92 Environmental Toxicology and Chemistry, Vol. 22, No. 1, pp. 92–100, 2003  2003 SETAC Printed in the USA 0730-7268/03 $12.00 + .00 OXIDATIVE STRESS BIOMARKERS IN BIVALVES TRANSPLANTED TO THE GUADALQUIVIR ESTUARY AFTER AZNALCO ´ LLAR SPILL ANTONIO ROMERO-RUIZ,² O SCAR AMEZCUA,‡ MANUEL J. RODRI ´ GUEZ-ORTEGA,² J OSE ´ L. MUN ˜ OZ,‡ JOSE ´ ALHAMA,² A NTONIO RODRI ´ GUEZ-ARIZA,² J OSE ´ L. GO ´ MEZ-ARIZA,§ and JUAN LO ´ PEZ-BAREA²* ²Department of Biochemistry and Molecular Biology, University of Co ´rdoba, Campus of Rabanales, Severo Ochoa Building, 14071 Co ´rdoba, Spain ‡CICEM El Torun ˜o, P.O. Box 16, Puerto de Santa Marı ´a, 11500 Ca ´diz, Spain §Department of Chemistry and Materials Sciences, University of Huelva, Palos de la Frontera Road, La Ra ´bida, 21819 Huelva, Spain ( Received 17 December 2001; Accepted 6 June 2002) Abstract—Biological effects of metals were studied in clams (Scrobicularia plana) transplanted to Guadalquivir estuary (Spain) at several times after the spill of toxic metals from Aznalco ´llar pyrite mine (southwest Spain) (April 1998) using biochemical biomarkers responsive to reactive oxygen species. Significant As, Cd, Fe, Mn, Ni, and Pb uptake was found in clams living for seven months at the estuary (from July 1999). Increased activity of antioxidant (catalase, glucose-6-phosphate, and 6-phosphoglu- conate dehydrogenase) and glutathione-related (glutathione reductase and glyoxalase I and II) enzymes was also found after short exposures; the levels of malondialdehyde and metallothionein increased also, particularly with long exposures. Clams living four weeks at the estuary (from March 2000) but not at a reference site also accumulated metals. The higher malondialdehyde and lower reduced-glutathione levels and the more oxidized glutathione status confirmed the oxidative stress of clams living at the estuary, while no marked increase of antioxidant activities was found this time. Lower metal availability along the second transplant could explain the limited responses in this shorter experiment. Although the status of Guadalquivir estuary has recovered since Aznalco ´llar spill, continuous monitoring is needed to confirm its progress and to be alert to possible deterioration after heavy rains. Keywords—Aznalco ´llar accident Bivalve mollusks Metal uptake Antioxidant defenses Oxidative damages INTRODUCTION Bivalve mollusks are used worldwide as marine pollution sentinels due to their sessile nature, filter-feeding habits, and ability to concentrate pollutants [1]. Molecular and cellular changes, the earliest and most sensitive responses to pollutants, have been validated in bivalves as biomarkers for pollution biomonitoring [2]. Biochemical biomarkers are powerful tools to detect the exposure and biological effects of environmental pollutants [3]. Several organic contaminants and metals convert O 2 into reactive oxygen species (ROS) that are highly toxic and mu- tagenic. Metals catalyze the reaction of superoxide anion and hydrogen peroxide to form hydroxyl radical, a highly dele- terious form of reactive oxygen [4]. Reactive oxygen species damage oxidatively various biomolecules; thus, fatty acids generate oxidized by-products such as malondialdehyde [5,6]. Oxidative stress arises when the ROS production rate exceeds that of quenching detoxification and biomolecule repair [7]. Several defense lines protect aerobic organisms from oxidative stress [7]. Primary antioxidant enzymes, such as catalase, de- toxify ROS and ancillary enzymes, such as glucose-6-phos- phate or 6-phosphogluconate dehydrogenases, and recycle NADP + into the reduced form of this coenzyme (NADPH). Subsequently, glutathione reductase uses NADPH to turn ox- idized glutathione (GSSG) into its reduced form (GSH), a soluble thiol that keeps cytosol reduced and is oxidized by ROS [7]. Finally, glyoxalases I and II use GSH to detoxify - oxoaldehydes, products of cellular oxidative processes [8]. Assessment of the biological effects of ROS allows eval- * To whom correspondence should be addressed (bb1lobaj@uco.es). uating exposure and effects of chemicals yielding prooxidant effects. In marine organisms, antioxidant defenses are useful biomarkers of pollution by metals and organic compounds that generate oxidative stress [8–13]. The glutathione content and redox status and the malondialdehyde level respond also to oxidative stress [10,11]. Metallothioneins, small inducible pro- teins that bind metals with their cysteine residues [14], have antioxidant properties and are also useful metal-pollution bio- markers [15]. Since 1979, the activity of Aznalco ´llar pyrite mine (Fig. 1) produced 7  10 7 tons of acidic waste, mud, and tailings that were stocked in a dam (25 m high, 3.6 km 2 area) [16]. On April 25, 1998, the dam released into Guadiamar River 2  10 6 m 3 of mud and 4  10 6 m 3 of acidic water with high metal content [17], threatening Don ˜ana Park and Guadalquivir es- tuary. Mud spread 400 m at both Guadiamar banks and 40 km downstream; mud removal started immediately and ended by October, before the rainy season. Acidic water went 20 km further downstream, being retained before entering Don ˜ana, though it was diverted in part to Guadalquivir River; acidic water was depurated in situ and poured through the Guadiamar course [17]. Of the over 10 6 metal tons released by this ac- cident, less than 2.5% reached Guadalquivir estuary due to mud deposit and efficient removal, precipitation of metals by the alkaline water, and lack of weathering and mobilization of mud by the dry 1998 conditions [17–19]. Possible biological effects of Aznalco ´llar spill in terrestrial ecosystems were stud- ied in wild mice (Mus spretus). Animals collected close to the Guadiamar river course had higher antioxidant and detoxifying activities than controls [20]. Significant DNA breakage was also found in storks from spill-affected Don ˜ana areas [21].