1012 Environmental Toxicology and Chemistry, Vol. 23, No. 4, pp. 1012–1018, 2004  2004 SETAC Printed in the USA 0730-7268/04 $12.00 + .00 INFLUENCE OF CHLORIDE ON SILVER UPTAKE BY TWO GREEN ALGAE, PSEUDOKIRCHNERIELLA SUBCAPITATA AND CHLORELLA PYRENOIDOSA DAE-YOUNG LEE,CLAUDE FORTIN, and PETER G. C. CAMPBELL* INRS-Eau, Terre et Environnement, Universite ´ du Que ´bec, 2800 rue Einstein, C.P. 7500, Sainte-Foy, Que ´bec G1V 4C7, Canada ( Received 13 March 2003; Accepted 3 September 2003) Abstract—Silver bioavailability in the presence of chloride was estimated from short-term (60 min) uptake experiments with two green algae, Pseudokirchneriella subcapitata and Chlorella pyrenoidosa. In the first experiment, silver uptake was monitored under two concentration regimes in which total dissolved silver ([Ag] tot ) and [Cl] were manipulated to maintain [Ag + ] at a constant value (10 nM). Comparable uptake rates were measured for both treatments despite the dramatic changes in [Cl] and [Ag] tot . In the second experiment, ambient [Ag] tot was held constant (10 or 115 nM), but [Cl] was varied (0.005–50 mM) to explore the whole range of silver chloro-complexes. Intracellular silver varied markedly along the [Cl] gradient and exhibited a clear, positivecorrelation with ambient [Ag + ] for both algae. We conclude that the biotic ligand model reliably describes silver bioavailability in the presence of chloride for the two test algae and that its applicability depends on the relative magnitudes of silver fluxes through the unstirred diffusion layer and across the cell membrane, with the latter being affected by the presence or absence of a Cu(I) transporter. In the presence of chloride, no evidence was found for the internalization of silver via anion transport or passive diffusion of the neutral mono-chloro-complex, AgCl 0 . Keywords—Silver Speciation Diffusion Biotic ligand model Algae INTRODUCTION Freshwater algae play a key role in the biogeochemistry and ecotoxicology of silver. In surface waters, silver tends to associate with particulate matter, including algal cells (K d rang- ing from 10 4.5 to 10 6 [1]). Once assimilated by or adsorbed to algae, silver can be transferred either to the sediments by set- tling or to higher-level consumers through trophic pathways. In laboratory experiments, Ag uptake by consumption of var- ious algal cells accounted for 4 to 49% of the total silver assimilation in zooplankton and bivalves [2–4]. In addition, zooplankton exhibited obvious signs of silver toxicity when silver was accumulated by dietary consumption of algal cells grown with environmentally relevant concentrations of silver [5]. In these latter experiments, toxic symptoms were not ap- parent when silver was taken up directly from water, even though body burdens of Ag in zooplankton were higher than in the case of dietary Ag uptake. Given this involvement of phytoplankton in silver biogeochemistry and ecotoxicology, it clearly is important to consider how silver interacts with algal cells. In aquatic environments, three key factors should be known to fully comprehend a metal–organism interaction [6,7]: Metal speciation in the external environment, route of metal uptake by the organism, and biological effects of the metal on the organism. The speciation of silver is mostly influenced by the formation of strong complexes with simple inorganic ligands, such as thiosulfate or other sulfur(II)-containing ligands (for- mation constants, log K  8.2), a behavior that is consistent with its class B, soft-metal characteristics [1]. In the absence of reduced sulfur species, silver reacts with simple, ubiquitous inorganic ligands, such as chloride, and forms less robust chlo- ro-complexes (log K  3.0). The biotic ligand model (BLM), * To whom correspondence may be addressed (petercampbell@inrs-eau.uquebec.ca). a common model for metal–organism interactions, assumes that the free-ion concentration (or activity) of a metal ([M z+ ]) is the primary factor determining dissolved metal uptake and toxicity [8] and, thus, predicts that the bioavailability of silver will be positively related to the concentration of the free ion, [Ag + ], among various silver complexes in solution. The BLM, however, will apply only when a number of assumptions are satisfied. Two important assumptions are, first, that metal in- ternalization, or transport of metal across the plasma mem- brane (k int ) (Fig. 1), is slower than the advection or diffusion (k dif ) of the metal toward the plasma membrane, and, second, that metal internalization occurs at cation-transport systems in the plasma membrane (Fig. 1) [7]. Earlier studies of the influence of inorganic ligands on sil- ver–algae interactions raised some doubts about the ability of the BLM to predict silver uptake and toxicity [9–11]. For example, Fortin and Campbell [9] investigated the influence of chloride on silver uptake by a freshwater alga, Chlamy- domonas reinhardtii, and demonstrated that silver uptake in- creased with the concentration of total silver and chloride, even though the free Ag + concentration was fixed for all treatments. This result contradicted the BLM prediction that silver uptake rate would remain constant regardless of the [Cl] (or the total silver concentration) provided that the free Ag + concentration remained unchanged. In this case, the failure of the BLM appeared to result from a rate of silver internalization that was greater than the diffusional flux from the bulk solution to the algal surface, a situation that is incompatible with the first assumption of the BLM. The BLM also failed to predict silver uptake by C. reinhardtii in the presence of another inorganic ligand, thiosulfate. Silver uptake was significantly faster in the presence of thiosulfate than in the presence of chloride, even though the free Ag + concentration was the same in both treat- ments [10]. It was concluded that silver was accidentally trans- ported across the plasma membrane via a sulfate/thiosulfate-