Molecular biomarkers, like gene transcripts or enzyme activities, are potentially powerful tools for early warning assessment ofpollution. However, a thorough understanding of response and baseline variation is required to distinguish actual effects frompollution. Here, we assess the freshwater musselAnodonta anatinaas a biomarker model species for freshwater ecosystems, bytesting responses of six transcriptional (cat,gst,hsp70,hsp90,mt,andsod) and two biochemical (AChE and GST) biomarkers toenvironmentally relevant Cu water concentrations. Mussels (n=20), collected from a stream free from point source pollution,were exposed in the laboratory, for 96 h, to Cu treatments (< 0.2μg/L, 0.77 ± 0.87μg/L, and 6.3 ± 5.4μg/L). Gills and digestiveglands were extracted and analyzed for transcriptional and biochemical responses. Biological and statistical effect sizes from Cutreatments were in general small (mean log2fold-change≤0.80 and Cohen’sf≤0.69, respectively), and no significant treatmenteffects were observed. In contrast, four out of eight biomarkers (cat,gst,hsp70, and GST) showed a significant sex:tissueinteraction, and additionally one (sod) showed significant overall effects from sex. Specifically, three markers in gills (cat,mt,GST) and one in digestive gland (AChE) displayed significant sex differences, independent of treatment. Results suggest that sexor tissue effects might obscure low-magnitude biomarker responses and potential early warnings. Thus, variation in biomarkerbaselines and response patterns needs to be further addressed for the future use ofA. anatinaas a biomarker model species.
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