The sample prepared a t 800 “C for a 0.3-h period contains small, randomly distributed crystals which exhibit disorder. On the other hand, the 800 “C preparation, which was heated for 26 h, is found to contain crystals which have undergone some annealing of the disorder. Although there has been some crystal growth associated with the prolonged heating, the major difference between these two preparations that can be detected by the techniques used in this study is the extent of disorder. Thus, the sulfation kinetics show the characteristic effect of disorder. The 680 “C, 60-h preparation contains large calcite crystals which, by X-ray examination, appear to be disordered. If it is assumed that the nature of the disorder in the larger crystals is similar to that in the smaller crystallites, then the major difference between the 800 “C, 0.3-h and the 680 “C, 60-h preparations would be in the crystal size (surface area). The available surface will depend on the presence of cracks, voids, and various defects within the larger crystals. However, cracks and voids will contribute to defect formation so that separation of the surface area and disorder factors in the kinetics is not readily accomplished. However, it is believed that some effect of the surface area on the kinetics of sulfation is being observed in Figure 12. The yield curves of the 680 O C , 60-h and 800 “C, 0.3-h preparations approach each other after a prolonged reaction period. This may be explained by the tendency of the larger calcite pseudocrystals to break up into randomly oriented crystals of smaller sizes as the heating a t 750 “C progresses. The curves of Figure 12 demonstrate a relationship between kinetic behavior and crystal size and disorder but they also suggest that morphological changes which affect the progress of the reaction are occurring during the reaction itself. A detailed kinetic-morphological study of the sulfation process is currently under way to determine the nature of the mechanisms involved.
Acknowledgment
I t is a pleasure to acknowledge the contributions by Mr. B. S. Tani, Analytical Chemistry Laboratory, who carried out much of the X-ray diffraction studies pertaining to the crystal sizes and disorder. The participation in the X-ray investigations by Miss Mary Kolar, student aide, is also gratefully acknowledged.
L i t e r a t u r e Cited ( 1 ) Yang, R. T., Cunningham, P. T., Wilson, W. I., Johnson, S. A., Adu. Chern. Ser., No. 139,149-157 (1975). (2) Cunningham, P. T., Holt, B. D., Hubble, B. R., Johnson, S. A., Siegel, S., Wilson, W. I., Cafasso, F. A., Burris, L., “Chemical Engineering Division Environmental Chemistry Annual Report”, pp 44-90, USERDA Report ANL-75-51, July 1974-June 1975. (3) Hubble, B. R., Siegel, S., Fuchs, L. H., Cunningham, P. T., “Chemical, Structural, and Morphological Studies of Dolomite in Sulfation and Regeneration Reactions”, Proceedings of the Fourth International Conference on Fluidized-Bed Combustion, pp 367-91, the MITRE Corporation, McLean, Va., May 1976. (4) Hubble, B. R., Siegel, S., Cunningham, P. T., J . Air Pollut. Control Assoc. 25, 1256 (1975). (5) Hubble, B. R., Siegel, S., Fuchs, L. H., Hoekstra, H. R., Tani, B. S., Cunningham, P. T., ibid., 27,343 (1977). (6) Haul, R. A. W., Wilsdorf, H. G. F., Nature (London), 167,945 (1951). (7) Haul, R. A. W., Wilsdorf, H., Acta Crystallogr., 5,250 (1952). ( 8 ) Harvey, R. D., Steinmetz, J. C., Enuiron. Geol. Notes, Ill. State Geol. Suru , 50, (1971). (9) Borgwardt, R. H., Harvey, R. D., Enuiron. Sci. Technol., 6,350 (1972). (10) O’Neill, E. P., Keairns, D. L., Kittle, W. F., Thermochim. Acta, 14, 209 (1976).
Received for review October 31, 1977. Accepted July 18, 1978. This work was performed under the auspices of the U.S.Department of Energy.
Regulation of Copper Availability to Phytoplankton by Macromolecules in Lake Water Rene Gachter *, Joan S. Davis, and Antonin Mares Federal Institute for Water Resources and Water Pollution Control, CH-8600 Dubendorf, Switzerland
Approximately two-thirds of the copper in lake water remains in the nonfilterable residue of the following two-step procedure: 0.45 pm filtration followed by Diaflo UM-2 membrane (passing mol w t 1000) ultrafiltration. Examination of the effect of this residual copper on the photosynthesis rate of natural phytoplankton has shown that this residual copper is physiologically not available to the organisms. This fact can be utilized in a bioassay (as demonstrated with EDTA as a test substance) to gain information on the concentration of a ligand and its apparent association constant with copper. I t is concluded that nonultrafilterable ligands present in lake water form copper complexes as stable as the CuEDTA complex and can thus play an ecologically significant role in the regulation of copper availability and therefore its toxicity to phytoplankton. Organic ligands may play a significant role in the regulation of phytoplankton growth. The observed growth stimulation in their presence is usually considered to be the result of an 1416
Environmental Science & Technology
increase in the availability of required trace metals due to complex formation ( I ) . However, on the basis of observations made with copper, the complexation could as well lower the availability of toxic substances which then would also explain the increased photosynthesis. Strong organic chelators such as NTA ( 2 , 3 ) ,EDTA (4-7), extracellular polypeptides, or a zooplankton extract (8)are known to partially or completely counteract copper toxicity. Pagenkopf et al. (9)and Andrew et al. ( I O ) have indicated that by increasing the bicarbonate alkalinity or ortho- or pyrophosphate concentration the toxicity is reduced by the formation of nontoxic inorganic complexes. An addition of FeC13, which in alkaline solution forms negatively charged colloids capable of binding copper, also has the same detoxifying effect (7). To what extent in nature lake organics complex copper, and, therefore, the ecological significance of this effect, are as yet unknown. Sunda ( 1 1 ) and Bundi ( 1 2 ) have shown that in a chemical-thermodynamic model an algal surface can be treated as a ligand having a certain complex formation capacity (Atot) and a certain complex-formation constant ( K ) .Thus, we can assume, as a first approximation, that copper poisoning is due 0013-936X/78/0912-1416$01 .OO/O
@ 1978 American Chemical Society
to the formation of copper complexes a t important physiologically active sites, and that these complexes (CuA) are in chemical equilibrium with the copper aquo species (Cuaq2+) present in the experimental medium: K[Cu*,2+1 [Atotl 1 K[Cu,,2+] Since the physiological response of organisms to increased copper concentrations depends on the concentration of CuA, copper toxicity obviously cannot be correlated to total copper concentration in the medium, but rather to the concentration of the Cuaq2+species in equilibrium with the organisms. The works of Pagenkopf e t al. ( 9 ) ,Andrew et al. ( I O ) , and Anderson and Morel ( 1 3 ) demonstrate this relationship. In natural waters, copper forms various hydroxo and carbonato complexes; thus, only a fraction of the total copper exists as the copper aquo complex (14). In addition, several facts point indirectly to the possibility that apart from carbonato and hydroxo complexes in natural lake waters, organic copper complexes or surface complexes with inorganic colloids also have to be considered. For example, in undigested samples, part of the dissolved copper can neither be recovered by the diethyldithiocarbaminate method ( 1 5 ) nor by anodic stripping voltametry (16), nor can it be dialyzed (17). In lake water a significant fraction of the so-called dissolved organic material is probably present in macromolecular or colloidal form. Allen (18), Wheeler (191, and Gjessing (20) have shown that up to more than 70% of the dissolved organic carbon can be associated with molecules having a nominal molecular weight of more than 1000. If these macromolecules or colloids form stable copper complexes, then it follows that in natural lake water a significant fraction of the total copper also should be present in macromolecular form. Based on the above observations, this study was initiated with the threefold purpose: (a) to measure what fraction of the total dissolved copper is retained by a n Amicon UM-2 membrane; (b) to investigate to what extent nonultrafilterable ligands modify the availability of copper to phytoplankton; and (c) to describe the observed interactions between copper and these substances on a chemical-thermodynamic basis. [CuA] =
PRESSURE
TCF 10 SYSTEM
+
Met hods Membrane-filtered (0.45 wm) subsurface samples from the lake of Alpnach, a eutrophic alpine lake, were filtered through an Amicon Diaflo UM-2 membrane, with a cut-off limit for molecules with a mol wt of 1000. The Diaflo membranes were mounted on an Amicon T C F 10 thin channel system. The filtration pressure was 2 bars, giving a flow rate of about 1 mL/min. In this way nonultrafilterable substances dissolved in lake water were concentrated. In order to determine the complexing capacity and an average conditional complex formation constant of these substances, this residue was again diluted with the ultrafiltrate to a total copper concentration of low7M. (The [CuItot of original lake water and its ultrafiltrate were about 2 x 10-8 and 5 X M, respectively.) Then the ultrafiltrate as well as the ultrafiltrate enriched with nonultrafilterable ligands were spiked with varying amounts of copper sulfate. In order to reduce the risk of copper adsorption onto the glass, all glassware was rinsed several times and equilibrated overnight with the appropriate test solution. The following morning, these solutions were discarded and freshly prepared test solutions were filled in the preequilibrated 125-mL glass bottles. In order to reduce the risk of sudden exposure of the algae to ionic copper not yet equilibrated with the natural ligands, these solutions were equilibrated for 2 h before inoculation with natural phytoplankton (collected with a plankton net, mesh size 30 wm). After the addition of I4C-labeled sodium bicarbonate, the inoculated samples were exposed for 24 h in
LAKE WATER
ULTRAFILTRATE
Figure 1. Principle of ultrafiltration Copper species which pass the membrane. 0 Ligands and macromolecular which are retained by the membrane and therefore copper complexes (0.) become concentrated in the residue
an incubator a t the temperature of the lake water. Experiments were conducted at the natural pH of 8.1 if not otherwise indicated. Since the lake water is well buffered (alkalinity: 2 mval/L) and phytoplankton density was kept low, the pH did not change during the experiments. Calcium concentration was 1 X lop3 M. Primary production was measured as described by Schindler et al. (21).Phytoplankton concentration was kept low in order to avoid any detectable change in the concentration of total dissolved copper due to sorption. Thus, it might be concluded that the added phytoplankton did not influence the speciation of the dissolved copper. Copper concentrations were measured either with a Perkin-Elmer Model 400 atomic absorption spectrophotometer equipped with a graphite atomizer HGA-74 (if nothing else is indicated) or where indicated with the DDTC method described by Strickland and Parsons (15). Carbonate alkalinity was determined by titrating 100 mL of water with 0.1 N HC1, using methyl orange as an indicator. I t is expressed in milliequivalents per liter (mval/L) (1mval/L corresponds to 50 ppm of CaC03).
Results and Discussion Figure 1 illustrates the principle of the concentration of macromolecular copper species by ultrafiltration. Whereas most small species pass the membrane, larger ligands and complexes are retained and become concentrated in the residue. With respect to the residue, the following equation is valid for any 1:l complex:
[L], [CuL], and [Cuaq2+]denote the concentrations of the nonfilterable ligand, the copper complex, and the copper aquo ion, respectively. Since the concentration of the copper aquo ion is assumed not to be affected by the filtration process, it follows from Equation 2 that ultrafiltration does not change the ratio [CuL]/[L]. Ultrafiltration thus is considered to induce neither complex formation nor complex dissociation, as long as polymerization and microbial degradation of dissolved ligands can be neglected. As a result, during the concentration process, the total concentration of nonfilterable complexes ([CuL]) in the residue increases in proportion to the volume of the water filtered (Vfid:
3
( +-
[CUL] = [CULIO 1
(3)
where [CuL]o stands for the original concentration in the natural lake water. The total copper concentration in the residue is then: Volume 12, Number 13, December 1978
1417
Table 1. Estimation of Ultrafilterable ( [ C U ] ~and ) Nonultrafilterable Fractions ([CuLl0) of Total “Dissolved” Copper in Lake Water a date of collection
[ c u e
lake
[Cub
[CuLlo
[Cut~tlo
A 0.6 1.4 2.0 70 6/21/75 10/13/75 L 0.7 1.4 2.1 67 A 0.7 1.3 2.0 65 9/29/76 a Ultrafilter, Amicon UM2 membrane; A, Lake Alpnach L, Lake Lucerne;the concentrations are M. Copper concentrations were measured with the DDTC method( 15). cy,,,]^ = 100%.
[Cut,,]
in
MI
lake water+ EDTA
Figure 2. (a) Effect of EDTA on the inhibition of photosynthesis by copper. The X intercepts of the dotted lines give isotoxic concentrations of the two media. In this way the points were determined for graph b. (b) lsotoxic (with respect to inhibition of photosynthesis)copper conM centrations in lake water and lake water enriched with EDTA The right scale of the yaxis indicates the [Cu2+] in the isotoxic solutions. [Cu2+] = 2.56 X 10-3[CuL,] (pH 8.1; alkalinity 2 mval/L; [Ca2+] = M)
i
2)
[Cultot = [CULIO 1 + -
+ [CUI0
(4)
where [CUI, is the total concentration of all filterable species. Rearrangement of Equation 4 yields: [Cultot = [Cutotlo + [CULIO
(””’)
(5)
vres
From Equation 5 [ C U L ]can ~ be estimated if the total copper concentrations in the residue and in the original lake water are known. Table I indicates that up to 70% of total dissolved copper is retained by the Diaflo UM-2 membrane. It is well known that an increase in copper concentration in the nutrient medium can depress the phytoplankton photosynthesis. The extent of this decrease is related to the amount of copper absorbed by the algae, [CuA], assumed to be in equilibrium with the different copper species in solution. Thus, if a test medium differs from the control medium only by the presence of one or more unknown ligands which form physiologically nonavailable copper complexes (but otherwise not interfering with photosynthesis), then the observed differences in the decrease of photosynthesis in the two media give information regarding the complexation abilities of the unknown ligands. This is because if in two media copper causes an equal decrease of photosynthesis, then the amount of copper associated with the algae can be assumed to be equal. Equal concentrations of CuA necessarily require equal concentrations of all copper species present in both media. If, therefore, in two media different total copper concentrations exhibit the same effect on photosynthesis, then this difference must be due to the presence of one or more physiologically nonavailable copper complexes (CuL), present only in one of the media. Figures 2a, 3, 6, and 7 show that the slope of the curves representing photosynthesis as a function of [Cu]totdecreases 1418
Environmental Science & Technology
with increasing [Cu]tot.Thus, the estimation of [CuL] as the difference between the two curves becomes more inaccurate the higher the total copper concentration becomes. For this reason in our experiments we did not exceed total copper M. concentrations above 3 X A control experiment with EDTA as a test ligand was carried out. Filtered lake water, and filtered lake water enriched M EDTA, both spiked with varying amounts of with copper sulfate were inoculated with natural phytoplankton. If the added EDTA did not affect copper toxicity, then the two lines in Figure 2a would become identical and thus the points in Figure 2b would follow the dotted 1:l line. However, as shown in Figure 2, the detoxifying effect of EDTA is evident. Since, as mentioned above, equal toxic effects require equal concentrations of all copper species present in both media (CuL,), and in addition the concentration of the copper adsorbed by the algae (CuA) is negligibly small, [CuL,] and [CuEDTA] can be deduced from the experiment as shown in Figure 2b, e.g., [Cubt] in the medium “lake water EDTA” equals [CuL,] [CuEDTA]. With increasing [CuItotthe curve in Figure 2b becomes parallel to the 1:l line, indicating that above a total copper concentration of 2 X M practically all added EDTA is complexed by copper. Isotoxic total copper concentrations differed a t the most by approximately 1.05 x M, which is very close to the EDTA concentration used. In order to estimate an upper limit of the concentration of the copper aquo ion, we assume that [CuL,] = [Cuaq2+] [CuOH+] [Cu(OH)zO] [ C U ( C O ~ ) ~ ~ -[ ]C U C O ~ ~This ]. assumption, intended as a first approximation, does not consider a possible complexation of copper by lake organics. Using the computer program developed by Perrin and Agarwal(22) and stability constants from the compilations of Sillen and Martell (23), the equilibrium calculation shows that a t pH 8.1 and alkalinity of 2 mval/L the copper aquo ion conX [CuL,]. centration is equal to 2.56 X Thus, the experiment allows the estimation of the equilibrium concentration of the two species Cuaqz+and CuEDTA as a function of total copper concentration and, therefore, the determination of a conditional complex formation constant K * , which is defined as:
+
+
+
+
+
+
[CuEDTA] (6) K*C~EDTA = [C~aq~+]([EDTAlrnt - [CuEDTA]) From Table I1 it can be seen that the experimentally determined value of K*C~EDTA is of the order of magnitude of This is in excellent agreement with the result derived from computer calculation (lolo), which includes the inorganic copper species mentioned above and the organic species CUEDTA,CaEDTA, HEDTA, and H2EDTA. The experiment with EDTA thus confirms that the measurement of the copper-induced decrease of photosynthesis is a useful tool for estimking in lake water the total concentration of an additional ligand forming physiologically unavailable copper complexes and its conditional complex formation constant.
Table II. Estimation of Conditional Complex Formation Constant K’ for CuEDTA Complex a 10-7. 10-7. Cutot
10-7. [CuLi]
10-7. [CUEDTA]
7.2 9.7 11.7
1 2 3
6.2 7.7 8.7
:;0 1 [CU
([EDTAhot [CuEDTA])
]
4.3 2.8 1.8
2.56 5.12 7.68
K‘
109.8 109.7 109.8
M; alkalinity 2 mval/L; [CaZf] = lo3 M; pH
[EDTAItat = 1.05 X 8.1.
AL-A-A-A-
macromolecular Cu- complexes I
5
0
[cu,,,]
(
IO mole/l i
Figure 3. Effect of increased total copper concentration on phytoplankton photosynthesis Media: ultrafilteredlake water enriched with (a) macromoiecular copper complexes and (b) with CuS04(pH 8.1; alkalinity 2 mval/L; [Ca2+] = M)
Since ultrafiltration does not change the concentration of the ultrafilterable copper-aquo ion in the residue, an addition of the copper concentrate, obtained by ultrafiltration, to the ultrafiltered lake water increases its total copper concentra-
tion but has no effect on the concentration of the copper-aquo ion. The physiological experiment shows (Figure 3) that in contrast t o copper sulfate the naturally occurring nonultrafilterable copper compounds do not inhibit phytoplankton photosynthesis. Figure 4a shows in analogy to Figure 2b in two separate experiments the detoxifying effect of naturally occurring ligands. In both experiments the two test media were ultrafiltered lake water (UF) and ultrafiltered lake water enriched with ligands, retained in the residue during ultrafiltration. From Table I it can be deduced that the copper concentration in the ultrafiltrate was on the order of 5 X 10-9 M. The addition of residual copper to the ultrafiltrates increased their [Cu]totto lo-’ M. On top of these initial concentrations, [CuItot was further increased in both media by the addition of CuSO4. The strong deviation from the 1:1line, representing equal concentrations in the two media, clearly indicates the detoxifying effect of the M the macromolecular ligands. Up to [CuItotof about 3 X curve does not become parallel to the 1:l line, as it did in the experiment with EDTA. This shows that up to this [CuItotthe nonultrafilterable ligands were not completely complexed by copper. The experiment with EDTA as a test ligand has shown that the differences between isotoxic concentrations in the two media must be due to the presence of physiologically nonavailable copper complexes (CuL). Furthermore, it has shown that in unaltered lake water it is a reasonable approximation to consider only the carbonato and hydroxo complexes and to neglect other species. This indicates that a t the total copper concentrations used in the experiment, the Cu2+concentration has not been substantially influenced by other than inorganic ligands. This of course also holds true, if as done in this experiment, lake water is replaced by ultrafiltered lake water. Again, it can thus be assumed that [Cu2+]equals 2.56 X [CULLI.
EXPERIMENT 2
EXPERIMENT 1 M
10-9
10-7 M
M
-
[Cut,,]
ultrafiltrate t residue
(
a
12
Mi
.\/
/
/
IO-9~
/
1:
‘7
[Cut,,]
ultrafiltrate t residue ( i O - 7 M )
‘Or
o+
I
, t
I
1
2
3
[ C U , ~ ~ ~(10-9 ] MI
Figure 4. (a) lsotoxic copper concentrations (with respect to inhibition of photosynthesis) in ultrafiltered lake water (UF) and in ultrafiltered lake water enriched with macromolecular ligands (for further explanation see Figure 2).(b) Equilibrium concentration between [Cuaq2+] and two postulated complexes [CULI] and [CuL?] The value pairs ( 0 )are determined from Figure 4a. The lines represent the values obtained with the Perrin computer program for various [ L z ] ~7~X~ : M 1.4 X M (- -), and 2.1 X M (-) in experiment 1 and 6 X M .), 1.2 X M (- -), and 1.8 X M (-) in experiment 2. [L,],,, is 2.5 X and 7 X M in experiments 1 and 2, respectively; pH 8.1; alkalinity 2 mval/L; [Ca2+] = 2 X M
-
(e.
(a
-
Volume 12,Number 13,December 1978
a).
1419
m
’Figure 5. Influence of macromolecular compounds on the relationship between total copper and cupric ion activity M pH 8.1; alkalinity 2 mval/L; [CaZ+] =
In Figure 4b equilibrium concentrations of the physiologically nonavailable copper complexes CuL and the CuaqZ+ion are plotted. At higher concentrations CuL seems to be nearly linearly related to Cu2+.The fact that the extrapolated linear regression line does not pass through the origin but cuts the y-axis in the positive range indicates that a t least two groups of nonultrafilterable ligands have to be considered, whereas [Ll]t,t > Kz* (K* = conditional complex formation constant valid for alkalinity 2 mvalb, [Ca2+]= M, and pH 8.1). The complexing capacity of the L1 group can directly be deduced from the intercept with the y axis. In experiments 1 and 2 [L1ltot thus equals 2.5 X M and 7.0 X M, respectively. In order to estimate the average conditional complex formation constant of the CuL1-type complex, it is assumed that prior to the addition of CuS04 practically all the macromolecular complexed copper was present as the CuLl complex. [CuL1] therefore was about 10-7 M, and the concentration of the Cuaq2+ion has been estimated as 2.56 X 10-3 X 5 X 10-9 M = 1.3 X 10-11 M. Inserting these values in Equation 7 : (7) yields complex formation constants for the CuL1-type complexes of 1010.7and 1OlO.l in experiments 1and 2, respectively. Although the CuZ+ ion activity is only an estimate based on the results shown in Table I, a possible inaccuracy here would not change the order of magnitude of these conditional complex formation constants. From Figure 4a it was concluded that not all macromolecular ligands were complexed a t the maximum copper concentration used in the experiment. Thus, obviously, the concentration [Lz] cannot be determined exactly from such experiments. Nevertheless, an estimate is possible. Since K2*
