# Impact of pesticides, their mixture and transformation products on freshwater periphyton metabolism.

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

► To cite this version:

Mélissa Eon, Joséphine Veron, Chloé Bonnineau, Christelle Margoum, Nicolas Creusot. Impact of pesticides, their mixture and transformation products on freshwater periphyton metabolism. RFMF, Jun 2025, PARIS, France. 2025. [hal-05174676](https://hal.science/hal-05174676v1)

HAL Id: hal-05174676

https://hal.science/hal-05174676v1

Submitted on 21 Jul 2025

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

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

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

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

RÉPUBLIQUE
FRANÇAISE

Liberté
Égalité
Fraternité

INRAE

UR/1454
ECOSYSTEMES AQUATIQUES
& CHANGEMENTS GLOBAUX [EABX]

RiverLy

<figure><img src="image_3.png" /></figure>

AQUA

<figure><img src="image_4.png" /></figure>

BORDEAUX
METABOLOME

<figure><img src="image_5.png" /></figure>

METABOHUB

LIFe
IN FRESHWATERS
AND ESTUARIES

<figure><img src="image_6.png" /></figure>

AnaEE

# Impact of pesticides, their mixture and transformation products on freshwater periphyton metabolism.

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>

<figure><img src="image_8.png" /></figure>

### Structural, functional & adaptive descriptors

* Biomass (Ash Free Dry Weight)

* Photosynthetic activity (Yied)

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

* PICT (Pollution-Induced Community Tolerance)

## Results

### 1. Physiological, structural and adaptive response

<figure><img src="image_9.png" /></figure>

✓ Strong decrease of biomass with TBA150

Photosynthetic activity Yield (UA)

<figure><img src="image_10.png" /></figure>

✓ Strong decrease photosynthetic activity (TBA150)

✓ Effect on leucine aminopeptidase activity (TBA150 & gA)

<figure><img src="image_11.png" /></figure>

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

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

### 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)

<figure><img src="image_12.png" /></figure>

INRAE

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

## 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

### Experimental design in indoor channels

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

#### 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

### Metabolomics workflow

Sampling & quenching

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

Storage (-80°C)

<figure><img src="image_15.png" /></figure>

Freeze drying

<figure><img src="image_16.png" /></figure>

Biphasic Extraction

<figure><img src="image_17.png" /></figure>

UPLC-HRMS/MS analysis

<figure><img src="image_18.png" /></figure>

Data processing

<figure><img src="image_19.png" /></figure>

Chemometrics analysis

+ Annotation

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

NPC classifier

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

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

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

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

Phase
lipophile
MTBE

Phase
hydophile
MeOH/H2O

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

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

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

### 2. Meta-metabolome response

Overview all samples (PCA)*

<figure><img src="image_30.png" /></figure>

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

Focus on T4 (HCA)

<figure><img src="image_31.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)

Annotation of distinct features (NPC Pathways) **

<figure><img src="image_32.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)

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

### Perspectives

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

## Acknowledgement

TAPIOCA project (ECOPHYTOII - OFB Funding)

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

<figure><img src="image_34.png" /></figure>

OFB

OFFICE FRANÇAISE
DE LA BIODIVERSITE

ÉCOPHYTO

RÉDUIRE ET AMÉLIORER
L'UTILISATION DES PHYTOS