In vitro activities in mussel hemocytes as biomarkers of environmental quality: a case study in the Abra Estuary (Biscay Bay).

Mussels, Mytilus galloprovincialis, were collected from six coastal sites of dissimilar water quality (Zierbena, Santurtzi, Arrigunaga, Galea, Meñakoz, and Plentzia) at Biscay Bay in September 1991, January 1992, June 1992, and September 1992. The extent of hemocyte infiltration in connective tissue of the digestive gland was quantified by stereology on histological sections in terms of volume density of hemocytes (HVD). HVD was elevated in mussels collected from Plentzia (the less polluted site) in January 1992 and September 1992, while such increases occurred in January 1992 in Santurtzi and Arrigunaga and in September 1991 and September 1992 in Galea. Conversely, HVD was reduced in Arrigunaga in September 1991 and in Galea in January 1992. Moreover, HVD was kept unchanged through the year in mussels collected from Meñakoz and Zierbena. On the basis of this preliminary in vivo study, hemocytic activities of mussels collected in September 1994 from Arrigunaga and Plentzia were further investigated by means of four in vitro immunotoxicity assays: (a) the trypan blue exclusion assay, indicative of cell viability; (b) the zymosan phagocytosis assay, indicative of phagocytic activity; (c) the diaminobenzidine-manganese (DAB-Mn2+) oxidation assay for estimating reactive oxygen intermediate (ROI) production; and (d) the neutral red (NR) uptake assay, indicative of endocytic ability. These in vitro tests indicated some significant differences between Plentzia and Arrigunaga. Hemocytes from mussels collected in Plentzia exhibited a higher capability to phagocytose zymosan while, conversely, hemocytes from mussels collected in Arrigunaga endocytosed more NR and produced more ROI under nonstimulated conditions. These differences in the in vitro hemocytic activities of mussels from Plentzia (nonpolluted) and Arrigunaga (moderately polluted) suggest that in vitro assays may be used as biomarkers of environmental quality in coastal and estuarine areas.