1272 Environmental Toxicology and Chemistry, Vol. 19, No. 5, pp. 1272–1277, 2000 q 2000 SETAC Printed in the USA 0730-7268/00 $9.00 1 .00 TOXICITY OF TRI-n-BUTYLTIN TO SEA URCHIN EGGS AND LARVAE: RELATION TO BIOACCUMULATION AT THE NANOMOLAR LEVEL JEAN-PIERRE GIRARD,*² J OANNA SZPUNAR,‡ MARIA-LUIZA PEDROTTI,‡ and DANIELLE PESANDO§ ²Laboratoire de Physiologie et Toxicologie Environnementales, EA 2138, Faculte´ des Sciences, BP71, F-06108-Nice, France ‡CNRS EP 132, Helioparc, 2 av. Pr. Angot, F-64000-PAU, France §Laboratoire d’Oce´anographie Biologique et Ecologie du Plancton Marin, B.P. 28, F-06234-Villefranche-Sur-Mer,France ( Received 10 February 1999; Accepted 8 August 1999) Abstract—Nanomolar concentrations of tri-n-butyltin (TBT) from 5 3 10 210 M to 5 3 10 29 M, were assayed on sea urchin (Paracentrotus lividus) egg cleavage and on larval development. Preincubation enhanced TBT toxicity to first cleavage DNA and protein syntheses but not to intracellular calcium sequestration. Exposure to nanomolar TBT affected the larval development up to the ninth day by reducing arm length and increasing the diameter of the rudiment. Chromatographic analysis of TBT in eggs shows a dose-dependent biomagnification with a half-time of 5 min, which is much shorter than the length of preincubation necessary to provoke cytotoxicity at the same concentration (5 3 10 29 or 5 3 10 210 M). Our data suggest that nanomolar concentrations of TBT similar to those encountered in polluted waters could directly affect sea urchin egg development after fertilization and the larval cycle, these effects being independent of bioaccumulation. Keywords—Tributyltin Bioaccumulation Sea urchin Egg Larva INTRODUCTION Organotin compounds have been used in a variety of chem- ical and biological applications [1,2]. Trisubstituted organotin compounds such as tri-n-butyltin (TBT), which are widely present in antifouling paints, are responsible for many dele- terious effects to aquatic organisms [3] in spite of several countries having restricted their use. Organotins degrade slow- ly in the environment [4] but accumulate at a rapid rate in certain species, particularly in fishes and bivalves [5]. A recent survey conducted to investigate concentrations of antifouling agents in subsurface water in the Mediterranean showed that, despite restriction of the use of TBT in boat paint, the current contamination of seawater is still substantial, with a range of 100 to 700 ng TBT/L, i.e., an average concentration of 0.5 to 2 nM [6]. A wide spectrum of biological damage due to organotins at higher concentrations has been described. Nevertheless, nanomolar amounts of TBT also produce some harmful effects in marine organisms [7]. Furthermore, bioaccumulation of or- ganotin is high in aquatic animals, especially those subject to pollutant intake because of dietary or branchial filtration and a slow rate of elimination [5]. Organotins accumulate first in lipophilic compartments and particularly in membrane struc- tures where they provoke abnormal permeabilities to ions or solutes [8,9]. In addition to several aspects of organotin tox- icity demonstrated in marine invertebrates [10], a number of cellular effects have been described on various cell types that reveal that organotin cytotoxicity is the result of immunotoxic [11], teratogenic [12], or other deleterious effects. Cytotoxicity of TBT often results in an arrest of cellular dynamics, leading to apoptosis [13] or blocking of cell division [9], occurring mainly through alteration of macromolecular syntheses [9,14] or membrane-mediated processes controlling cell signaling, which consist mainly in disruption of calcium * To whom correspondence may be addressed (girard@unice.fr.). homeostasis [15,16] or calcium signaling [9,17]. In a previous article, assessment of the cellular targets of TBT in sea urchin eggs, on the basis of TBT-induced inhibition of egg cleavage after fertilization, showed that enzymatic activity and mac- romolecular syntheses were affected by TBT concentrations ranging from 10 to 100 nM, while alterations of membrane- associated processes occurred in the presence of 1 to 100 mM TBT [9], i.e., concentrations 1,000 times higher than those most commonly described in the literature [16–18]. The ob- jective of the present study was to provide an assessment of the chronic cytotoxicity of TBT by comparison with its acute toxicity [9] and its bioaccumulation in the sea urchin egg and larva when exposed to concentrations of TBT not exceeding a few nanomoles, an environmentally realistic scenario. MATERIAL AND METHODS Determination of cleavage rate in sea urchin eggs The sea urchin Paracentrotus lividus collected in the bay of Villefranche-sur-Mer, France, provided the eggs and sperm cells [19]. Eggs were placed in filtered (Millipore 0.22 mm; Milli Q, Medford, MA, USA) natural seawater (NSW) and dejellied by four successive passages through a 90-mm mesh nylon filter. Sperm was collected dry and kept at 48C. Shortly before use, it was diluted 1:50 in NSW, and 20 ml of this suspension were added per milliliter of egg suspension [20]. Experiments were carried out at room temperature with a 4% (v/v) egg suspension containing 20,000 eggs/ml in NSW stirred with a three-blade propeller. Tri-n-butyltin chloride was purchased from Sigma-Aldrich (St. Louis, MO, USA); all other chemicals were from Sigma (Milwaukee, WI, USA). Dilutions of TBT were made in ethanol. Control experiments showed that, at 0.5%, the highest concentration of ethanol used did not affect the eggs. When necessary, a 4% egg suspension was preincubated for 4 or 6 h with TBT. The rate of division was determined as described by Biyiti et al. [20]. At time intervals after fertilization, the number of