Combined effects of seawater acidification and diclofenac on immune parameters of the clam Ruditapes philippinarum.

Increased CO2emissions in the atmosphere are indicated as themain cause of global climate changes and result in ocean acidification, which in turn can affect marine organisms, such as bivalves. Reduced pH may alter bioavailability and toxicity of contaminants, displaying either cumulative, synergistic or antagonistic effects. Since information on combined effects of acidification and contaminants on immune parameters of bivalves is scarce, the present study was performed to address this lack of data. Clams (Ruditapes philippinarum) were held for 7 days (T0) at three pH values (8.1, 7.7 and 7.4), at 22 °C and 34 psu. Clams kept at each pH value were then exposed for 14 days (T1) to the non-steroidal anti-inflammatory drug diclofenac (0, 0.05, 0.5 μg/L). At T0 and T1 haemolymph was collected and total haemocyte count (THC), haemocyte volume(HV) and diameter (HD), Neutral Red uptake (NRU), haemocyte proliferation (HP) and cytotoxicity (assessed by lactate dehydrogenase assay, LDH) were measured. At T0, reduced pH significantly affected all immunomarkers, except for HD. THC increased significantly in clams maintained at 7.4 pH, suggesting increases in HP (as confirmed by cell proliferation assay results) and/or in movement of cells from tissues into haemolymph. Decreased NRU in animals kept at 7.7 and 7.4pHsuggestedweakeningof haemocyte pinocytotic capability, whereas increased LDH activity indicated that experimental conditions tested were cytotoxic to cells. At T1, differences in THC, HV and HP were not maintained. When comparing T1 and T0, NRU showed an opposite pattern of variation with decreasing pH, whereas cytotoxicity always increased. Significant increases in HD and cytotoxicitywere observed in diclofenac-exposed clams at 7.4 pH. Diclofenac also caused a significant reduction in NRU at 7.7 pH. Overall, acidification influenced haemocyte parameters in clams, the combination of lowered pH and diclofenac sometimes affecting them more markedly.