Indirect Aluminum Toxicity to the Green Alga Scenedesmus through Increased Cupric Ion Activity John G. Rueter, Jr.," Kirk T. O'Reiliy,+ and Richard R. Petersen
Department of Biology, Portland State University, Portland, Oregon 97207 Additions of aluminum and copper to chemically defined media resulted in inhibition of growth of Scenedesmus and of alkaline phosphatase activity. The alkaline phosphatase activity was assayed both on commercially available purified enzyme from bacteria and on the enzyme present in whole Scenedesmus cells. The effect of metal additions was compared to the total aluminum added and to the computed free ion activities for both copper and aluminum. In all three systems (algal growth, purified enzyme, and algal enzyme) the observed toxicity with increased total aluminum was mostly due to an increase in cupric ion activity. The algal growth response was affected for the range of cupric ion activities from lo4 to The toxic dose response of aluminum was largely due to indirect competitive effects of A1 in the medium that displaced copper from the chelator. Introduction Trace metal toxicity in aqueous environments may not be simply a dose response to a particular metal added but rather the net result of a shift in the equilibria among several metals and natural ligands. For phytoplankton it has been demonstrated that the toxicity and nutrition of trace metals depend on the free metal ion activities (1,2). These nutritional and toxic responses may interact and can characteristically be related to the ratio of the toxic to nutritional trace metal ion activities, for example, Cu/Zn (3),Cu/Mn (4), or Cd/Fe (5). Antagonistic interactions such as these may result from competition among aqueous metals for binding to cell surfaces or the biochemical effects of metals inside the cell due to displacement of a metal from an essential site. Aquatic animals, such as zooplankton and fish, have membranes exposed to aqueous solutions just like algae but also have metal uptake due to ingestion of food. Limited evidence with these higher trophic levels also indicates that their toxic response is related to free metal ion activities (rather than total metal in the water) (6-8). Perturbations in aquatic chemistry should affect all organisms to some extent, but the response of phytoplankton may be the simplest to observe. Aluminum toxicity appears to be an important component of the deleterious effect of acid precipitation in natural waters. A dose response has been observed between dissolved aluminum concentrations and the degree of toxic response (9,101. Addition of aluminum to acidified
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Present address: Department of Bacteriology and Biochemistry, University of Idaho, Moscow, ID 83843. 0013-936X/87/0921-0435$01.50/0
waters, however, not only increases the total amount of this metal but can also change the concentration of organic and inorganic complexes with other metals and consequently the activities of these other metals. Considering that aluminum is not usually found to be highly toxic in laboratory studies, the dose response observed for lakes could be due to indirect effects of aluminum on aquatic chemistry. In this research we examine the hypothesis that the toxic responses to increased aluminum can be due to dissolved aluminum competitively displacing copper from organic complexes. This displacement results in a higher free cupric ion activity, which would be the actual causitive agent of toxicity to the phytoplankton. To demonstrate this interaction, we used chemically defined algal growth media in which we varied the total aluminum and copper concentrations. Toxicity was determined from changes in overall growth characteristics and inhibition of the alkaline phosphatase, previously shown to be sensitive to cupric ion toxicity (11). The parallel inhibition of the growth rate and alkaline phosphatase activity of Scenedesmus is compared to the calculated equilibrium chemical speciation of copper and aluminum to demonstrate that the response correlates to the cupric ion rather than total aluminum concentration or free aluminum ion activity. Methods Algal Culture. Scenedesmus quadricauda (Chlorophyta) was grown in FRAQUIL medium (12) as modified by Petersen (13). This medium is prepared from three stock solutions (nutrients, salts, and a trace metal-EDTA solution). The nutrient and salt solutions were passed through a preequilibrated Chelex-100 column (Bio-Rad) to remove contaminant trace elements from the reagents. After addition of known quantities of trace metals, the medium was filtered through 0.4-pm polycarbonate filters (Nuclepore) to remove any fine particulate material and most bacteria that might be present. All labware was acid washed and rinsed with ultra-pure water (Barnstead NANO-PURE), and manipulations were carried out in a laminar flow hood to minimize possible contamination. Cultures were grown at 18 "Cwith continuous light (100 kE-m-2-S-1).Growth experiments were performed in 30-mL polycarbonate tubes (Nalgene, Oak Ridge type) with 20 mL of medium. Although adsorption losses from natural water samples have been reported for polycarbonate (14), the metal content of our medium is buffered at much higher total metal concentrations by the use of synthetic chelators than in those studies. Also, polycarbonate tubes
0 1987 American Chemical Society
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were used instead of Teflon because they can be placed directly into a filter fluorometer (Turner Designs, Model 10) for daily measurements of in vivo chlorophyll fluoresence. We used these fluorescence values to calculate the growth rate for the exponential-phase growth during 7-day experiments (15). Aluminum and Copper Chemistry. Experimental media were prepared 24 h beforehand to allow equilibration between metal species and EDTA. The pH was adjusted to 7.3. A matrix of copper and aluminum concentrations was tested ranging from 0 to 5 X 10+ M Cu and M Al. The growth rate was determined from 0 to 8 X for 31 combinations of these metal concentrations. The chemical speciation in the media was estimated with the computer program MINEQL (16). Alkaline Phosphatase Activity. The activity of alkaline phosphatase was determined by reaction with the artificial substrate p-nitrophenyl phosphate (Sigma) (11). Purified bacterial enzyme (Sigma) or whole cells were exposed to different combinations of A1 and Cu concentrations. Purified bacterial enzyme was assayed in a M NaHCO,, M minimal medium that contained NTA, and lo-* M ZnClz. In these enzyme experiments only, 3-phosphoglycerate (Sigma) was added as a P source to produce high-density Scenedesmus cultures with increased alkaline phosphatase activity. The algal enzyme activity was determined in FRAQUIL medium. Results
The growth rate of Scenedesmus varied with both copper and aluminum additions to FRAQUIL (Figure 1). There was a region of Cu and A1 concentrations (0 < Cu I 2 pM and 0 I A1 5 40 pM)that forms a plateau of optimal growth rate, outside of which the growth rate declined. The growth rate with no added copper was inexplicably affected by increased aluminum. A crosssection through this figure at any particular Cu concentration will result in a dose-response curve for Al, but the curves will be different depending on the Cu concentration chosen. Presenting the above data in terms of the cupric and aluminum ion activities of the media portrays quite a different picture. The decline in the growth rate is generally correlated to the cupric ion activity (Figure 2a), but the same aluminum ion activity can result in a wide range of growth rates depending on the cupric ion activity (Figure 2b). For example, with 3 p M Cu and 20 p M Al, the computed pCu2+is 11.4 and the growth rate was 0.8, but with the same total copper (3 p M ) and 3 times the aluminum (60 p M ) the computed pCu2+is 6.4 and the growth rate drops to 0.4. Aluminum on its own does not appear to be very toxic; 60 p M Al total ( P A P 5.7) is required to produce any inhibitory effect if the medium is free of copper, and high aluminum ion activities can support near optimal growth rates (Figure 2b). The majority of the toxic response of Scenedesmus to aluminum in this medium is due to indirect chemical interactions that result in higher cupric ion activities. 436
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Figure 2. (a) Growth rate as a function of the negative log of the computed cupric ion activity (pCu). This is the same experimental data as for Figure 1. Total AI concentrations are noted by symbols: 0 (e), 20 (o),30 (a),40 (O), 50 (A),60 (A),and 80 p M (X). (b) Growth rate as a function of the negative log of the computed free aluminum ion activity (pAl). Same data as for Figure 1 and (a). Total copper 4 (a),4.5 (n), concentrations are noted by symbols: 0 (e),3 (O), and 5 pM (A).
Purified bacterial alkaline phosphatase assayed in defined buffer systems was inhibited by cupric ion activity and was only slightly inhibited by aluminum ion activity. Changing the aluminum concentration with no added copper only reduced the activity by 22% (Figure 3a) whereas the same change in total aluminum in assay buffer with some added copper had dramatic inhibitory effects (Figure 3a). The enzyme activity was correlated to cupric ion activity but showed no relationship with aluminum ion activity (compare panels b and c of Figure 3). These results, in simple chemical systems, demonstrate that the apparent toxic effects of aluminum additions are largely a result of the displacement (and consequent increase in activity) of cupric ions. Aluminum also produces the same indirect toxic effect with alkaline phosphatase obtained from Scenedesmus (Figure 4). Increased aluminum alone has little effect, but in the presence of copper the toxic effect is magnified (Figure 4a). As with both the cultures and the purified enzyme, the best predictor of toxicity is the free ion activity of copper rather than aluminum (Figure 4b,c). Discussion
Aluminum can cause toxicity through the displacement of copper from ligands. We have demonstrated this indirect toxicity of aluminum for both algal growth and for enzyme activity in artificial media with synthetic ligands. Additionally, computer models of the chemical speciation in these media support our contention that the toxic response is related to the cupric ion activity rather than the total copper or any of the aluminum species. Naturally occurring humic and fulvic acids bind copper and aluminum in a competitive manner (17),and we feel that our system mimics a mechanism that could be important in natural waters.
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Flgure 3. Purified bacterial alkaline phosphatase activity as a function of trace metal conditions. Duplicate points are shown. Four replicates of the control were all within f5%. (a) Enzyme activity vs. the total aluminum added at different copper concentrations: 0 (e),2 ( o )4, (H), and 6 pM (0). (b) Enzyme activity vs. cupric ion activity for different aluminum concentrations: 0 (e),20 (O), 40 (H), and 60 pM (0). (c) Enzyme activity vs. aluminum ion actlvity for different total copper concentrations, as in (a).
The assay for alkaline phosphatase activity was a valuable biochemical indicator of toxicity, but the results are not meant to imply that inhibition of this enzyme was the cause of the growth inhibition of Scenedesmus. In some cases, the inhibition of alkaline phosphatase might impair the phosphorus nutrition of algae, leading to reduced primary productivity. For our purposes, however, it is important to show that in chemically defined media, in which the metal ion activities are controlled by the buffering capacity of the ligands, cells and enzymes respond in a consistent manner to changes in the aqueous chemistry. Chemical speciation models may be particularly useful in toxicity studies that involve possible chemical interactions. Studies of fish and algal toxicity as a function of copper and hardness or humic acid content of the water are good examples. Investigators have generally observed that increased hardness or humic acid concentration decreases the toxicity of copper (18-21). These observations could be straightforwardly explained by the decreased cupric ion activity due to binding of copper to carbonates, organics, or hydroxides (if the pH also increases). Pa-
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Flgure 4. Actlvity of alkaline phophatase in Scenedesmus cultures as a function of the trace metal conditions. (a) Enzyme activity vs. total aluminum concentration. Copper concentration is given symbols: 0 (e),4 (O), 8 (H),and 12 pM (0). (b) Enzyme activity vs. pCu. Total aluminum concentration is given by symbols: 0 (e),5 (o),10 (M), and 20 pM (0). (c) Enzyme activity vs. pAl. Copper concentration is given by the same symbols as in (a).
genkopf (22) has reviewed some of these studies and concluded that some toxicity is modified by competitive ions at the gill surface of fish. Similar “more-than-additive’’ effects of metals on toxicity here and by others (13) with zinc imply that these toxic effects can be largely explained by metal competition for ligands and resultant changes in the free metal ion activities of the active (Cu2+)species. The toxicity of aluminum species (and its effects on other metals) has not been adequately addressed. For example, the demonstration that aluminum hydroxide [Al(OH),+] was the toxic species to Chlorella (23) ignored possible interactions with other metals in solution. Recent work however has shown that the toxicity of A1 and Cu depends on the availability and transport of these ions (24). Aluminum may be more toxic to other organisms than to Scenedesmus. Evidence on the molecular nature of direct aluminum toxicity is scarce (251, and what little there is suggests that the binding to macromolecules (DNA for example) in aqueous solutions has “little bearing on in vivo systems”. A common symptom of aluminum toxicity in plants, chlorosis, is believed to be a competition with iron causing a deficiency (26,271; similar indirect competitive mechanisms may occur with respect to calcium Environ. Sci. Technol., Vol. 21, No. 5, 1987
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or magnesium nutrition. Aluminum has been observed to be toxic to algae (28,29)and animals such as zooplankton (29,30),insects (31),and fish (32). These studies demonstrate that there is a toxic dose response to total aluminum concentration. Chemically controlled media would have to be used to isolate the causitive ion in these above studies. The destructive effects of acid precipitation and increased aluminum on natural waters are well-known, but many of the mechanisms causing the deterioration are little understood. Accelerated weathering of watersheds will lead to increased concentrations of several metals in lakes, not just elevated aluminum (33). In addition, acidification and increased aluminum concentrations in lake waters will cause displacement of metals from humic acids due to proton and aluminum ion competition. All of these processes will happen in concert. A full understanding of the mechanism of toxicity observed during the acidification of natural waters has implications for the design of toxicity studies as well as short-term amelioration measures or long-term lake recovery projects. Registry No. Al, 7429-90-5; Cu, 7440-50-8; alkaline phosphatase, 9001-78-9.
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(10) Dillon, P. J.; Yan, N. D.; Harvey, H. H. CRC Crit. Rev. Environ. Control 1984, 13, 167-194. (11) Rueter, J. G. Limnol. Oceanogr. 1983, 28, 743-748. (12) Morel, F. M. M.; Rueter, J. G.; Anderson, D. M.; Guillard, R. R. L. J. Phytol. 1979, 15, 135-141. (13) Petersen, R. Environ. Sci. Technol. 1982, 16, 443-447. (14) Fitzwater, S. E.; Knuaer, G. A,; Martin, J. H. Limnol. Oceanogr. 1982,27, 544-551. (15) Brand, L. E.; Guillard, R. R. L.; Murphy, L. S. J . Plankton Res. 1981, 3, 193-201. (16) Westall, J. C.; Zachary, J. L.; Morel, F. M. M. Technical Note 18;R. M. Parsons Laboratory, Massachusetts Institute of Technology: Cambridge, MA, 1976. (17) Perdue, E. M.; Lytle, C. Environ. Sci. Technol. 1983, 17, 654-660. (18) Howarth, R. S.; Sprague, J. B. WaterRes. 1978,12,455-462. (19) Chakoumakos, C.; Russo, R. C.; Thurston, R. V. Environ. Sci. Technol. 1979, 13, 213-219. (20) Winner, R. W. Aquatic Toxicol. 1984, 5, 267-274. (21) Winner, R. W. Water Res. 1985, 19, 449-455. (22) Pagenkopf, G. K. Environ. Sci. Technol. 1983,17,342-347. (23) Helliwell, S.; Bately, G. E.; Florence, T. M.; Lumsden, B. G. Enuiron. Technol. Lett. 1983, 4, 141-144. (24) Folsom, B. R.; Popescu, N. A,; Wood, J. M. Environ. Sci. Technol. 1986, 20, 616-620. (25) Haug, A. CRC Crit. Rev. Plant Sci. 1984, 1, 345-373. (26) Foy, C. D.; Chaney, E. L.; White, M. C. Annu. Rev. Plant Physiol. 1978, 29, 511-566. (27) Foy, C. D.; Fleming, A. L. J. Plant Nutr. 1982,5,1313-1333. (28) Foy, C. D.; Gerloff, G. C. J. Phycol. 1972, 8, 268-271. (29) Burrows, W. D. CRC Crit. Rev. Environ. Control 1977, 7, 167-216. (30) Havas, M.; Hutchinson, T. C. Can. J. Fish. Aquat. Sei. 1982, 39, 890-903. (31) Witters, H.; Vangenechten, J. H. D.; Van Puyumbroeck, S.; Vanderborght, L. L. J. Bull. Environ. Contam. Toxicol. 1984,32, 575-579. (32) Freemand, R. A,; Everhart, W. H. Trans. Am. Fish. SOC. 1971,100,644-658. (33) Nordstorm, D. K.; Ball, J. W. Science (Washington,D.C.) 1986, 232, 54-56.
Received for review December 13,1985. Accepted November 10, 1986. This work was supported in part by a grant from the PSU Research and Publication Committee to J.G.R. This is Environmental Sciences and Resources Program Publication 193.
