Using lysosomal membrane stability of haemocytes in Ruditapes philippinarum as a biomarker of cellular stress to assess contamination by caffeine, ibuprofen, carbamazepine and novobiocin

Although pharmaceuticals have been detected in the environment only in the range from ng/L to mu g/L, it has been demonstrated that they can adversely affect the health status of aquatic organisms. Lysosomal membrane stability (LMS) has previously been applied as an indicator of cellular well-being to determine health status in bivalve mussels. The objective of this study is to evaluate LMS in Ruditapes philippinarum haemolymph using the neutral red retention assay (NRRA). Clams were exposed in laboratory conditions to caffeine (0.1, 5, 15, 50 mu g/L), ibuprofen (0.1, 5, 10, 50 mu g/L), carbamazepine and novobiocin (both at 0.1, 1, 10, 50 mu g/L) for 35 days. Results show a dose-dependent effect of the pharmaceuticals. The neutral red retention time measured at the end of the bioassay was significantly reduced by 50% after exposure to environmental concentrations (p < 0.05) (caffeine = 15 mu g/L; ibuprofen = 10 mu g/L; carbamazepine = 1 mu g/L and novobiocin = 1 mu g/L), compared to controls. Clams exposed to these pharmaceuticals were considered to present a diminished health status (retention time <45 mm), significantly worse than controls (96 min) (p < 0.05). The predicted no environmental effect concentration (PNEC) results showed that these pharmaceuticals are very toxic at the environmental concentrations tested. Measurement of the alteration of LMS has been found to be a sensitive technique that enables evaluation of the health status of clams after exposure to pharmaceuticals under laboratory conditions, thus representing a robust Tier-1 screening biomarker.