CHARACTERIZATION AND IN VITRO RESPONSIVENESS OF ENZYMATIC BIOMARKERS IN MARINE PELAGIC COPEPODS

• Baseline biomarker profiles for four marine copepod species • The studied species revealed very distinct enzymatic fingerprints • Carboxylesterases were highly sensitive to organophosphate flame retardants • AChE inhibition was species- and compound-dependent, and significant for TBBPA • A multi-species multi-biomarker approach improves ecological risk assessment Plastic-derived additives are recognized as emerging pollutants, yet their effects on marine zooplankton remain largely unexplored. This study provides baseline activities of nine pollution-related biomarkers and it also examines the in vitro inhibition of B-esterase activities by environmentally relevant plastic additives in four marine planktonic copepod species ( Acartia tonsa, Centropages typicus, Oithona davisae and Paracartia grani ). Biomarkers included acetylcholinesterase (AChE), carboxylesterases (CEs), glutathione S-transferase (GST), catalase (CAT), lipid peroxidation (LPX), electron transport system (ETS), lactate dehydrogenase (LDH), and N-acetyl-β-D-glucosaminidase (NAGase). The four copepod species exhibited distinct enzymatic fingerprints, with A. tonsa showing high AChE and GST activities, C. typicus elevated CE activity, O. davisae distinct CAT and ETS activities, while P. grani displayed the highest NAGase activity. In vitro inhibition assays confirmed CEs as primary targets for organophosphate flame retardants and bisphenol derivatives causing up to 73% reduction in baseline enzymatic activity. In contrast, AChE inhibition was compound- and species-dependent, reaching up to 90% inhibition in O. davisae with the halogenated flame retardant tetrabromobisphenol A. Ranked by sensitivity, largely driven by CE inhibition patterns, O. davisae appeared as the most enzymatically resilient species, whereas P. grani and C. typicus were the most responsive to plastic additive exposure under the tested in vitro conditions. These findings provide mechanistic insights into additive-induced enzymatic disruption and the potential of B-esterases as markers of these emerging chemical exposures. Finally, a multi-species multi-biomarker approach to conduct pelagic risk assessments is recommended.