Seawater acidification and emerging contaminants: A dangerous marriage for haemocytes of marine bivalves

The combined effects of seawater acidification and the non-steroidal anti-inflammatory drug diclofenac on haemocyte parameters of the mussel Mytilus galloprovincialis and the clam Ruditapes philippinarum were investigated for the first time. Animals were maintained for one week (TO) in natural pH condition (8.1) and two reduced pH values (pH -0.4 units and pH -0.7 units). Bivalves were then exposed for additional 14 days (T1 and T2) to the three experimental pH values in both the presence and absence of environmentally realistic concentrations of diclofenac (0.05 and 0.50 mu g/L). To assess potential impairment in immunosurveillance, haemocyte parameters (total haemocyte count, haemocyte volume and diameter, Neutral Red uptake, haemocyte proliferation and lysozyme activity) were measured after 7, 14 and 21 days of exposure to differing pH value or pH/diclofenac combinations. In both species, pH affected the whole haemocyte data set at all sampling times, influencing most of the parameters measured at T0 and T1 in clams, and at T2 in mussels. Conversely, in both species diclofenac affected the overall haemocyte response at T2 only. However, in R. philippinarum a higher number of haemocyte parameters were significantly influenced even at T1. A significant interaction between pH and diclofenac was mainly evident in mussels, affecting haemocyte size and lysozyme activity at both T1 and T2. Overall, the results obtained demonstrated that the experimental conditions tested can alter markedly haemocyte parameters in marine bivalves.