# Contamination procedure for chemical risk assessment in periphyton v2

Lin Zi, Mélissa Eon, Zahrasadat Alavikakhki, Chloé Bonnineau, Nicolas Creusot

► To cite this version:

Lin Zi, Mélissa Eon, Zahrasadat Alavikakhki, Chloé Bonnineau, Nicolas Creusot. Contamination procedure for chemical risk assessment in periphyton v2. INRAE EABX; Bordeaux Metabolome Platform. 2026. hal-05519485

HAL Id: hal-05519485

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Submitted on 19 Feb 2026

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Feb 17, 2026

Version 2

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# Contamination procedure for chemical risk assessment in periphyton V.2

DOI

dx.doi.org/10.17504/protocols.io.bp2l6zjpdgqe/v2

Lin Zi¹, Mélissa EON¹, Zahrasadat Alavikakhki¹, Chloé Bonnineau¹, Nicolas Creusot¹

¹INRAE EABX

MetaboHUB-Bordeaux

Lin

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Lin Zi
INRAE

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METABOHUB

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BORDEAUX
METABOLOME

OPEN ACCESS

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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6zjpdgqe/v2

**Protocol Citation:** Lin Zi, Mélissa EON, Zahrasadat Alavikakhki, Chloé Bonnineau, Nicolas Creusot 2026. Contamination procedure for chemical risk assessment in periphyton. protocols.io

https://dx.doi.org/10.17504/protocols.io.bp2l6zjpdgqe/v2 Version created by **Lin Zi**

**License:** This is an open access protocol distributed under the terms of the **Creative Commons Attribution License**, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

**Protocol status:** Working

We use this protocol and it's working

**Created:** February 17, 2026

**Last Modified:** February 17, 2026

**Protocol Integer ID:** 243441
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**Keywords:** Chemical risk assessment, periphyton, contamination, contamination procedure for chemical risk assessment, throughput chemical risk assessment, standardized contamination procedure, chemical risk assessment, using periphyton, contamination procedure, simultaneous evaluation of multiple chemical, multiple chemical, laboratory efficiency, periphyton this document, laboratory

**Funders Acknowledgements:**

Agence nationale de la recherche

Grant ID: ANR-11-INBS-0010

Agence nationale de la recherche

Grant ID: ANR-21-ESRE-0035

PARC: Partnership for the Assessment of Risks from Chemicals

Grant ID: 101057014

PHARM-ERA

Grant ID: 101119261

Abstract

This document outlines a standardized contamination procedure for high-throughput chemical risk assessment using periphyton in a 48-well plate format. This miniaturized approach allows for the simultaneous evaluation of multiple chemicals and concentration gradients, bridging the gap between field-scale complexity and laboratory efficiency.

Materials

<figure><img src="image_8.png" /><figcaption>48-well plate</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>HPLC amber glass vial to prepare chemical stock solution</figcaption></figure>

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periphyton colonized at 9 mm glass discs
# Troubleshooting

## Safety warnings

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**Safety:** Wear a lab coat, gloves, and safety glasses; utilize the fume hood and follow chemical safety rules.

**Waste:** Recycle all waste according to quality document EABX_GEN_706.

**Traceability:** Optimize labeling and storage for all samples, reagents, and stock solutions.

**Monitoring:** Perform gravimetric monitoring for all stock solutions.

**Documentation:** Record the weight, volume, and chemical nature of all internal standards in the laboratory notebook.

## Before start

Periphyton has already been colonized at 9 mm diameter glass discs which are suitable for 48-well plates. The colonization procedure has been described in protocol "Periphyton growing and sampling in situ or in mesocosm for metabolomic analysis".

<figure><img src="image_12.png" /><figcaption>Periphyton colonized at 9 mm diameter glass discs</figcaption></figure>
# Setting-up discs in 48-well microplates and contaminants preparation

## 1 Draw up a 48-well plate plan

* 48-well plates allow to have 8 concentrations (including control) in row, and each concentration have 6 replicates in column.

* Define the chemical concentration range: 1-2 environmentally realistic concentrations, 2-3 intermediate concentrations aimed at detecting metabolomic responses, and 2-3 high-range concentrations aimed at inducing functional changes. Algal EC₅₀ can be used as a reference for functional disruption.

* Six replicates per concentration is required to account for the inherent biological heterogeneity of periphyton communities.

* In parallel with each batch of experiment, include terbuthylazine (TBA) as a positive control. This facilitates the comparison of results across different experimental batches, accounting for the inherent biological variability of periphyton. TBA was chosen for its well-characterized dose-response relationship, ensuring a reliable reference for validating experimental sensitivity.

<figure><img src="image_13.png" /><figcaption>Plate 1: TBA 24 h</figcaption></figure>

## 2 Transfer the discs into the 48-wells microplates

* Before transfer the discs into 48-well plate, add 1 mL of 6x diluted DAUTA into each well.
* Transfer the disc into the microplate. Ensure that the colonized periphyton remains face-up throughout handling.

* Acclimate the microplates for 3 days

## 3 Prepare serial dilution of chemicals

* Prepare chemical stock solution in organic solvent (e.g. 100% Ethanol) or ultra-pure water according to the solubility of the chemical. In case of stock solution in organic solvent, ensure the final content of organic solvent applied to the periphyton is 0.05%.

* Example of prepare the serial dilution of chemicals

1. If you want to achieve the final concentration that applied to the periphyton as below:

<table><thead><tr><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>F</th><th>G</th><th>H</th></tr></thead><tbody><tr><td>Final concentration of Bisphenols (mg/L) contain 0.05% EtOH</td><td>10.00</td><td>3.00</td><td>1.00</td><td>0.30</td><td>0.10</td><td>0.03</td><td>0.01</td></tr></tbody></table>

2. The concentration of stock solution in 100% EtOH would be: $10 (mg/L)/0.05\% = 20 (mg/mL)$. Prepare at least 100 µL of stock solution.

3. Prepare the serial dilution as follows: in this way, after 2000 times dilution, the final concentration of EtOH applied to periphyton is 0.05%, and all the concentrations contain same amount of EtOH. Using the cultivation solution of periphyton in the experiment to prepare the serial dilution (here using 6 x diluted DAUTA)

<table><thead><tr><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>F</th><th>G</th><th>H</th></tr></thead><tbody><tr><td>Serial dilution in 100% EtOH (mg/mL)</td><td>20.00</td><td>6.00</td><td>2.00</td><td>0.60</td><td>0.20</td><td>0.06</td><td>0.02</td></tr><tr><td>100 x dilution (mg/L): 10 µl serial 1+990 µl 6x DAUTA</td><td>200.00</td><td>60.00</td><td>20.00</td><td>6.00</td><td>2.00</td><td>0.60</td><td>0.20</td></tr><tr><td>20 x dilution (mg/L): 400 µl serial 2+7600 µl 6x DAUTA</td><td>10.00</td><td>3.00</td><td>1.00</td><td>0.30</td><td>0.10</td><td>0.03</td><td>0.01</td></tr></tbody></table>

* Store the stock solution at -20 °C. Prepare the final serial of dilution at earliest 3 days before use.

## Apply chemicals to the periphyton: contamination

### 4 Applied the chemicals well by well to avoid desiccation

* Discard all media of one well

* Gently add 1 mL of contaminants solution as fast as possible

* Duration of exposure: 4 h, 24 h, 72 h, 1 week: to be adapted to the scientific question. According to the duration think if you need to change the solution in the middle.

* For metabolomic analysis, 1 plate for 1 time-point. In case of several time points, start to contaminate for the longer exposure and finish by the shortest in order to be at the
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same level of growth.

Protocol references

For the periphyton colonization, please refer to the protocol "Periphyton growing and sampling in situ or in mesocosm for metabolomic analysis".