Twenty exposure concentrations, zero dose-response for subcellular biomarkers: Unravelling this paradox in cadmium and copper exposed Dreissena polymorpha

• No dose response for biomarkers despite internal Cd/Cu accumulation and dose-dependent mortality • Different response pattern between cadmium and copper in mussels • Inter-individual variability masks biomarker responses to Cd/cu • Prospective analysis aids future experimental design optimisation The assessment of aquatic ecosystem quality under the Water Framework Directive suffers from a gap between chemical measurements and observed effects at the community level, with a critical lack of direct measurements of organism health. Biomarkers, situated at intermediate levels, could bridge this gap but cause-effect relationships remain poorly understood, limiting their operationality. This study implements an innovative experimental protocol with 20 exposure concentrations in order to answer 4 questions: (i) Is there a dose-response relationship for both subcellular biomarkers and tissue concentrations? (ii) Does exposure duration influence the response of biomarkers? (iii) Does response pattern of biomarkers differ between cadmium and copper? The results obtained and used to answer the first three questions gave rise to another one: (iv) How did the inter-individual variability and statistical power of our experimental design influence our ability to detect biological effects? Dreissena polymorpha was exposed to cadmium and copper for 4 and 8 days, with measurement of 8 biomarkers (SOD, GPx, TAC, GST, CSP3, LOOH, ETS, LDH) and tissue metal concentrations. Results reveal a paradox: while dose-response relationships were confirmed for bioaccumulation and mortality, subcellular biomarkers showed no dose-response relationships. Exposure duration had no influence, but correlation networks revealed different biomarker response patterns between cadmium and copper. Prospective analysis demonstrated that a sample size of 6-10 individuals per group would optimise statistical detection. This study reports on the difficulties associated with applying biomarkers under controlled conditions, provides data on the model species Dreissena polymorpha and contributes to the debate on the operationality of biomarkers.