In Vitro Activities in Mussel Hemocytes as Biomarkers of Environmental Quality: A Case Study in the Abra Estuary (Biscay Bay) M. P. CAJARAVILLE, 1 I. OLABARRIETA, AND I. MARIGOMEZ Zitologi eta Histologi Laborategia, Biologia Zelularra eta Zientzia Morfologikoen Saila, Euskal Herriko Unibertsitatea, 644 PK, E-48080 Bilbo, Basque Country, Spain Received January 9, 1996 Mussels, Mytilus galloprovincialis, were collected from six coastal sites of dissimilar water quality (Zierbena, Santurtzi, Arrigunaga, Galea, Men ˜ akoz, and Plentzia) at Biscay Bay in Sep- tember 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 Janu- ary 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 re- duced in Arrigunaga in September 1991 and in Galea in January 1992. Moreover, HVD was kept unchanged through the year in mussels collected from Men ˜ akoz and Zierbena. On the basis of this preliminary in vivo study, hemocytic activities of mussels col- lected 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 vi- ability; (b) the zymosan phagocytosis assay, indicative of phago- cytic activity; (c) the diaminobenzidine–manganese (DAB–Mn 2+ ) 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 (nonpol- luted) and Arrigunaga (moderately polluted) suggest that in vitro assays may be used as biomarkers of environmental quality in coastal and estuarine areas. © 1996 Academic Press INTRODUCTION In vitro toxicology has bloomed during very recent years and the examination of cell cultures has partially replaced the rou- tine sacrifice of experimental animals (Fry, 1993). Such a trend is also noted in the field of environmental toxicology and tox- icity tests with fishes or invertebrates are currently being sub- stituted by in vitro testing of the toxicity of chemicals (Alvarez and Friedl, 1992; Zahn and Braunbeck, 1993; Seibert et al., 1994). Moreover, the in vitro testing approach could be also valuable for environmental monitoring programs based on the use of sentinel organisms, which could provide primary cul- tures of target cells whose functional responses could be used as measures of environmental quality (Anderson, 1988; Dunier and Siwicki, 1993). Bivalve molluscs and specially mussels are widely used as sentinels in monitoring programs of marine environmental quality (Goldberg, 1986). Chemical analysis of their tissues, measurement of biomarkers of exposure to specific chemicals, and measurement of pollution effects are increasingly per- formed in environmental monitoring programs (Widdows and Donkin, 1989; Rainbow, 1993). In most cases, sentinel mussels are killed to conduct these analyses and relatively large samples are required to attain feasible conclusions on water quality. Alternatively, in vitro assays might provide useful bio- markers of either exposure to pollutants or their biological effect (Anderson, 1993). Particularly, some functional responses of hemocytes and brown cells of molluscs have been proposed to develop in vitro toxicity assays indicative of the condition of sentinel mussels (Cheng, 1988a,b; Zaroogian et al., 1992; Zaroogian and Yev- ich, 1993) and, therefore, of the environmental quality of their habitat. Although the field of immunotoxicology of bivalve molluscs was in its infancy one decade ago (Anderson, 1988), it has grown up during the past years due to its interest for fisheries, aquaculture, and ecotoxicology (Anderson, 1993). Cell viability, phagocytic and endocytic activities, lysosomal enzyme activities, production of oxygen free radicals, and rela- tive proportion of hemocyte types have been quantified in vitro after in vivo exposure to chemical pollutants (McCormick-Ray, 1987; Cheng, 1988a,b; 1990; Sami et al., 1992; Coles et al., 1994). Nevertheless, different chemicals or even different doses of the same chemical may provoke different hemocytic responses which, additionally, might be modified by either environmental factors or concurrence of other chemicals (Anderson, 1993; Auffret and Oubella, 1994). 1 To whom correspondence should be addressed. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 35, 253–260 (1996) ARTICLE NO. 0108 253 0147-6513/96 $18.00 Copyright © 1996 by Academic Press All rights of reproduction in any form reserved.