# Response of freshwater periphyton to pesticides, their mixture and transformation products: New insight from untargeted metametabolomics

Mélissa Eon, Joséphine Veron, Chloé Bonnineau, Christelle Margoum, Nicolas Creusot

► To cite this version:

Mélissa Eon, Joséphine Veron, Chloé Bonnineau, Christelle Margoum, Nicolas Creusot. Response of freshwater periphyton to pesticides, their mixture and transformation products: New insight from untargeted metametabolomics. SETAC, May 2025, Vienne, Austria. 2025. hal-05067371v2

HAL Id: hal-05067371

https://hal.science/hal-05067371v2

Submitted on 15 May 2025

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# Response of freshwater periphyton to pesticides, their mixture and transformation products: New insight from untargeted metabolomics

Mélissa EON 1,2
Joséphine VERON 3
Chloé BONNINEAU 1,3
Christelle MARGOUM 3
Nicolas Creusot 1,2

1 INRAE, UR EABX, France

2 Bordeaux Metabolome, MetaboHUB

3 INRAE, UR RIVERLY, France

## Introduction

* Pesticides can be degraded by abiotic processes and generate transformation products (TPs) for which the identity and the ecotoxicity is mostly unknown [1]

* Freshwater periphytic communities are increasingly used in ecotoxicology due to their ecological relevance to assess the effect(s) of environmental stress at the community level [2,3].

* Facing the lack of sensitivity of usual descriptors, untargeted metabolomics is an approach of choice by providing a comprehensive sensitive picture of the molecular phenotype in response to changing environmental conditions [4]

**Aim :** To evaluate the toxicity of the herbicide terbuthylazine (TBA) and its transformation products on periphyton through the implementation of a multi-descriptors approach

## Materials & Methods

### Biological model : periphyton

<figure><img src="image_7.png" /><figcaption>3D structure of periphyton</figcaption></figure>

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### Experimental design in indoor channels

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#### 4 weeks exposure

* ✓ Terbuthylazine at 5 (TBA5) and 150 (TBA150) µg/L

* ✓ TBA-desethyl commercially available degradation product at 5 (TP5) and 150 (TP150) µg/L

* ✓ Weathered TBA, homemade by photodegradation from TBA at 5 (TBAv5) and 150 (TBAv150) µg/L

* ✓ TBA-desethyl in mixture with 2 ubiquitous contaminants, glyphosate 0.1µg/L and AMPA 0.3 µg/L (gA TP5 and gA TP150)

* ✓ A control (without any contaminants) and gA conditions

### Structural, functional & adaptive descriptors

* ✓ Biomass (Ash Free Dry Weight)

* ✓ Photosynthetic activity (Yied)

* ✓ Enzymatic activity (Beta-glucosidase, Phosphatase, leucine aminopeptidase)

* ✓ PICT (Pollution-Induced Community Tolerance)

### Metabolomics workflow

Sampling & quenching

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Storage (-80°C)

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Freeze drying

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Biphasic Extraction

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UPLC-HRMS/MS analysis

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Data processing

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Chemometrics analysis

+ Annotation

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NPC Classifier

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Phase
lipophile
MTBE

Phase
hydophile
MeOH/H2O

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UPLC-ToF
(XevoG2-S ToF,
WATERS)
ESI+& - MSe
Full HRMS
50-1500 m/z
Coll 6V vs 10-40V

<figure><img src="image_25.png" /></figure>

## Results

### 1. Physiological, structural and adaptive response

<figure><img src="image_26.png" /></figure>

✓ Strong decrease of biomass with TBA150

#### Overview all samples (PCA)*

<figure><img src="image_27.png" /></figure>

✓ Clear separation between T0 control vs T4 control vs exposed samples

#### Focus on T4 (HCA)

<figure><img src="image_28.png" /></figure>

✓ Effect of all TPs (i.e. TBAv vs TPs) that differs from TBA

✓ Clear mixture effect (e.g. gA vs TP5 vs gA TP5)

✓ Specific features of each condition

<figure><img src="image_29.png" /></figure>

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✓ Strong decrease photosynthetic activity (TBA150)

✓ Effect on leucine aminopeptidase activity (TBA150 & gA)

<figure><img src="image_31.png" /></figure>

✓ Sensitivity to TBA shift only in community pre-exposed to TBA

<figure><img src="image_32.png" /></figure>

✓ Sensitivity to gA shift only in community pre-exposed to gA and gA TP5

#### Annotation of distinct features (NPC Pathways) **

<figure><img src="image_33.png" /></figure>

✓ No marked apparent discrepancy in the modulated « pathways » between conditions but clear differences at lower level (i.e. NPC superclass) of classification (data not shown)

* Following sample median transformation, data cube root transformation and data pareto scaling

**, Following volcano plot control vs the tested conditions (Fold >1.5, RAW pvalue <0.05)

## Take home message

* ✓ After 4 weeks exposure, **biomass, photosynthetic yield, heterotrophic enzymatic activities** were strongly affected by TBA150 and not or slightly by its TPs, being the TBA-desethyl (TP) or the TBAv and the mixture with glyphosate

* ✓ Controversy, multivariate analyses (PCA, HCA) showed discrepancies in the **meta-metabolome** profile between each conditions, TBA 150µg/L being the most discriminated while both 5 and 150 µg/L of TBA-desethyl (TP) and TBAv were also separated from the controls. Volcano (not shown) and Venn diagram further highlighted specific features associated to gA, gA TP5, gA TP150, TBA150, TBAv150 and TP150

* ✓ Putative annotation did not showed marked discrepancy in the responses of metabolites classes at the NPC_pathway level whereas all the modulated features differed between all conditions. NPC superclass level showed clear differences (not shown)

## Perspectives

✓ To Link meta-metabolome response to apical endpoints to gain understanding of adverse outcome pathways of TBA and its TPs

INRAE

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50 avenue de Verdun
F-33612 Cestas Cedex
melissa.eon@inrae.fr

## Acknowledgement

TAPIOCA project (ECOPHYTOII - OFB Funding)

XPO facility INRAE & MetaboHUB (ANR-11-INBS-0010)

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ÉCOPHYTO
RÉDUIRE ET AMÉLIORER
L'UTILISATION DES PHYTOS

OFB

DE LA BIODIVERSITE

## References :

[1] Mitra et al., 2024 Pesticides in the environment: Degradation routes, pesticide transformation products and ecotoxicological considerations https://doi.org/10.1016/j.scitotenv.2024.173026

[2] Creusot et al., Metabolomics insight into the influence of environmental factors in responses of freshwater biofilms to the model herbicide diuron. Environmental Science and Pollution Research, 2022, 29 (20), pp.29332-29347. (10.1007/s11356-021-17072-7). (hal-03691889)

[3] Guasch, H., et al., Microbial Ecotoxicology, 2017. p. 251-281

[4] Bedia et al. (2018) Applications of Metabolomics Analysis in Environmental Research in Comprehensive Analytical Chemistry Volume 82, 2018, https://doi.org/10.1016/bs.coac.2018.07.006