Biochemical stress responses in Procambarus clarkii exposed to pristine and aged glitter

Glitter is a primary microplastic typically composed of a polyethylene terephthalate or polyvinyl chloride core coated with a reflective metallic layer, usually containing aluminium, titanium, iron or bismuth. Despite its widespread use and direct release into aquatic environments, its biological effects on freshwater organisms remain poorly understood. This study investigated sub-lethal biochemical responses to glitter exposure in the non-native crayfish Procambarus clarkii. Adult males and females were exposed for 30 days to pristine or lake-aged glitter at 0.067, 0.134 and 0.268 g/L, with an unexposed control group. Tested concentrations were selected from previous microplastic exposure studies, due to the limited availability of environmental concentration data for glitter. Lake-aged particles were obtained through in situ conditioning in a lacustrine environment to simulate environmental weathering. A multi-biomarker approach was applied to the hepatopancreas. Catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase were analysed as oxidative stress-related biomarkers, while carboxylesterases, lipase and N-acetyl-β-D-glucosaminidase were assessed as hydrolase activities. The electron transport system was included as a marker of energy metabolism. Gamma generalized linear models were used to evaluate the effects of glitter type, concentration and type-specific dose-response patterns. After false discovery rate correction, significant responses were mainly restricted to carboxylesterase activity. The interaction between glitter type and concentration was significant for carboxylesterase measurements with three substrates tested: p-nitrophenyl butyrate, α-naphthyl acetate and α-naphthyl butyrate. Pristine glitter generally increased carboxylesterase activity with concentration, whereas lake-aged glitter induced stable or slightly decreasing responses. Oxidative stress biomarkers did not show robust generalized effects. Overall, these findings indicate that glitter may induce targeted biochemical alterations rather than broad oxidative stress activation. Carboxylesterase activity appears to be a sensitive endpoint for detecting exposure to synthetic particulate materials, while the contrasting effects of pristine and aged glitter highlight the importance of environmental weathering in modulating biological responses.