Envlron. Sci. Technol. 1993, 27, 1132-1 138
Surface Complexation of Aluminum on Isolated Fish Gill Cells Kevin J. Wllklnson,t Paul M. Bertsch,* Charles H. Jagoe,* and Peter G. C. Campbell'*t
INRS-Eau, Universite du Quebec, CP 7500, Sainte-Foy, Quebec,Canada G1V 4C7, and Savannah River Ecology Laboratory, University of Georgia, Drawer E, Aiken, South Carolina 29802 Cells from the gills of largemouth bass (Micropterus salmoides) were isolated and exposed to dilute solutions of Al, A1 in the presence of fluoride, or A1 plus dissolved organic matter (DOM) to determine the cells' metal binding potential in an acidic medium. Microelectrophoresis was employed to monitor the extent of aluminum sorption to cells in the presence of added ligand. In the absence of Al, the gill cells exhibit an appreciable negative charge; A1 binding to the cell surface increases the electric potential at the shear plane and leads to a reduction in the cell's (negative) electrophoretic mobility. In the presence of both A1and F, aluminum complexation at the gill surface is only marginally reduced; the formation of a mixed ligand complex, {F-Al-L-cell), is proposed to account for the observed results. The presence of such ternary complexes was subsequently verified by 19Fnuclear magnetic resonance spectroscopy and by potentiometry. Addition of DOM increased the negative electrophoretic mobility of the isolated gill cells both in the presence and absence of aluminum (7.4 pM). Introduction
In watersheds characterized by thin soils and resistant bedrock geology (e.g., parts of eastern North America, Scandinavia), marked increases in the geochemical mobility of aluminum are frequently observed in response to environmental acidification (1). This mobilization of A1 is often episodic in nature, associated with pH depressions occurring during spring snowmelt or specific storm events (2-4). Coincident with these increases in [All, A1 speciation also varies, changing as a function of the ligand concentrations (i.e., dissolved organic matter (DOM), F-, S042-, H4Si04) and pH. Fluoro-A1 complexes are particularly sensitive to pH changes, with the relative proportion of A1F2+and AlF2+increasing markedly in the pH range 4.5-5.5 (5). Equilibrium calculations indicate that mixed ligand Al(OH),F, complexes are important in slightly acidic waters (6). In addition, the concentration of organically bound A1 has been demonstrated to increase during episodic acidification (6). Elevated A1 concentrations, together with the associated low pH, can be harmful to fish and other forms of aquatic life (7-9).In a field setting, these biological consequences will depend on the duration of the episode, the A1 concentrations attained, the prevailing physicochemical conditions (e.g., [H+l, [Ca2+]),and the specific chemical forms of A1 present (A1speciation). With regard to this latter point, considerable research effort has been devoted to relating the speciation of metals in solution to their biological effects. For metals commonly linked to anthropogenic activities (e.g.,Cd, Cu, Zn),the free-ionactivity has often proven to be the best predictor of metal
* Author to whom correspondence should be addressed. Universit6 du QuBbec.
* University of Georgia. +
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Environ. Sci. Technol., Vol. 27, No. 6, 1993
availability toward aquatic biota (10-12).Simple models involving the surface complexation of metals at the biological interface have been developed to explain these results (eq 1 and 2) (10, 13). In such "free-ion" toxicity models, the surface complex is represented by (MZ+-Lcell), where L-cell is a cellular ligand, and the biological response is proportional to the concentration of surface complexes [MZ+-L-celll. M"
+ L-cell
K
M"-L-cell
(1)
biological response CY [M"+-L-cell] = K[L-cell] [MZ+l (2)
By analogy, A1 toxicity would be expected to vary as a function of the free AP+activity. At any biological surface, only a fraction of the sites could be expected to play a role in the toxicological response of the organism. In our conceptual model, we assume that the high-affinity sites (those sites affected at low [MZ+l)are equivalent to the sensitive sites. Given the central role accorded to the surface complexation of metals at biological interfaces, there is a clear need to relate A1 speciation in solution to A1 accumulation/ speciation at the biological surface. The primary objective of the present investigation was to demonstrate the influence of added ligands (F, DOM) on the binding of aluminum at the surface of fish gill cells. We have determined A1 speciation at the surface of isolated fish gills using microelectrophoresis and 19Fnuclear magnetic resonance (NMR) spectroscopy, two methods well-suited to examine the properties of a homogeneous suspension of particles. The decision to work with suspended cells was guided by our appreciation of the complexity of the microenvironment of intact gills (e.g., variable mucous secretion; pH gradients: see refs 14 and 15); in addition, the gill surface itself likely varies over time (16). Shortterm uptake of A1 occurs not at the gill surface but primarily in the fish mucus (17). Diffusion across the mucous layer, followed by A1 binding at the gill surface, likely precedes the biological response. By employing isolated gill cells, we simplified the experimental system and eliminated complicating factors such as variable ventilation and mucous secretion rates. Block and Plirt used a similar approach to examine cadmium uptake across monolayers of respiratory epithelial gill cells from rainbow trout (18). Experimental Section
Choice of Methods. Photon correlation spectroscopy and laser doppler velocimetry were employed to determine the electrophoretic mobility of suspended gill cells. Cell size and surface charge contribute to cell mobility in an electrical gradient; the charge of the cell surface will be influenced by the specific sorption of ions. In a carefully controlled experimental medium, a simple relationship can be deduced between the ionization parameters of a particle surface and its electrokinetic behavior (19,20). 0013-936X/93/0927-1132$04.00/0
0 1993 American Chemical Society
Specificadsorption of small ions will occur inside the Stern plane and necessarily inside the surface of shear (which is generally situated outside the Stern plane). In our experiments, mobilities were always referenced to a control value (absence of metal or ligand or both), thus allowing an unequivocal measurement of whether or not sorption had occurred. Sorption of A1 should be distinguishable from that of H+ or that of A1 in the presence of F or organic matter. Additionally, the immediate environment of the surface-associated fluoride ion can be probed using 19F NMR spectroscopy. This technique is sensitive to changes in the chemical speciation of F in solution and at surfaces; investigations included model systems (cation-exchange resins) as well as isolated gill cells. In addition, a fluorideselective electrode was employed to quantify changes in free [F-1 in the presence of the solid phases. Cell Separation. Adult (ca. 1 kg) largemouth bass (Micropterus salmoides) were captured from a circumneutral pH, low A1 reservoir on the U.S.Department of Energy's Savannah River Site. Whole gill arches were removed, blotted, and then immediately rinsed in an icecold wash solution composed of 100 mM NaC1, 10 mM HEPES, 1mM glucose, 2 mM EGTA, 2 mM EDTA, and 100 units-mL-l of heparin (medium no. 1). A second volume of medium no. 1 (50 mL.gl of wet wt gill tissue) was added to remove excess blood, mucus, and cell debris. Gills were subsequently shaken gently for 5 min in this fresh medium. This wash procedure was repeated six times; Le., until the turbidity of the supernatant had been substantially reduced. Filaments were then excised from arches, blotted, and transferred to a warm (30 "C) collagenase digestion medium which was stirred for 45 min to promote the breakdown of intracellular adhesion (medium no. 2: 100 mM NaC1, 10 mM HEPES, 1 mM glucose, 1mM CaC12,200units.mL-l collagenase; 5 mL of mediumg' of wet w t gill tissue). Following the digestion of the gill filaments, cells were separated by centrifugation: the solution was decanted or spun very slowly (20g, 1 min) to separate gill arches and debris from the cell suspension and then centrifuged (lOOOg,5 min) five times to separate cellular fragments (supernatant) from intact cells (pellet). Medium no. 2 without collagenase was used for the initial washes, whereas a simplified solution ([NaClI = 0.1 M; pH 4.5) was employed for the two final washes to minimize the complexation of A1 by other than the studied ligands. Following the final wash, the cells were chilled in a minimum volume of the salt solution. Ten minutes before the mobility measurements, the cells were diluted 50-fold into the A1 solutions, which were in all other respects identical to the final wash solutions, to give a final cell density of about 3000 cells-mL-l. The experiments were performed quickly and in random order to minimize bias due to cell deterioration; all experimental manipulations of the cells were complete in
