Environ. Sci. Technol. 1996, 30, 1713-1720
Influences of Natural Dissolved Organic Matter on the Interaction of Aluminum with the Microalga Chlorella: A Test of the Free-Ion Model of Trace Metal Toxicity LISE PARENT,† MICHAEL R. TWISS, AND PETER G. C. CAMPBELL* INRS-Eau, Universite´ du Que´bec, CP 7500, Ste-Foy, Quebec, Canada G1V 4C7
The free-ion model of trace metal interactions with aquatic microorganisms states that the biological response to a metal is proportional to the activity of the free-ion {Mz+} in solution. The applicability of the free-ion model, as it applied to the toxicity of aluminum to the green alga Chlorella pyrenoidosa in the presence of a soil fulvic acid (SFA), was tested in defined media (pH 5) designed to limit Al interactions to algae and SFA. Toxicity was not proportional to the activity of Al3+, an apparent failing of the freeion model. Fulvic acid adsorbed to cell surfaces (17 mg m-2, pH 5) and increased membrane permeability (as measured with [14C]sorbitol) whereas Al decreased membrane permeability. In addition, SFA may act as a source of phosphorus to P-deficient algae. These results emphasize the importance of considering not only the metal-complexing properties of natural dissolved organic matter but also its direct metabolic and physiological influences on algae.
Introduction In the area of aquatic toxicology, much qualitative evidence exist to show that the total aqueous concentration of a trace metal is not a good predictor of its bioavailability, i.e., that the metal’s speciation will greatly affect its availability to organisms (1-3). In effect, a convincing body of evidence has been developed to support the tenet that the biological response elicited by a dissolved metal is usually a function of the free-metal ion concentration, Mz+(H2O)n. The freeion model for metal-organism interactions was developed to rationalize these experimental observations and to explain what was initially perceived as “the universal * Author to whom correspondence should be addressed; telephone: (418)654-2538; fax: (418)654-2600; e-mail address: Campbell@ UQuebec.ca. † Present address: Te ´ le´-universite´, Universite´ du Que´bec, 1001 rue Sherbrooke est, 4e e´tage, CP 670, Succ. C., Montre´al, Que´bec, Canada H2L 4L5.
0013-936X/96/0930-1713$12.00/0
1996 American Chemical Society
importance of free-metal ion activities in determining the uptake, nutrition, and toxicity of all cationic trace metals” (4). To elicit a physiological response from a target organism or to accumulate within this organism, a metal must interact with a cell membrane. Within the construct of the free-ion model, this interaction of the metal with the cell surface, involving either the free-metal ion (Mz+) or a metal complex (ML) as the reactive species, is represented in terms of the formation of M-X-cell surface complexes, where -X-cell is a cellular ligand present at the cell surface and L is a metal-binding ligand in solution. In the simplest case, L + Mz+
+ –X–cell
k1 k–1
Mz+–X–cell + L
(1)
ML
where a pseudoequilibrium is established between the cell surface and the exposure solution, the biological response is assumed to vary as a function of the concentration of surface complexes {Mz+-X-cell}:
biological response ∝ {Mz+-X-cell} ) K[Mz+]{-X-cell} (2) where K is the formation constant for the surface complex (eq 1: k1/k-1). According to this approach, the role of ligands (L) in solution is limited to their participation in various complexation equilibria in solution, as represented on the lefthand side of eq 1. By complexing the metal and reducing the concentration of the free-metal ion, Mz+, the ligands will tend to reduce the concentration of surface complexes, {Mz+-X-cell}, and thus attenuate the biological response. The decrease in metal toxicity generally observed in the presence of chelating agents is thus attributed to the chelation of the metals in the medium, rather than to a direct physiological effect of the chelating agent on the organism (4). It should be appreciated, however, that virtually all the experiments supporting the free-ion model have been carried out with divalent metals, at constant pH and hardness, in the presence of low molecular weight synthetic ligands, such as nitrilotriacetic acid (NTA) or ethylendiaminetetraacetic acid (EDTA), that bear little resemblance to naturally occurring organic ligands. Indeed, there is a striking scarcity of studies suitable for testing the applicability of the free-ion model in the presence of natural dissolved organic matter (3). Numerous reports can be found in the literature of the effects of natural dissolved organic matter (DOM) on trace metal bioavailability (e.g., refs 5-8), but virtually all of these studies are qualitative in nature since metal speciation was undefined and are thus unsuitable for testing the validity of the free-ion model. Among the rare exceptions to this generalization are several earlier studies involving copper toxicity (9-11), in which the Cu-selective electrode was used to monitor the free Cu2+ ion in systems containing natural DOM. Given the differences between natural DOM and the typical synthetic ligands normally used as metal buffers in defined exposure media and the relative scarcity of
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quantitative experiments run in the presence of DOM, we designed an experiment to test the implicit free-ion model assumption that the only function of DOM is to complex metals in solution. This test involved Al as the test metal, Chlorella pyrenoidosa as the test organism, and a fulvic acid as a source of DOM. Such a test of the applicability of the free-ion model is relevant considering the ubiquity of DOM in natural waters (12) and the toxicological and nutritive importance of trace metals in phytoplankton ecology (1). Our experiments clearly demonstrate that the action of DOM is not limited to its participation in complexation reactions with aluminum in solution. On the contrary, we present clear evidence for fulvic acid interaction with the algal surface (adsorptive losses of fulvic acid from solution in the presence of algal cells and changes in algal membrane permeability) and for fulvic acid acting as a nutrient for P-deficient algae (as indicated by phosphatase status).
Materials and Methods The experimental approach was designed to determine the growth response of Chlorella pyrenoidosa to a constant concentration of Al3+, in the absence of a soil-derived fulvic acid (SFA) or in the presence of increasing concentrations of SFA and total Al. According to the free-ion model, the algal response should reflect the (constant) free-Al3+ concentration and should thus be constant across the range of experimental conditions. Growth experiments were carried out in pH-buffered, chemically defined media and were accompanied by concurrent measurements of Al speciation in solution and of short-term accumulation of Al by the alga. In addition, various complementary diagnostic measurements were used to probe possible direct effects of the SFA on cell physiology (e.g., membrane permeability, phosphatase activity). Test Organism and Culture Conditions. An axenic culture of C. pyrenoidosa (University of Toronto Culture Collection of Algae and Cyanobacteria; UTCC 89) was maintained on 100% Bold Basal Medium agar slants (13) under dim light. Prior to toxicity tests, cells were transferred into a semicontinuous culture system under controlled and constant temperature (20-22 °C) and light conditions (100115 µmol of photons m-2 s-1). These stock cultures were grown in AAP liquid medium, as modified by Chiaudani and Vighi (14), under constant bubbling with filtered air in Teflon containers (1-2 L). Cell densities in these cultures were monitored daily with an electronic particle counter (Coulter Electronics). Cultures were diluted daily to maintain a cell concentration of ≈105 cells mL-1; this corresponded to the early exponential growth phase. Axenicity of the stock culture was monitored regularly. Reagents and Glassware. Deionized water used for preparing stock solutions and culture media was obtained from a commercial system employing mixed-bed ion exchange, charcoal adsorption, and filtration (0.2 µm) steps. To minimize metal contamination, contact with glassware was avoided; laboratory manipulations were carried out in acid-washed polyethylene, polypropylene, or Teflon containers. Manipulations that required precautions against possible contamination by airborne particulates were performed in a laminar flow hood under a positive pressure of filtered air. All filtrations of were conducted using polycarbonate membrane filters (Nuclepore; Nuclepore Corp., Pleasanton, CA) and a low applied vacuum (
