Environ. Sci. Technol. 2005, 39, 2067-2072
Toward a Biotic Ligand Model for Freshwater Green Algae: Surface-Bound and Internal Copper Are Better Predictors of Toxicity than Free Cu2+-Ion Activity When pH Is Varied K A R E L A . C . D E S C H A M P H E L A E R E , * ,† JENNIFER L. STAUBER,‡ KARYN L. WILDE,§ SCOTT J. MARKICH,§ P A U L L . B R O W N , §,| N A T A S H A M . F R A N K L I N , ‡,⊥ 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 Cu2+ ion (as pCu ) - log{Cu2+ activity as molarity}), and as external (surface-bound) Cu and internal Cu in the algal cells. With increasing pH, EC50dissolved 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 Cu2+ activity. The EC50pCu 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 Cu2+ 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 (EC50external) and as internal copper (EC50internal) did not vary substantially when pH was increased. External Cu was operationally defined as the Cu fraction removable from the algal cell by shortterm contact with ethylenediaminetetraacetic acid; internal copper was defined as the nonremovable fraction. For Chlorella sp. the EC50external varied between 5 and 10 fg of Cu/ * Corresponding author phone: +32 9 264 37 64; fax: +32 9 264 37 66; e-mail:
[email protected]. † Ghent University. ‡ CSIRO Energy Technology. § Australian Nuclear Science and Technology Organization. | Monash University. ⊥ McMaster University. 10.1021/es049256l CCC: $30.25 Published on Web 02/04/2005
2005 American Chemical Society
cell (factor of 2 difference) and the EC50internal between 25 and 40 fg of Cu/cell (factor of 1.6 difference). For P. subcapitata the EC50external varied between 10 and 28 fg of Cu/cell (factor of 2.8 difference) and the EC50internal between 42 and 71 fg of Cu/cell (factor of 1.7 difference). Because the observed variation in EC50external and EC50internal is much less than the variation in EC50Cu2+, it is concluded that both external and internal copper are better predictors of copper toxicity than Cu2+ 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 Ca2+, Mg2+, 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 bioavailability 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 Cu2+ 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 {Cu2+ activity as molarity}). On the basis of this observation, which is supported by several earlier studies (15, 17-19), De Schamphelaere et al. (12) hypothesized that the number of deprotonated binding sites at the algal surface continuously VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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increases with increasing pH; i.e., protons can effectively compete with metal ions for a range of binding sites on the cell surface with different pKa (e.g. see refs 20 and 21) and consequently more copper can bind to this surface at higher pH levels. This eventually leads to an increased toxicity at higher pH, be it either directly (at the surface) or indirectly (after transport into the cell) (22). In support of this, Franklin et al. (15) measured intracellular and surface-bound copper in Chlorella sp. (an isolate different from the one used in the present study) at pH 5.7 and 6.5 and showed that both surfacebound and intracellular copper, as well as copper toxicity, increased as the external pH increased. The relationship between surface-bound and intracellular copper and effects on microalgal growth inhibition have since been wellestablished for both Chlorella sp., P. subcapitata, and Scenedesmus subspicatus (S. subspicatus) (14, 15, 23). However, the ability to predict copper toxicity based on surface bound (external) copper and/or internal copper when pH is varied has never been investigated over a wide pH range. In this paper, the effect of pH on the toxicity of copper to Chlorella sp. and P. subcapitata was investigated, and in parallel, external and internal copper in the algal cells were determined to test the potential use of the cell surface as the biotic ligand for freshwater microalgae.
Materials and Methods Origin and Culture of Algae. Chlorella sp., a tropical chlorophyte, was isolated by J. Stauber from Lake Aesake, Strickland River, Papua New Guinea (isolate 12). The alga was maintained in JM/5 media at pH 7.3 (24) at 27 °C on a 12 h:12 h light:dark cycle with Philips TL 40 W cool white fluorescent lighting (75 µmol of photons m-2 s-1). P. subcapitata (Korschikov) Hinda´k 1990 (formerly Selenastrum capricornutum) was obtained from the Culture Collection of Algae and Protozoa (CCAP 278/4, Culture Collection of Algae and Protozoa, Ambleside, United Kingdom) and was maintained at pH 8.3 in carbon-filtered and 0.45-µm-filtered aerated tap water (Gent, Belgium) to which the modified Provasoli’s ES enrichment (25) at half-strength, and, additionally, 1.4 mg L-1 FeSO4‚7H2O, 15 mg L-1 NaH2PO4‚2H2O, 150 mg L-1 NaNO3, and 2.35 mg L-1 MnCl2.4H2O were added. This culture was kept at 20 °C under continuous light (240 µmol of photons m-2 s-1). Culture media for Chlorella sp. and P. subcapitata contained 0.5 and 1.5 µg of Cu L-1, respectively. Experiments with Chlorella sp. were conducted at the Commonwealth Scientific and Industrial Research Organization (CSIRO; Lucas Heights, Australia); those with P. subcapitata, at Ghent University (Gent, Belgium). Cells in the exponential growth phase were used for all experiments. Algal Bioassays. Algal toxicity tests with copper were conducted at 27 °C at pH 6.0, 6.5, and 7.5 with Chlorella sp. and at 25 °C at pH levels 5.9, 6.5, 7.6, and 8.5 with P. subcapitata. Test media used were a synthetic freshwater (26) for Chlorella sp. and standard OECD test water (27) for P. subcapitata (see Supporting Information for composition of these test waters). The pH was controlled by adding one of the pH buffers 2-[N-morpholino]ethanesulfonic acid (MES) or 3-[N-morpholino]propanesulfonic acid (MOPS) or through daily manual pH adjustments with dilute HCl or NaOH. Algal growth was monitored during 48 (Chlorella sp.) or 72 h (P. subcapitata). In parallel, external (surface bound) and internal copper concentrations were determined on/in the algal cells at the end of the bioassays, i.e., after 48 (Chlorella sp.) or 72 h (P. subcapitata). More details on the test waters and the test conditions are summarized as Supporting Information. More detailed information about the exact test and analysis methods is given in De Schamphelaere et al. (12) for P. subcapitata and in Franklin et al. (23) for Chlorella sp. Immediately prior to the inoculation with algae, dissolved (
