Sub-lethal toxicity of five disinfection by-products on microalgae determined by flow cytometry - Lines of evidence for adverse outcome pathways

Théo Ciccia; Pascal Pandard; Philippe Ciffroy; Nastassia Urien; Léo Lafay; Anne Bado-Nilles

doi:10.1016/j.ecoenv.2023.115582

Sub-lethal toxicity of five disinfection by-products on microalgae determined by flow cytometry - Lines of evidence for adverse outcome pathways

Ecotoxicol Environ Saf. 2023 Nov 1:266:115582. doi: 10.1016/j.ecoenv.2023.115582. Epub 2023 Oct 20.

Authors

Théo Ciccia¹, Pascal Pandard², Philippe Ciffroy³, Nastassia Urien³, Léo Lafay³, Anne Bado-Nilles²

Affiliations

¹ Laboratoire National d'Hydraulique et Environnement (LNHE), Division Recherche et Développement, Electricité de France (EDF), 6 Quai de Watier, 78401 Chatou Cedex 01, France. Electronic address: theo.ciccia@edf.fr.
² Institut National de l'Environnement Industriel et des Risques (INERIS), Parc Technologique Alata, BP 2, 60550 Verneuil-en-Halatte, France.
³ Laboratoire National d'Hydraulique et Environnement (LNHE), Division Recherche et Développement, Electricité de France (EDF), 6 Quai de Watier, 78401 Chatou Cedex 01, France.

PMID: 37862747
DOI: 10.1016/j.ecoenv.2023.115582

Abstract

Standardised tests are often used to determine the ecotoxicity of chemicals and focus mainly on one or a few generic endpoints (e.g. mortality, growth), but information on the sub-cellular processes leading to these effects remain usually partial or missing. Flow cytometry (FCM) can be a practical tool to study the physiological responses of individual cells (such as microalgae) exposed to a stress via the use of fluorochromes and their morphology and natural autofluorescence. This work aimed to assess the effects of five chlorine-based disinfection by-products (DBPs) taken individually on growth and sub-cellular endpoints of the green microalgae Raphidocelis subcapitata. These five DBPs, characteristic of a chlorinated effluent, are the following monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), bromochloroacetic acid (BCAA) and 1,1-dichloropropan-2-one (1,1-DCP). Results showed that 1,1-DCP had the strongest effect on growth inhibition (EC₅₀ = 1.8 mg.L^-1), followed by MCAA, TCAA, BCAA and DCAA (EC₅₀ of 10.1, 15.7, 27.3 and 64.5 mg.L^-1 respectively). Neutral lipid content, reactive oxygen species (ROS) formation, red autofluorescence, green autofluorescence, size and intracellular complexity were significantly affected by the exposure to the five DBPs. Only mitochondrial membrane potential did not show any variation. Important cellular damages (>10%) were observed for only two of the chemicals (BCAA and 1,1-DCP) and were probably due to ROS formation. The most sensitive and informative sub-lethal parameter studied was metabolic activity (esterase activity), for which three types of response were observed. Combining all this information, an adverse outcome pathways framework was proposed to explain the effect of the targeted chemicals on R. subcapitata. Based on these results, both FCM sub-cellular analysis and conventional endpoint of algal toxicity were found to be complementary approaches.

Keywords: Biomarkers; Disinfection by-products; Flow cytometry; Growth inhibition; Microalgae.

MeSH terms

Adverse Outcome Pathways*
Dichloroacetic Acid / analysis
Disinfection / methods
Flow Cytometry
Microalgae*
Reactive Oxygen Species
Trichloroacetic Acid / analysis
Trichloroacetic Acid / toxicity

Substances

Reactive Oxygen Species
chloroacetic acid
bromochloroacetic acid
Trichloroacetic Acid
Dichloroacetic Acid