The present acute and subchronic laboratory study (96 h and 30 days) aimed to provide for the first time the possible adverse effects of two experimental concentrations, based on Water Framework Directive 2000/60/EC (WFD) - maximum allowable concentration in water and maximum allowable concentration in biota of the sum of six PBDE congeners (PBDE-28 (tri-brominated), PBDE-47 (tetra-brominated), PBDE-99, PBDE-100 (penta-brominated), PBDE-153, PBDE-154 (hexa-brominated)), further referred as ∑PBDE, in zebra mussels (Dreissena polymorpha Pallas, 1771). Therefore, we studied the process of bioaccumulation in the treated with ∑PBDE mussel samples and calculated the bioaccumulation factor, analysed the activity of various cellular biomarkers - the metabolic enzymes alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), cholinesterase (ChE), and lactate dehydrogenase (LDH); the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) in the digestive gland. We also evaluated lysosomal membrane stability by applying the neutral red retention time assay to the hemocytes of the adductor muscle in exposed individuals. Overall, the results showed that all applied concentrations of ∑PBDE altered the cellular indicators in both the acute and subchronic treatment compared to the control (p < 0.05), with PBDE-28 showing the highest accumulation levels in mussels on the 96th hour, and PBDE-100, PBDE-153, and PBDE-154 on day 30, thus suggesting they could have more adverse effects on the health of zebra mussel. In light of the above, our study significantly contributes to a deeper understanding of the biological relevance and practical implications of regulatory thresholds. It also aligns with the precautionary principle embedded in the European water legislation by providing experimental evidence on whether supposedly "safe" concentrations are indeed biologically benign in key aquatic organisms.
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