EFFECTS OF FREE CU 2 AND ZN 2 IONS ON GROWTH AND METAL ACCUMULATION IN FRESHWATER ALGAE

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1 Environmental Toxicology and Chemistry, Vol. 16, No. 2, pp , SETAC Printed in the USA /97 $ EFFECTS OF FREE CU 2 AND ZN 2 IONS ON GROWTH AND METAL ACCUMULATION IN FRESHWATER ALGAE KATJA KNAUER,* RENATA BEHRA and LAURA SIGG Swiss Federal Institute for Environmental Science and Technology, 8600 Duebendorf, Switzerland (Received 31 January 1996; Accepted 9 June 1996) Abstract Five species of unicellular green algae were exposed to a broad range of Cu 2 and Zn 2 concentrations to examine the relationship between the free Cu 2 and Zn 2 ion concentrations and algal growth at metal concentrations relevant for freshwater phytoplankton. We estimated extra- and intracellular metal concentrations and characterized the adsorption of copper and zinc on algal surfaces. The optimal growth rate of Scenedesmus subspicatus occurred in a broad range of Cu 2 and Zn 2 concentrations (from to 10 7 and from to M, respectively). Chlamydomonas reinhardtii reacted more sensitively toward copper, optimal growth was achieved only at a pcu ( log[cu 2 ]) around 11, whereas growth was optimal over a broad range of free Zn 2 concentrations (from to 10 6 M). The optimal range for growth of Chlorella fusca occurred over about three orders of magnitude of the free Cu 2 concentration (from to M). Chlamydomonas cultures isolated from Lake Constance tolerated Cu 2 concentrations over seven orders of magnitude (pcu 7 to 14). The growth of algae showed a high tolerance toward high intracellular copper and zinc concentrations. This suggests that the cells may immobilize the metals intracellularly. The affinity of copper for algal surfaces is higher than that of zinc in the experimental concentration range: adsorption constants log K Cu (11.06) log K Zn (6.49) (ph 7.9). These freshwater algae tolerate higher Cu 2 and Zn 2 concentrations than marine algal species. In comparison to our lake data, the results obtained from the culture algae indicate a possible role for copper as a limiting factor for certain algal species in eutrophic lakes. Keywords Copper Zinc Algae Limitation Freshwater INTRODUCTION The chemical speciation of certain trace metals and the growth of phytoplankton are tightly linked, as shown for a number of oceanic and coastal algal species [1]. Experimental evidence indicates that growth and toxicity are mostly a function of the activity of the free metal cation [2 6]. Trace metals, such as copper and zinc, are important micronutrients for algae but are toxic at high concentrations. In freshwater ecosystems the total copper and zinc concentrations are usually higher than in ocean waters [7,8]. In a study of Lake Greifen, a eutrophic lake in Switzerland, total dissolved copper was in the range of 0.5 to M [9], and total dissolved zinc was in the range of 1 to M [10]. The corresponding free Cu 2 and Zn 2 concentrations ranged from to M and from 10 8 to 10 9 M, respectively [9,10]. The very low Cu 2 concentrations and the ratio of the Cu 2 concentration to total dissolved copper (10 6 to 10 7 ) indicate the presence of very strong complexing ligands, probably of biological origin. Seasonal variations of the Cu 2 concentration in this and other lakes suggest a direct relationship between the presence of strong ligands and algal productivity [11]. Zinc appears to be complexed to a much lesser extent than copper. The ratio of the Zn 2 concentration to total dissolved zinc was about 0.02 to 0.1 in Lake Greifen. These observations lead to the question of growth requirements of freshwater phytoplankton with regard to copper and zinc. In particular, the optimal copper and zinc ion concentrations for freshwater algae need to be determined. Studies on marine algae have indicated that pcu 11 to 12 (where pcu log[cu 2 ]) is toxic for sensitive species [3,4] and that pzn * To whom correspondence may be addressed. ( log[zn 2 ]) 8 is toxic to some species, whereas pzn 11 may be limiting [6]. No data about copper limitation are available for marine algae. Previous studies [12,13] showed that growth of freshwater algae was inhibited at high Cu 2 ion concentrations. Few data are available regarding the growth of freshwater algae, particularly at environmentally relevant metal ion concentrations. In this study we investigated the growth of freshwater algae in a trace metal buffer system designed to regulate and quantify the free metal concentration in the culture medium over a wide concentration range. Our objectives were to examine the relationship between the free Cu 2 and Zn 2 ion concentrations and algal growth at concentrations relevant for freshwater situations and at higher concentrations. Moreover, we determined the cellular content of copper and zinc in the green alga Scenedesmus subspicatus and attempted to determine characteristic cellular contents of copper and zinc under conditions close to the natural ones and at higher concentrations. For a more complete understanding of the uptake process, we attempted to distinguish between the adsorption of metal ions on algal surfaces and the intracellular uptake. The results from the algae cultures are interpreted with respect to field data in lakes. MATERIALS AND METHODS Preparation of culture medium The growth medium used in these experiments was based on the Organisation for Economic and Co-operative Development (OECD) guideline medium for testing chemicals [14]. The medium contained M NaNO 3, M KH 2 PO 4 when using a M N-2-hydroxyethylpipera- 220

2 Effects of copper and zinc on freshwater algae Environ. Toxicol. Chem. 16, zine-n -2-ethane-sulfonic acid (HEPES) buffer or MKH 2 PO 4 and MNa 2 HPO 4 2H 2 O when phosphate was buffering the medium, M CaCl 2 2H 2 O, M MgSO 4 7H 2 O, M NaHCO 3, MH 3 BO 3, and MNa 2 MoO 4 2H 2 O. It was modified as described below and therefore had a defined composition with respect to the metals copper, zinc, manganese, and iron. Departures from standard OECD medium included the addition of 10 4 Mor10 5 M ethylenediaminetetraacetic acid (EDTA) or nitrilotriacetic acid (NTA) to regulate the free concentration of the trace metal ions. The medium was buffered with a HEPES buffer ( M) or with phosphate ( MNa 2 HPO 4 and MKH 2 PO 4 ), and the ph was finally adjusted to 7.9 with NaOH. The medium was prepared from reagent-grade chemicals (Merck, Dietikon, Switzerland). Solutions as HNO 3 and NaOH used for the preparation of the medium were suprapure chemicals. To minimize contamination, all flasks were presoaked in 0.1 M HNO 3 for 6 d, rinsed with nanopure water, and dried at 100 C. The medium (100 ml) was sterilized in 250-ml polycarbonate flasks in a microwave oven [15]. The sterile, filtered trace metal solutions (Fe, Mn, Cu, and Zn) were then added to the medium to obtain specific free metal concentrations. Iron was added as FeEDTA. The medium was then pre-equilibrated for at least 24 h for chemical equilibrium to be achieved [16]. Copper and zinc experiments The concentrations of free Cu 2 and Zn 2 ions in the medium were computed with the chemical speciation program MICROQL [17,18]. The equilibrium constants were taken from Martell and Smith [19] and corrected for ionic strength. The total concentrations of copper and zinc in the medium were varied according to these calculations. In the copper experiments only the free Cu 2 concentration was varied. The other free metal concentrations (Fe, Mn, and Zn) were kept constant ([Fe 3 ] M, [Mn 2 ] 10 6 M, and [Zn 2 ] 10 9 M) by varying the corresponding total concentrations. The free Cu 2 concentration was varied between 10 7 and M, which, in the presence of 10 4 M EDTA, corresponded to levels of total dissolved copper of 10 4 Mto 10 9 M. We did not add other trace metals (e.g., Co) to the medium. Nevertheless, the concentration of cobalt in the medium was M because of contamination from the chemicals used. However, cobalt had no influence on the speciation, even at the lowest free Cu 2 concentration. In other experiments, we achieved the same free Cu 2 ion concentration for a 10 times lower total copper concentration by using an EDTA concentration of 10 5 M. Additionally, NTA was used at high free Cu 2 concentrations to check the growth rates in a different medium. In the zinc experiments the free Zn 2 concentration was varied between 10 5 and M. As for the other metals, the free Cu 2 concentration was kept constant (10 14 M). The actual levels of total dissolved zinc ranged between and M using 10 4 M EDTA. The working range of pcu and pzn was limited by contamination and determination limits on the low side (Cu total 10 9 M and Zn total M) and by precipitation on the high side ([Cu 2 ] 10 7 M and [Zn 2 ] 10 5 M). Consequently, we did not add copper to the medium to obtain the lowest free Cu 2 concentration. We determined the metal concentrations in each culture replicate and calculated again the free ion concentrations in the media. From these calculations we determined the standard deviation of the free Cu 2 and Zn 2 concentrations in the replicates. Furthermore, samples of the media were taken daily during the growth experiments, and the concentrations of copper and zinc were measured after filtration so that the actual concentrations were known. Test organisms and culture conditions Stock cultures on agar of three green algae Scenedesmus subspicatus SAG 86.81, Chlamydomonas reinhardtii 11-32a, and Chlorella fusca 211-8b were obtained from the algal collection in Göttingen (Institute of the University of Göttingen, Germany). The two strains of Chlamydomonas tremulans (Skuja) and Chlamydomonas clinobasis were obtained from the culture collection of the Institute of Limnology of Lake Constance in Germany. These two strains were isolated from Lake Constance. The cultures were grown in modified OECD medium at 25 C. An illumination of 52.2 E m 2 s 1 was provided by OSRAM L 20 fluorescent lights under a 12 h light:12 h dark regime. Exponentially growing cells were inoculated into each flask (5 ml of the preculture) to provide initial cell densities of approx. 2 to cells/ml. Inocula were obtained from an axenic stock culture grown in the described medium, and we took into account the transfer of trace metals with the inoculum. We did not add copper to the preculture for the copper experiments; the calculated free Cu 2 concentration in the preculture was around M. The free Zn 2 concentration in the preculture for the zinc experiments was around to M. We followed the growth for 5 d, the time needed until steady state of growth was achieved. The first transfer from the preculture to the different media was named culture 1. One to five milliliters in volume was removed from each flask one to four times each day for optical density measurements at 685 nm. Previous experiments have shown a good correlation between optical density and cell counts. Determination of algal cell size with a Galai CIS-1 particle counter showed that copper and zinc concentrations did not cause a change in cell size. The specific growth rates were calculated by plotting the natural log of cell numbers against time and calculating a linear regression for the exponential growth phase. The final cell density was determined as soon as cell division started to decrease in the late exponential growth phase. At the end of the exponential growth phase, the cell density was around 10 6 cells/ml. To control whether the cultures were acclimated to the experimental trace metal regime, we transferred 5 ml from these cultures into fresh culture medium with the same chemical composition and followed growth over 5 additional d. The second transfer was named culture 2. We did this procedure four times at the lowest pcu of 14 to confirm the growth rate. Each growth experiment was done 12 times. The growth rates were reproducible. Cellular metal content The cellular metal content of the algae was determined at the end of the exponential growth phase. The cultures (300 ml) were filtered onto m filters (Sartorius) and washed with 50 ml of medium without EDTA and trace metals to

3 222 Environ. Toxicol. Chem. 16, 1997 K. Knauer et al. Fig. 1. Relationship between the growth rate (d 1 )ofscenedesmus subspicatus and the calculated free Cu 2 concentration (M) in culture medium containing 10 4 or 10 5 M EDTA of the first (culture 1) and second (culture 2) transfers. The vertical error bars correspond to the standard deviation of the growth rate, and the horizontal error bars correspond to the standard deviation of the free Cu 2 concentration (M) in the medium (n 12). Where not shown the error bar is within the symbol. remove metal-containing adhering medium. After drying the filter for 15 h at 50 C, the dry weight of cells was determined. The filters were then placed in digestion flasks, 1 ml of H 2 O 2 (30%) and 5 ml of HNO 3 (65%) were added, and digestion was performed in a microwave oven for 14 min. After cooling, the solutions were transferred into 25-ml graduated flasks, and these were filled with nanopure water. The metal concentrations were determined by inductively coupled plasma spectrometry mass spectrometry (Perkin Elmer Sciex Elan 5000). Data were corrected for the metal content of blank filters. Blank values were mol Cu/filter and mol Zn/filter. The filters had previously been washed twice with HNO 3 for 2 h at 90 C. To differentiate between extracellular adsorption and intracellular uptake, the cells were washed with EDTA [20]. Preliminary experiments showed that M EDTA and 10 min were good conditions for this differentiation at ph 8. No cell breakage and leakage were observed under the microscope after the EDTA extraction, and cell growth continued at the same rate when algae were placed in a new culture medium. Algae were filtered on a m filter, washed with 20 ml of M EDTA for 10 min, and again filtered. The intracellular metal concentration was defined as the cellular metal content determined after the EDTA wash. We determined the adsorbed metal content by measuring the metal concentrations in the extraction solution. Additionally, adsorbed metal was calculated as the difference between the metal content before and after washing with EDTA. RESULTS Relationship between growth rate and free Cu 2 and Zn 2 concentrations The dependence of algal growth on free the Cu 2 concentration was determined for three typical culture species as well as for two isolates from Lake Constance. The shape of the growth curves of S. subspicatus were similar at all tested Cu 2 concentrations with respect to the lag phase and the final cell density. In contrast, the growth curves of C. reinhardtii showed differences in final cell density at low (10 12 M) and high (10 10 M) free Cu 2 concentrations, the highest cell density occurring at M. The cell density of C. fusca was reduced at low (10 14 M) and high Cu 2 concentrations (10 9 M) (not shown). Growth rates are presented as a function of the Cu 2 concentration as calculated from the initial composition of the medium. When plotted against the free Cu 2 concentration in the media containing EDTA concentrations of 10 4 Mor10 5 M (Fig. 1), growth rates were comparable. The optimal growth rate of S. subspicatus occurred within a broad range of Cu 2 concentrations. Growth was not limited even at the lowest free Cu 2 ion concentration (10 15 M), which corresponded to a total copper concentration of 10 9 M. Working at such a low Cu 2 concentration is difficult because of the background metal contaminations originating from the chemicals used. These background contaminations ranged between 10 8 to 10 9 M total copper. Also, the algae tolerated up to 10 7 M free Cu 2 without a significant reduction in the division rate (Fig. 1). Subsamples of each culture were inoculated again in culture medium to confirm growth rate and cellular content. The specific growth rate did not change, especially at the lowest free Cu 2 concentration (Fig. 1). This was tested with two additional transfers in culture medium at the lowest Cu 2 concentration. The growth rate was maintained at 0.9 d 1. We confirmed the growth rate ( d 1, N 3) at high free Cu 2 concentrations (10 7 M) in a medium with NTA. Growth rates were comparable in both media, buffered with HEPES or with phosphate. Chlamydomonas reinhardtii was found to grow optimally at a pcu around 11. Its growth rate was limited by 20% at a pcu value of 13 and inhibited by 20% at a pcu of 7 (Fig. 2).

4 Effects of copper and zinc on freshwater algae Environ. Toxicol. Chem. 16, Fig. 2. Growth rates (d 1 )ofscenedesmus subspicatus, Chlamydomonas reinhardtii, and Chlorella fusca in relation to free Cu 2 concentration (M). The vertical error bars correspond to the standard deviation of the growth rate, and the horizontal error bars correspond to the standard deviation of the free Cu 2 concentration (M) in the medium (n 12). Where not shown the error bar is within the symbol. The specific growth rate of C. fusca was about 1 d 1 when grown under optimal conditions. At pcu concentrations higher than 10 and lower than 13 growth rate decreased by 25%, reflecting the inhibition and limitation of algal growth (Fig. 2). The growth of two algal isolates from Lake Constance, C. tremulans and C. clinobasis, were also investigated under various free Cu 2 concentrations. No effect on growth was observed within a pcu range of 7 to Inhibition of growth occurred when the free copper concentration was higher than 10 7 M (Fig. 3). Growth of S. subspicatus and C. reinhardtii was investigated at different free Zn 2 concentrations within a range of Mto10 5 M. Optimal growth of S. subspicatus occurred at a pzn of 12 to 5.5 (Fig. 4). A nutritional deficiency resulting in a 90% reduction of growth rate appeared to occur at a free Zn 2 concentration of M. At zinc values above the optimal pzn range, inhibition of growth occurred as shown in Fig. 4. The growth of C. reinhardtii was optimal over a broad range of pzn (from 12 to 6). Chlamydomonas reinhardtii tolerated a concentration up to 10 6 M and was inhibited at higher concentrations (Fig. 4). Relationship between cellular content and free metal ion concentration The cellular metal content of S. subspicatus was determined at the end of the exponential growth phase at different copper concentrations and in the presence of 10 4 M and 10 5 M EDTA. This allowed a comparison of cultures grown at similar free metal ion concentrations but different total metal concentrations. We found a hyperbolic relationship between the total copper content of the algae and the free Cu 2 concentration in the medium. These results are in good agreement at the two EDTA concentrations (Fig. 5). The total cellular copper concentration increased from to mol Cu/g algae with an increasing free copper concentration of to 10 7 M. The maximal intracellular copper content was mol Cu/g algae ( mol Cu/cell) at a Cu 2 concentration of 10 7 M (Fig. 6). Thus, up to 80% of the total cellular copper content was located intracellularly, and 20% was located on the surface of the cells. The two buffers (phosphate and HE- PES) had no influence on the cellular metal content. By measuring daily the concentrations of copper and zinc in the growth medium, we checked that copper was not actually depleted in our media after uptake by the algae. Copper in the algae represented only 1% or less of the total added copper. We calculated again the free ion concentrations in the media at the end of the growth experiments. The speciation of the media did not change during the growth of the algae. Binding isotherms were used to describe the adsorption process on the surface of S. subspicatus. The dependence of copper adsorbed on cell surfaces on the Cu 2 concentration in solution could be described by the Langmuir adsorption isotherm in form of the van den Berg equation [21] (Fig. 7): 2 2 [Cu ] 1 [Cu ] [CuL] K [L ] [L ] L total total The equation was used to determine L total, the complexing capacity, which represents the number of binding sites on the cell walls (Cu max mol/g algae, Cu max mol/cell), and K L, the adsorption equilibrium constant (log K Cu 11.06), conditional at ph 7.9. CuL is the concentration of copper bound to the algae surfaces. In the pzn range of 9 to 13 the cellular content was nearly independent of the Zn 2 ion concentration in the medium (Fig. 8). In this range the cellular content increased only slightly (from to mol Zn/g algae). At higher free Zn 2 concentrations (10 9 to 10 5 M), a nearly linear relationship was observed in the log-log plot, with the total cellular zinc content increasing from to mol Zn/g

5 224 Environ. Toxicol. Chem. 16, 1997 K. Knauer et al. Fig. 3. Growth rates (d 1 ) of the two isolates of Lake Constance, Chlamydomonas tremulans and C. clinobasis, in relation to the free Cu 2 concentration (M) in culture medium containing 10 4 M EDTA. The vertical error bars correspond to the standard deviation of the growth rate, and the horizontal error bars correspond to the standard deviation of the free Cu 2 concentration (M) in the medium (n 4). Where not shown the error bar is within the symbol. algae. The highest intracellular zinc content per cell was mol Zn/cell. In the pzn range of 9 to 16 the total and intracellular cellular content were equal. Only at higher Zn 2 concentrations ( 10 9 M) was zinc adsorbed to the cell surfaces. With increasing free Zn 2 concentration (from 10 9 to 10 5 M), the amount of zinc adsorbed to the cell walls increased from 5 to 80% of the total cellular content. As for the copper adsorption data, we used the van den Berg equation [21] to analyze the zinc data (Fig. 9). The complexing capacity of zinc to algal surfaces was Zn max mol Zn/g algae or Zn max mol Zn/cell, and the equilibrium constant was log K Zn 6.49 (ph 7.9). DISCUSSION We examined the growth and accumulation of copper and zinc by various algae species by using a metal ion buffer system that allows calculation of the trace metal speciation in the medium. The copper and zinc concentrations used in this study encompass the naturally occurring range of copper and zinc observed in freshwater lakes and higher ranges. The relationship between growth rate and free copper and free zinc concentrations in the medium shows very well the dual nature of essential elements like copper and zinc. Copper and zinc are required at low concentrations and can be toxic at higher concentrations, as shown for C. fusca, which was Fig. 4. The growth rates (d 1 )ofscenedesmus subspicatus and Chlamydomonas reinhardtii in relation to free Zn 2 concentration (M). The vertical error bars correspond to the standard deviation of the growth rate, and the horizontal error bars correspond to the standard deviation of the free Zn 2 concentration (M) in the medium (n 12). Where not shown the error bar is within the symbol.

6 Effects of copper and zinc on freshwater algae Environ. Toxicol. Chem. 16, Fig. 5. Relationship between cellular copper content (mol Cu/g algae) of Scenedesmus subspicatus and the free Cu 2 concentration (M) in the medium containing 10 4 or 10 5 M EDTA. limited at free Cu 2 concentrations lower than M and inhibited at concentrations higher than M, and for S. subspicatus, which was limited at free Zn 2 concentrations lower than M and inhibited at concentrations higher than M. Each alga had its own tolerance range for copper and zinc. The tolerance range was defined by the range of free Cu 2 or Zn 2 concentrations in which growth was found to be optimal. The optimal growth for S. subspicatus occurred over eight orders of magnitude of free Cu 2 and six orders of magnitude of free Zn 2 ion concentrations. This alga showed the highest tolerance toward copper and zinc. Chlamydomonas reinhardtii reacted more sensitively toward copper; optimal growth was only achieved at a pcu around 11, whereas growth was optimal over a broad range of free Zn 2 concentrations (from to 10 6 M). Thus, C. reinhardtii showed the highest sensitivity toward copper. The tolerance range for C. fusca was found to span about three orders of magnitude of the free Cu 2 concentration. The isolates of Lake Constance tolerated Cu 2 concentrations over seven orders of magnitude. In general, all species studied showed a high tolerance toward copper and zinc, growth being only slightly reduced even at the highest concentrations. Peterson et al. [12] and Rueter et al. [13] described a similar Fig. 6. Surface-bound, intracellular, and total cellular content of copper (mol Cu/g algae) in the green alga Scenedesmus subspicatus in relation to free Cu 2 concentrations (M).

7 226 Environ. Toxicol. Chem. 16, 1997 K. Knauer et al. Fig. 7. Adsorption of copper to Scenedesmus subspicatus, measured (points) and calculated with parameters obtained by linear regression according to van den Berg [21] (Cu max mol Cu/g algae, log K Cuads 11.06, ph 7.9). tolerance behavior toward copper in Scenedesmus quadricauda. At high concentrations of Cu 2 ( M), growth rate was reduced by half, whereas growth was optimal at a Cu 2 concentration lower than M. No limitation of growth occurred in the experiments at the lowest tested free Cu 2 concentration ( M) [12,13]. Our data showed that the presence of low free Cu 2 ion concentrations, close to environmental concentrations, can limit the growth of algae, although only to a small extent. In contrast to the freshwater data, Brand et al. [3] demonstrated that an average pcu value of caused 50% inhibition of the reproduction rate of several marine species. Similar results were reported by Gavis et al. [22]. Most marine species seem to be more sensitive than freshwater algae toward copper, as neritic clones have shown a greater tolerance to Cu 2 than have the oceanic species [22]. With some exceptions, few marine species show the same tolerance toward copper as species tested in this work [4,22,23]. Morel et al. [23] demonstrated that cupric ion concentrations up to M had no effect on growth of the marine diatom Skeletonema costatum. Our zinc data are consistent with the results of Peterson et al. [12], who found that S. quadricauda grew optimally over a broad range of free Zn 2 concentrations (from to 10 6 M). Inhibition of growth occurred at very high concentrations Fig. 8. Surface-bound, intracellular, and total cellular content of zinc (mol Zn/g algae) in the green alga Scenedesmus subspicatus in relation to free Zn 2 concentrations (M). The error bars correspond to the standard deviations (n 4).

8 Effects of copper and zinc on freshwater algae Environ. Toxicol. Chem. 16, Fig. 9. Adsorption of zinc to Scenedesmus subspicatus, measured (points) and calculated with parameters obtained by linear regression according to van den Berg [21] (Zn max mol Cu/g algae, log K Znads 6.49, ph 7.9). ( M). Bates et al. [20] also showed that high concentrations of zinc ( M) did not alter the division rate of Chlamydomonas variabilis. Studies on the growth of marine algae indicated that a pzn of 8 is toxic to several species, whereas a pzn of 11 is limiting [6]. The studied marine algae were mainly diatoms, whereas the studied freshwater algae are green algae. Considering these differences, we suggest that the tolerance range of marine algae toward zinc (10 8 to M) is much smaller than the range observed for freshwater algae in this study ( to M). The determinations of the metal content of the algae confirm the hypothesis that copper and zinc uptake is related to the free and not to the total copper or zinc concentration in the culture medium [3,6]. In contrast to the specific growth rate of S. subspicatus, cellular metal content was a more sensitive physiological parameter in verifying this hypothesis. The alga S. subspicatus accumulated up to mol Cu/g algae intracellularly. Compared to the content at the lowest Cu 2 concentration in the medium, intracellular copper was higher by a factor of 1,000. The adsorption data showed that after 5 d of exposure at a pcu of 7, only a small fraction (20%) of the total cellular copper concentration was located on the surfaces, the main fraction (80%) being found in the cells. No limitation of growth occurred at the lowest intracellular content (2 to mol Cu/g algae). The cellular copper contents of cultures 1 and 2 were the same within experimental error, indicating that the algae became acclimatized to the growth medium. Even at the highest intracellular concentrations, algal growth was not affected. Thus, optimal growth is maintained over an intracellular content range of mol Cu/g algae to mol Cu/g algae. This tolerance suggests that cells regulate the intracellular Cu 2 concentration, presumably by immobilization of excess copper. Silverberg et al. [24] showed that a copper-tolerant strain of S. subspicatus detoxified the metal by forming intracellular complexes. Phytochelatin, an intracellular metal chelator, was found to be induced by copper in freshwater [25,26] and in marine algae [27]. Another detoxification mechanism in algae is the complexation of toxic metals by polyphosphate [28]. In our experiments growth was comparable when cultures were grown in HEPES- or phosphate-buffered medium, which indicates that phosphate may not play an important role in the detoxification of copper and zinc in the algae studied. Our results show that the main fraction of total cellular zinc was intracellular. In the pzn range of 9 to 13 the intracellular zinc concentration was nearly independent of the free Zn 2 concentration in the media. The content was only slightly increased by a factor of 5. Thus, S. subspicatus may regulate its intracellular zinc uptake in this pzn range. At pzn values from 9 to 5 the intracellular zinc content increased by a factor of 200 in comparison to the content at the lowest free Zn 2 concentration in the medium. Comparing this to the growth results, growth was limited by up to 90% when the intracellular concentration was below mol Zn/g algae, whereas a toxic effect occurred when the intracellular concentrations were higher than mol Zn/g algae. Between these intracellular concentrations optimal growth was maintained. In short-term experiments Bates et al. [29] showed that the main fraction of zinc was bound to the cell surfaces when algae were incubated for 10 min with radiolabeled zinc. Those and our results (K. Knauer, in preparation) indicate two different uptake processes, a fast adsorption process complete within a few minutes and a slow intracellular uptake. Converting the total cellular copper concentations to molar copper/carbon ratios by dividing them by the mean values of experimentally measured cellular carbon for the green alga S. subspicatus (0.041 mol C/g algae), cellular copper/carbon ratios of the freshwater alga S. subspicatus were comparable to data from marine algae [30]. At pcu values closed to environmental concentrations (pcu 13 to 13.91), we determined copper/carbon ratios of 0.96 to 3.4 mol Cu/mol C. Our results are consistent with those of Kiefer [31] for the freshwater species C. reinhardtii (pcu 14, 0.45 to 2.3 mol Cu/C mol). For Thalassiosira oceanica at a pcu of and for Emiliania huxleyi at a pcu of 14.02, similar ratios (Cu/C ratios of 1.67 and 2.02 mol Cu/mol C, respectively) were deter-

9 228 Environ. Toxicol. Chem. 16, 1997 K. Knauer et al. mined [30]. At environmentally relevant Zn 2 concentrations (pzn 8 to 9), zinc/carbon ratios ranged from 10 to 100 mol Zn/mol C for S. subspicatus. Similar results were determined for C. reinhardtii (pzn 10, 69 to 110 mol Zn/mol C) [31]. These data are in good agreement with zinc/carbon ratios (10 to 70 mol Zn/mol C) of marine algae at a pzn value of 9 [5]. Despite large interspecies variation, it is possible to consider similar nutritional requirements for the trace elements copper and zinc for freshwater and seawater algae. Reflecting the concentration of naturally available free Cu 2 and Zn 2 in lakes, the algae examined need more cellular zinc than copper for optimal growth [9,10]. Under conditions similar to lake water the zinc/copper ratio in S. subspicatus ranged from 10 to 40. The affinity of copper for algal surfaces is much higher than that of zinc in the experimental concentration range, logk Cu (11.06) log K Zn (6.49). These results agree with the known complexing tendency of these trace metals with regard to organic ligands. Other studies [31,32] showed similar K Cu adsorption equilibrium constants for C. reinhardtii. Furthermore, our results are supported by Mahan et al. [33], who showed that the affinity of copper was higher than of zinc for different algal species. The maximal adsorption capacity for copper on algal surfaces (Cu max mol/g algae) is lower than that for zinc (Zn max mol/g algae). These results correspond to those of Kiefer [31], who found a complexing capacity of Cu max mol Cu/g algae for C. reinhardtii at a ph 6.9, and Ting et al. [34], who found a saturation value of approx mol Zn/g algae for the adsorption of zinc to Chlorella vulgaris at ph 6.8. For the uptake of iron, Hudson et al. [35] observed around mol Fe/cell binding site on the surface of the diatom Thalassiosira weissflogii grown under iron limitation. We determined here the maximum binding sites for copper and zinc on the surfaces of S. subspicatus (Cu max mol/cell, and Zn max mol Zn/cell). Application to field data in lakes These results on freshwater algae enable us to better understand the interactions between the metal ions copper and zinc and algae in lakes. The results on the dependence of growth rates and of cellular contents of copper and zinc may be related to our observations on copper and zinc speciation and on metal content of lake algae. Our results also have implications for the role of algae in removing metal ions to the sediments. Very low free copper ion concentrations have been measured in Lake Greifen and in other lakes (10 14 to M) [9], which correspond to the lowest range that could be reached in the growth experiments (Figs. 1 and 2). At this very low range, growth limitation is possible for some sensitive species, as indicated by the decrease in growth rate of C. fusca. This very low range of Cu 2 concentrations may therefore have some influence on the competition among various algae species in lakes, favoring species able to grow at very low levels. It may be expected at these low levels that copper bound to the algae corresponds to the minimal nutritional requirements of copper. In the case of S. subspicatus, the cellular content at this level was 2 to mol Cu/g algae. Algae collected from Lake Greifen appeared to contain a somewhat higher level of copper, namely 1.6 to mol Cu/g algae [31]. The higher content in the lake algae may be due to differences among various algal species. In comparison to the total copper present in Lake Greifen (approx. 1 to M), only a relatively small amount of copper is contained in the algae in the water column, namely about 2 to M, with 1 to 2 mg algae/l (dry weight). Zinc is complexed to a much lesser extent than copper in Lake Greifen; Zn 2 concentrations range from about 1 to M. In comparison to the data given in Fig. 4 for the growth rates, these concentrations appear to be in an optimum range for the two algal species examined. With regard to the cellular content (Fig. 8), these Zn 2 concentrations are at the upper end of the range in which the cellular content is regulated within narrow limits. The corresponding levels in S. subspicatus are about 0.5 to mol/g algae. Very comparable results were observed in algae collected from the lake, namely 0.6 to mol Zn/g algae (K. Knauer, in preparation). Using these values and the same estimates as above for algae dry weight, the amount of zinc bound to algae in the water column is estimated to be 1 to M, which is close to the range of total concentrations (1 to M). The results for zinc obtained in the algal cultures are therefore consistent with the results from the lakes and indicate that at similar Zn 2 concentrations, various algal species regulate their cellular content of zinc to similar values. These results are also consistent with observations of the concentrations of zinc in the water column of lakes that are being depleted by algal productivity [36,37]. The results obtained in algal cultures provide new insights into the processes regulating the concentrations of metal ions in lakes and raise new questions concerning the possible role of copper as a limiting factor for certain algal species in eutrophic lakes. Acknowledgement We thank HanBin Xue for valuable discussions about speciation and Werner Stumm for critical review of the manuscript. REFERENCES 1. Sunda, W.G Trace metal interactions with marine phytoplankton. Biol. Oceanogr. 6: Sunda, W.G. and R.R.L. 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