Toward a Biotic Ligand Model for Freshwater Green Algae: Surface-Bound and Internal Copper Are Better Predictors of Toxicity than Free Cu 2+ -Ion Activity When pH Is Varied KAREL A. C. DE SCHAMPHELAERE,* ,† JENNIFER L. STAUBER, ‡ KARYN L. WILDE, § SCOTT J. MARKICH, § PAUL L. BROWN, §, | NATASHA M. FRANKLIN, ‡, ⊥ NICOLA M. CREIGHTON, § AND COLIN R. JANSSEN † Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University, J. Plateaustraat 22, B-9000 Gent, Belgium, Centre for Environmental Contaminants Research, CSIRO Energy Technology, PMB 7, Bangor NSW 2234 Australia, ANSTO Environment, Australian Nuclear Science and Technology Organization, PMB1, Menai NSW 2234 Australia, Australian Sustainable Industry Research Centre, Monash University, Gippsland Campus, Churchill VIC 3842 Australia, and Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 The freshwater green microalgae Chlorella sp. and Pseudokirchneriella subcapitata (P. subcapitata) were chronically (48 and 72 h, respectively) exposed to copper at various pH levels, i.e., pH 6-7.5 and pH 5.9-8.5, respectively. Concentrations resulting in 50% inhibition of exponential growth rate (EC50) were determined as dissolved Cu, estimated chemical activity of the free Cu 2+ ion (as pCu )- log{Cu 2+ activity as molarity}), and as external (surface-bound) Cu and internal Cu in the algal cells. With increasing pH, EC50 dissolved decreased from 30 to 1.1 μg of Cu L -1 for Chlorella sp. and from 46 to 18 μg of Cu L -1 for P. subcapitata. The pH effect on copper toxicity was even more obvious when expressed as Cu 2+ activity. The EC50 pCu increased on average 1.4 pCu unit per pH unit for Chlorella sp. and 1.1 pCu unit per pH unit for P. subcapitata, thus indicating a marked increase of Cu 2+ toxicity at higher pH (more than 1 order of magnitude per pH unit). In contrast, it was found that EC50 values expressed as surface bound or external copper (EC50 external ) and as internal copper (EC50 internal ) did not vary substantially when pH was increased. External Cu was operationally defined as the Cu fraction removable from the algal cell by short- term contact with ethylenediaminetetraacetic acid; internal copper was defined as the nonremovable fraction. For Chlorella sp. the EC50 external varied between 5 and 10 fg of Cu/ cell (factor of 2 difference) and the EC50 internal between 25 and 40 fg of Cu/cell (factor of 1.6 difference). For P. subcapitata the EC50 external varied between 10 and 28 fg of Cu/cell (factor of 2.8 difference) and the EC50 internal between 42 and 71 fg of Cu/cell (factor of 1.7 difference). Because the observed variation in EC50 external and EC50 internal is much less than the variation in EC50 Cu 2+ , it is concluded that both external and internal copper are better predictors of copper toxicity than Cu 2+ when pH is varied. From the perspective of toxicity modeling, this observation is the first step toward considering the use of the cell surface as the algal biotic ligand for Cu in a similar way as fish gills fulfill this role in the biotic ligand model for predicting metal toxicity to fish species. Introduction It is widely recognized that the toxicity of metals to freshwater biota is dependent on physicochemical water characteristics, particularly dissolved organic carbon concentration, pH, and hardness (1, 2). The recently developed biotic ligand model (BLM) has been demonstrated to be successful in predicting metal bioavailability and toxicity as a function of water chemistry (3, 4). The model predicts metal binding to sites on the organism-water interface based on metal speciation and competitive binding between toxic metal ions and cations such as Ca 2+ , Mg 2+ , Na + , and H + . Those binding sites are commonly termed the “biotic ligand”. From the amount of metal accumulated at the biotic ligand, the BLM then predicts the toxic effect, assuming that the accumulation of metal to the biotic ligand determines the toxic effect, independent of water chemistry, and assuming thermodynamic equilibrium. For fish it was demonstrated that copper and nickel concentrations on the fish gill were related to the toxic effect, independent of water chemistry (5, 6). Subsequently, the fish gillsor gill-like structures in invertebrate speciesshas been treated as the biotic ligand in various metal bioavail- ability studies (3, 4, 7-10). Because of their key position as primary producers in ecological systems, algae are important test species for regulatory assessments of metals (11). Although empirical bioavailability models predicting metal toxicity to algae are available (12, 13), only limited evidence exists that the cell surface can be used as the biotic ligand for unicellular green algae. Ma et al. (14) demonstrated that the amount of copper bound to the algal surface at 50% growth inhibition was independent of added concentrations of the metal chelating agent ethylenediaminetetraacetic acid (EDTA) and freshwater fulvic acid. Although promising, the cited study alone does not verify the use of the algal surface as the biotic ligand. Indeed, besides the presence of copper complexing agents (e.g. fulvic acid), pH is at least equally important in determining copper toxicity to algae (12, 15). Moreover, the effect of complexing agents on copper toxicity to algae can easily be explained by differences in the chemical activity of the Cu 2+ ion (12, 16), whereas this is not the case for the effect of pH. Indeed, free cupric ions are much more toxic at high than at low pH levels, as illustrated by a 1.4 pCu unit increase of the EC50pCu per pH unit for Pseudokirchneriella subcapitata (P. subcapitata)(12) (pCu )-log {Cu 2+ activity as molarity}). On the basis of this observation, which is supported by several earlier studies (15, 17-19), De Scham- phelaere et al. (12) hypothesized that the number of deprotonated binding sites at the algal surface continuously * Corresponding author phone: +32 9 264 37 64; fax: +32 9 264 37 66; e-mail: Karel.Deschamphelaere@Ugent.be. † Ghent University. ‡ CSIRO Energy Technology. § Australian Nuclear Science and Technology Organization. | Monash University. ⊥ McMaster University. Environ. Sci. Technol. 2005, 39, 2067-2072 10.1021/es049256l CCC: $30.25  2005 American Chemical Society VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2067 Published on Web 02/04/2005