability to rework sediment, and to produce and irrigate biogenic structures (galleries). By their bioturbation activities, *tubificid* worms can stimulate microorganisms and associated biogeochemical processes occurring at the water-sediment interface.

However, the presence of contaminants in sediments can significantly modify these biogeochemical processes by impacting the survival, the physiology and the bioturbation activities of *tubificid* worms. Among these contaminants, **microplastic pollutants** are predominant in most sedimentary environments and can affect deposit-feeding organisms like *tubificid* worms. Indeed, the impacts of microplastic ingestion by *Tubificidae* have been reported in literature (Hurley et al., 2017, EST). Nevertheless, the consequences of microplastic ingestion on bioturbation activities and biogeochemical processes have never been studied.

Therefore, the **objective** of this study was to investigate the **effects** of sediment contamination with a **heterogeneous mixture of different shapes, sizes and polymer types of microplastic pollutants** (i.e. polymers of polystyrene and polyamide) on the survival, the physiological state and the bioturbation activity of *tubificid* worms.

## Acknowledgement

This work has been funded by the IDEXLYON project 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 (ZABR).

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IDEX
LYON

UNIVERSITÉ DE LYON

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INVESTIR
L'AVENIR

ZA
LTSEF FRANCE

Zones
Ateliers
BASSIN DU RHÔNE

AGENCE NATIONALE DE LA RECHERCHE
ANR

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H₂O'Lyon
Université de Lyon

## Material and Methods

### Microplastics (MPs)

<figure><img src="image_8.png" /><figcaption>Polystyrene
fragments</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Polyamide
fibers</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Polyamide
fragments</figcaption></figure>

Irregular shapes, sizes & polymers

### Experimental Setup

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<figure><img src="image_12.png" /><figcaption>Glass bottle (2 L)</figcaption></figure>

### Measurements

#### Impact on worms

* Survival

* Superoxide dismutase activity

#### Impact on bioturbation activity

* Sediment reworking (fecal pellet deposition)

* Water exchanges at the water-sediment interface

#### Impact on fluxes

* CO₂ fluxes

* NO₃⁻ fluxes

## Results

### Worm survival during the experiment

<figure><img src="image_13.png" /></figure>

* Although non significant, worm survival tended to decrease with MPs contamination

### Sediment reworking activity

<figure><img src="image_14.png" /></figure>

* Although non significant, sediment reworking through worm feeding activity tended to decrease with MPs contamination

### CO₂ degassing rates

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* In sediments without MPs addition, *tubificids* increased CO₂ flux by around 2 fold (W0 vs C0)

* The stimulation of CO₂ fluxes by worms was inhibited with moderate contaminations with MPS (W1 vs C1 & W10 vs C10)

### Superoxide dismutase (SOD)

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* Oxidative stress (SOD activity) increased with MPs addition

* The highest SOD activity was measured in W10 treatment (Different letters means significant differences among treatments) (Tukey test at p ≤ 0.05)

### Bio-irrigation

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* Worms stimulated water exchange between sediment and water

* No effect of MPs (Different letters means significant differences among treatments) (Tukey test at p ≤ 0.05)

### N-NO₃⁻ fluxes

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* Tubificids increased NO₃⁻ efflux rates in all MPs treatments

* Worm effect on NO₃⁻ flux was reduced by MPs contamination (Different letters means significant differences among treatments) (Tukey test at p ≤ 0.05)

## Discussion & Conclusion

* An increase in oxidative stress was observed in *tubificid* worms exposed to MPs. Significant cellular damage indicated by SOD activity was considered as an early warning for worm population because of the inverse trend on *Tubifex* survival depending on MPs exposure.

* Worm feeding activity (fecal pellet production) was apparently more affected by MPs contamination than bio-irrigation activity.

* In link with feeding activity decreasing trend, the roles played by *tubificid* worms on organic matter mineralization (CO₂ flux) and nutrient cycling (NO₃⁻ flux) were affected by MPs contamination. Surprisingly, at the highest MPs concentration (W100), worms tended to be less affected than at a moderate MPs concentration (W10). One explanation of this interesting result would be that, at the highest concentration, MPs were more easily detected and avoided by worms than at lower concentrations..