Environ. Sci. Technol. 1986, 20, 616-620
Comparative Study of Aluminum and Copper Transport and Toxicity in an Acid-Tolerant Freshwater Green Alga Brian R. Folsom, Nicolae Andre1 Popescu, and John M. Wood*
Gray Freshwater Biological Institute, Navarre, Minnesota 55392
w A comparative study of the transport and toxicity of one nonessential metal (aluminum), and one essential metal (copper), has been performed with the acid-tolerant green alga Chlorella saccarophila. This organism was isolated from a naturally acidified lake and grows well in laboratory cultures at pH 3.0. Our results show that the fast-exchange ions Ca2+,Mg2+,and Na+ offer some protection against both AP+ and Cu2+toxicity whereas K+ protects against A13+toxicity but enhances Cu2+toxicity. Plasma emission spectroscopy shows that complexation of A13+and Fe3+to cell surfaces is important in preventing toxic cytoplasmic levels of these metals, both in culture media and in acid mine water. The aqueous ion chemistry for toxic metal uptake is simplified considerably in acidic conditions, where competing hydrolysis and precipitation reactions are eliminated. Therefore, simple competitive experiments can be performed quantitatively. Introduction The biochemical basis for metal toxicity is complicated by the great variety of reactions a t the molecular and cellular levels. The exact chemical species responsible for the various toxic effects are generally the most chemically reactive forms, thereby making identification and quantification difficult. Even so, several strategies for biological resistance to metal intoxication have been identified (1-4). Strategies have evolved to maintain low intracellular concentrations of toxic substances. One general mechanism involves the active extrusion of toxic substances after they have entered the cell. A second mechanism involves the complexation of metals to biologically produced ligands either outside the cell, at the cell surface, or within the cell. A third mechanism involves oxidation, reduction, or chemical modification of the toxic species resulting in precipitation, volatilization, or immobilization. All of these mechanisms result in a lowering of the effective concentration of those toxic chemical species that can react at the cellular level. In addition, such mechanisms also require the expenditure of energy either directly, as with active transport, or indirectly, as with the synthesis of enzymes or compounds involved in chelation reactions directed at toxic metals. Metal ion interactions in biology can be classified into three major groups (5). (1)The first group is ions in fast exchange with biological ligands such as Na, K, Mg, Ca, Li, Cs, Rb, Sr, and Ba. These elements tend to complex to oxygen ligands, forming relatively unstable complexes which undergo rapid ligand exchange in water. (2) Ions in intermediary exchange such as Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II), and Pb(I1) demonstrate strong affinity for ligands containing 0,N, and S. These ions form stable complexes with a variety of ligands found in aquatic systems and are generally not found as free ions in water. (3) Ions in slow exchange such as Cu(I), Tl(I), Cd(II), Ag(I), Au(I), and Hg(I1) possess strong affinities for nitrogen- and sulfur-containing ligands. In natural systems, the concentrations of these last two groups of transition metals are generally less than total ligand concentrations and therefore usually form stable insoluble complexes in water. 616
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One current concern centers around the effects of acidification of aquatic systems by acid rain. Acidification of natural waters leads to dramatic changes in both biological diversity and abundance with many ecological changes occurring with a drop of only 1-2 pH units. One of the direct effects of acidification is an increase in the solubility of many metal ion complexes. One such metal, aluminum, is the third most abundant crustal element (I) and becomes available for uptake by living organisms as the pH is lower than about 6.0 ( 6 ) . The median background concentration of aluminum in the rivers of North America is 230 pg f L (7). An EPA study of the continuing acidification of 1050 lakes on the Laurentian shield in northeastern Minnesota, north central Wisconsin, and the upper peninsula of Michigan shows increasing concentrations of A13+ in water with increased acidification (7). Similar results have been found in acidified lakes in the Adirondacks (6) and by Hutchinson and Stokes for most acidified lakes in the Province of Ontario, Canada (7). All of this work supports the data first published in Sweden and Norway (7). Upon lowering the pH of natural waters, aluminum becomes more soluble and therefore available for uptake by living organisms. The chemical speciation of the aluminum changes upon acidification from an insoluble, nontoxic Al(OH), to toxic species including the kinetically favored Al(OH&+ which exists between pH 5.0 and pH 6.0 (9) and the predominant A13+ a t lower pH values which has a very low LD,, dose in rats (6). Another metal of interest is copper. This element in the form of copper sulfate has been added in large quantities to lakes as an algicide to control algal blooms. As with aluminum, the speciation of copper and the subsequent availability to organisms is greatly altered by changes in PH. As noted above, the most reactive chemical species are generally the most toxic forms, which are often those compounds in the lowest concentration and therefore the hardest species to detect. For these reasons, studies of toxicity are difficult due to the complexity of the related chemical reactions in aqueous systems. When the pH of the environment is lowered, many of the hydrolysis reactions surrounding metal ion chemistry are minimized, and the dominant chemical species is a soluble free ion. Consequently, the use of acid-tolerant strains of organisms simplifies experimentation concerning the toxic effects of metal ion pollution. Materials and Methods The green alga used for this study, Chlorella saccarophila, was isolated by T. C. Hutchinson (University of Toronto). Chlorella was grown in liquid culture of bold basal media (BBM) with 0.1% glucose added at pH 3.0 (8). For the metal toxicity studies, the ethylenediaminetetraacetic acid (EDTA) component on the BBM was eliminated. Individual cultures were grown in Ehrlenmeyer flasks with side arms which fit directly into a klett colorimeter. Cultures were grown with agitation into the exponential growth phase to a density of about 100 units and then inoculated with the various chemicals used to stress
0013-936X/86/0920-0616$01.50/0
0 1986 American Chemical Society
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Flgure 1. Concentration dependence for aluminum toxlclty. Optical density of Chlorella cultures vs. time in days. The dashed line at day 10 signifies where the AICI, was added to 0 (control), 10, 25, and 50 ppm of AI,+.
growth. Cultures reach the stationary growth phase at about 300 units. Samples taken for plasma emission spectroscopy included (1)a homogeneous liquid sample of the total growth media, (2) a media supernatant following centrifugation, (3) a lyophilized algal pellet following centrifugation, and (4)a lyophilized algal pellet washed with 0.1 % EDTA. Elemental analysis was performed by Atlantic Richfield Co. Samples for electron microscopy were taken at the same time as those for elemental analysis.
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Flgure 2. Partial suppression of aluminum toxicity by fast-exchange Ions. Optical density of Chlorella vs. time in days after stressing with metal ions. Cultures were grown up to 100 units when stressed with 50 ppm of AI3+. At the same time various fast-exchange ions were added. Curves were for no additions, no fast exchange ions, 150 ppm of K, 150 ppm of Na, 150 ppm of Ca, and 150 pprn of Mg.
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Aluminum shows concentration-dependent inhibition on the growth of Chlorella. At a pH of 3.0, A13+ speciation in water is essentially as the free ion, and therefore, relatively high concentrations can be readily attained. Aluminum expresses toxic effects at 10 ppm with increasing inhibition of growth to above 50 ppm (Figure 1). Higher AP+ concentrations result in similar rates of decline in culture density. Addition of fast-exchange ions demonstrates competitive protection against AP+ toxicity. At an aluminum concentration of 50 ppm, addition of 150 ppm of either Na+, K+, Ca2+,or Mg2+provides some reversal of toxic effects to AP+ (Figure 2). Calcium elicits the greatest protective effect at the concentrations chosen, with magnesium showing slightly better protection than sodium and potassium. The fast-exchange ions, Na, K, Ca, and Mg, apparently give some nonspecific protection from the toxic effects of aluminum by competing for ligands at the toxic site. This protective effect is further illustrated in experiments designed to alter the ratio of calcium to aluminum. In Figure 3, the ratio of calcium to aluminum is plotted vs. the growth rate in cultures of Chlorella. When aluminum is in excess over calcium (Le., a ratio less than 1) growth is inhibited, with cellular density actually declining at large excesses of aluminum over calcium. At a ratio of about 1:l (Ca:Al) the growth rate is close to that of algae in controls of BBM. As the ratio of calcium to aluminum
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Figure 3. Effect of the ratio of calcium to aluminum on growth rate. Growth rate of liquid alga cultures vs. the ratio of calcium to aluminum added to the culture. Dashed line indicates the growth rate of control cultures, and solid line indicates no change in culture density with time.
increases from 1to 2, growth rates remain close to control values and then decline as the ratio Ca:A1 is increased further. Similar toxicity profiles are found upon the addition of copper, but at lower concentrations than aluminum. As noted earlier, at a pH of 3.0, the predominant ionic species for copper will be free Cu2+. As seen in the growth curves in Figure 4, under these conditions, copper expressed toxicity in the 1-10 ppm range (i.e., about 1 order of magnitude lower concentration as that observed for inhibition by aluminum). Protection by the fast-exchange ions is also observed with copper. At a copper concentration of 10 ppm, 150 ppm of calcium, magnesium, or sodium demonstrate similar protection as was found with aluminum (Figure 5). In Environ. Sci. Technol., Vol. 20, No. 6, 1986 617
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Flgure 4. Concentration dependence for copper toxicity. Optical density of Chlorella cultures vs. time in days. The dashed line at day 10 signifies where the CuSO, was added to 0 (control), 1, 5, and 10 ppm of cu2+.
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Flgure 6. Effect of the ratlo of calcium to copper on growth rate. Growth rate of liquid alga cultures vs. the ratio of calcium to copper added to the culture. Dashed line indicates the growth rate of control cultures and solid line indicates no change in culture density with time.
Table I. Stable Complexation of Cu, Al, and Fe by Chlorella saccarophilla
copper"
aluminumn
ironn
200 ppm of aluminum
cells EDTA-washed cells 60 ppm of copper cells EDTA-washed cells 700 ppm of iron cells EDTA-washed cells 50% acid wine water cells EDTA-washed cells control cells EDTA-washed cells
50.32 39.74
1.17 0.57
11.57 3.43 0.38
1.88 0.90
82.99 46.65 4.85 0.45
0.21
180.90 168.40 0.94 0
Micrograms of metal per gram of lyophilized cells.
0 AY
Figure 5. Partial suppression of copper toxicity by fast-exchange ions. Optical denslty of Chlorella vs. time in days after stressing with metal ions. Cultures were grown up to 100 units and then stressed with 10 ppm of Cu". At the same time various fast-exchange ions were added. Curves are for no additions, no fast-exchange ions, 50 ppm of K, 50 ppm of Na, 50 ppm of Ca, and 50 ppm of Mg.
contrast, 150 ppm of potassium appears to enhance copper toxicity, resulting in a sharper decline in cell density. Variations in the ratio of calcium to copper exhibits different results than those observed for aluminum (Figure 6). In a ratio up to 6:l (Ca:Cu), there is a steady decline in culture density. At a ratio of 6:l (Ca:Cu), there appears to be a discontinuity in the relationship where the growth rates approach those of the controls. Above a ratio of 6:l (Ca:Cu), the growth rates rapidly decrease. The bioaccumulation of metals by Chlorella was determined by growing a thick suspension of algae for a period of 5 days in the presence of the various metals, 618
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followed by collection of the cells by centrifugation and then by using plasma emission spectroscopy to determine precise metal concentrations. Table I contains the results of stressing the algae with 60 ppm of Cu, 200 ppm of Al, or 50% acid mine water (taken from a copper-nickel mine) (I). Chlorella accumulates Al, Cu, and Fe at different levels, showing very significant iron accumulation. Both copper and aluminum are partially leached out by washing the cellular pellet with 0.1% EDTA. The amounts of metal removed from the culture medium due to algal bioaccumulation is minimal for all elements monitored (e.g., Al, Ca, Cu, Fe, Mg, Mn, and Ni). Physiological changes resulting from metal stress appear to primarily affect the structure of both algal cell walls and chloroplasts. Electron micrographs indicate a shrinking of the cell wall in the presence of both copper and aluminum. Since growth is inhibited, these morphological changes are probably not a result of changes in cell wall biosynthesis. Iron accumulation in the region of the cell wall is indicated as evidenced by the electron density surrounding cells stressed either with acid mine water or with 700 ppm of iron. Other changes include increased vacuolation of the cytoplasm and chloroplasts. These
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Flgure 7. Electron micrographs 01 Chlorella saccarophila grown for 5 days in the presence of the ions indicated. (A) A control culture grown at pH 3.0. The cell wall k clearly resolved. and me cell membranes lw Chlwwlasts. nucleus. and mitochondria are apparem. Very lime vacuolation is evident. Magnificationof 26100X. (E) A CuIture with 200 ppm of AI3+ showing the disruption 01 the cell wall with much vacuolation with these vacuoles containing electrondense debris. Magnification01 8700X. (C) A culture with 60 ppm of Cu2+ showing no cell wall and almost total vacuolation with membrane disruption. Intracellular electrondense debris is evident. Magnificationof 17400X. (D) A culture grown with 700 ppm 01 Fe3+which is nontoxic to h s e a b e . The cell wall is intact with evidence lw heavymetal precipitaton at the cell wall surface. Magnification of 13050X. (E) A cuhure grown with 5 0 % acid mine water from a copper-nickel mine (1). Vacuolation and cell wail complexation are evident. Magnification of 10440X.
cytoplasmic effects are difficult to fully understand since they are only subtle morphological events which become more severe in the advanced stages as the cells die (Figure
7). Discussion Both aluminum and copper demonstrate concentration-dependent growth inhibition and even death to the acid-tolerant strain of Chlorella used in this study. It appears that these metal ions are transported into the cells by nonspecific mechanisms since Na, K, Ca, and Mg offer some protection to AI toxicity and Na, Ca, and Mg to Cu toxicity. This is in contrast to a previous study of the toxicity of Ni in Chlamydomonas in which Mg directly competes for Ni transport (11). This lack of specificity hy Chlorella is further illustrated by our results which show the relationship between the growth rate and the ratio of calcium to the metals AI or Cu. For aluminum in Figure 3, it appears that calcium can directly compete with aluminum since equimolar levels of calcium can nearly eliminate growth inhibition, The toxicity by copper results in
quite different behavior as seen in Figure 6. It takes significantly more calcium to reverse the toxic effects of copper (Le., a ratio of 6 1 (Ca:Cu)). Further increases on the calcium to copper ratio again produce growth inhibition even though the absolute calcium concentrations are comparable within the two experiments. We cannot explain the reason for this discontinuity in the growth at this time. This anomalous behavior indicates that either the resistance mechanisms or the toxic sites are different for the two metals. Though aluminum is an abundant crustal element, its concentration in natural water near neutral pH is extremely low. This is true for many of the transition metals since insoluble, stable oxo and hydroxo complexes are readily formed. It is unlikely that organisms evolved specific resistance mechanisms for aluminum because they may never have experienced toxic levels of this metal historically. With the increase in the combustion of fossil fuels, acidification of natural waters has lowered the pH, thereby mobilizing not only aluminum, hut also many other metals from the sediments and the surrounding soils Envlron. Sci. Technol.. Vol. 20, No. 6. 1986
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Environ. Sci. Technol. 1986, 2 0 , 620-627
as observed in the Smoking Hills region of Canada (10). Organisms, including Chlorella saccarophilla, have to adapt to these higher concentrations of metals in the water in order to survive. Unfortunately, resistance to metals that historically may not have been present in aquatic systems dictates that either the evolution of new mechanisms or adaptation of those old mechanisms is primarily used for the transport of essential elements. This process is slow as evidenced by the loss of species diversity in highly acidified waterways (12). One resistance mechanism involves a change in the chemical speciation of toxic metal ions to inert species. This has been observed previously for C y a n i d i u m cald a r i u m which utilizes the reduction of sulfate to sulfide as a method of complexing and removing metals before they can enter the cell (1). This acid tolerant organism bioaccumulatessignificant amounts of several metals from liquid suspension at a pH of 1-3. This is not the case with Chlorella, since very little metal is removed from solution and relatively little remains inside cells. The mechanism by which Chlorella is resistant apparently involves keeping the metals from entering the cell. Though this organism may not be useful for biological mining or detoxification of metal-polluted water, it is a useful organism for studying the basis for metal ion toxicity which remains to be elucidated at this time. However, these acid-tolerant algae are very useful for studies of the biochemistry of inhibition because the acid environment simplifies the chemistry of free metal ions as they are transported.
Registry No. AI, 7429-90-5; Cu, 7440-50-8; Ca, 7440-70-2; Mg, 7439-95-4; Na, 7440-23-5; K, 7440-09-7; Fe, 7439-89-6.
Literature Cited (1) Wang, H. K.; Wood, J. M. Enuiron. Sci. Technol. 1983,17, 582A-590A. (2) Wood, J. M. Chem. Scr. 1983,21, 155-162. (3) Wood, J. M. Metal Ions i n Biological Systems; Sigel, H., Ed.; Marcel Dekker: New York, 1984; Vol. 18, pp 223-237. (4) Wood, J. M. Metal Ions i n Biological Systems; Sigel, H., Ed.; Marcel Dekker: New York, 1984; Vol. 18, pp 333-351. (5) Buffel, J. Metal Ions i n Biological Systems; Sigel, H., Ed.; Marcel Dekker: New York, 1984; Vol. 18, pp 165-221. (6) Wood, J. M. Enuiron. Health Perspect. 1985,63,115-121. (7) Jorgenson, S. E.; Jensen, A. Metal Ions i n Biological Systems; Sigel, H., Ed.; Marcel Dekker: New York, 1984; Vol. 18, pp 61-100. (8) Nichols, H. W.; Bold, H. C. J . Phycol. 1965, 1 , 34-38. (9) Helliwell, S., et al. Enuiron. Tech. Lett. 1983, 4, 141. (10) Havas, M.; Hutchinson, T. C. Nature (London) 1983,301, 23-27. (11) Wood, J. M. Proceedings of a Conference on I n situ Sediment Contamination, Aberystwyth, Wales, June 20,1984; Reynoldson, T., Ed.; International Joint Commission, in press. (12) Stokes, P. M.; Bailey, R. C.; Groulx, G. R. Enuiron. Health Perspect. 1985, 63, 79-89.
Received for review September 18,1985. Accepted January 27, 1986. This research was supported by a grant from Atlantic Richfield Co.
Quinoline Sorption to Subsurface Materials: Role of pH and Retention of the Organic Cation John M. Zachara,” Calvin C. Alnsworth, Larry J. Felice, and Charles T. Resch Pacific Northwest Laboratory, Richland, Washington 99352
The sorption of quinoline (pK, = 4.94) was investigated on low-organic-carbon subsurface materials that varied in pH. Sorption isotherms were measured from lo-’ to M quinoline and were found to be nonlinear. The resulting Freundlich constants (KF),based on total aqueous quinoline concentration, were poorly correlated with subsoil properties, including organic carbon. Higher sorption in the acidic subsoils and favorable coefficents for regression of KF, normalized to cation-exchange capacity vs. the ionization fraction ( Q ) , point to the importance of ion exchange of the protonated compound. When the subsoil pH is adjusted, it is shown that sorption parallels the ionization fraction and retention of the organic cation far exceeds that of the neutral species. Calculations of surface speciation and thermodynamic parameters of sorption (AHo,ASo) point to ion exchange and/or surface protonation at pH levels exceeding pK, by greater than 2 log units. It is suggested that in subsurface materials of low carbon content, quinoline sorption is controlled by pH, the nature and capacity of the exchange complex, and groundwater ion composition. Introduction
Basic nitrogen-heterocyclic compounds (NHC’s) are common to many products and waste materials from energy development technologies. Within this general compound class, quinoline is especially important from an environmental health perspective, because it exhibits both high solubility in water and the potential to induce liver 620
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carcinoma. Quinoline and numerous alkylated isomers have been identified in energy-derived waste materials slated for in-ground disposal, particularly solid and liquid wastes from coal gasification (1-4) and shale oil extraction (5). The water solubility of the compound suggests that, in the event of intruding groundwater, quinoline may be leached from the wastes and enter the subsurface environment. In fact, quinoline has been observed in groundwaters proximate to underground coal gasification sites (6, 7). To a large degree, the subsurface dissemination of quinoline is controlled by sorption processes that may reduce the rate of compound movement relative to the transporting water front. The soil or subsurface adsorption of multiring basic NHC’s has not been investigated in great detail, and information on quinoline sorption by natural heterogeneous adsorbents is absent. As observed for many organic compounds, limited research with higher molecular weight quinoline analogues attests to the importance of organic carbon in sorption. In soils and sediments at pH values above pK,, acridine and biquinoline sorption follow the Kw-KOwand K,-S relationships (8,9)commonly observed for “neutral” aromatic compounds (10, 11). In contrast, an investigation of the soil sorption of benzidine, an aromatic amine, has demonstrated the important influence of organic compound protonation and the presence of the organic cation in the overall sorption process (12). Similarly, studies with layer silicates and oxides demonstrate the high sensitivity of quinoline adsorption to pH and
0013-936X/86/0920-0620$01.50/0
0 1986 American Chemical Society
