Influence of Copper and Zinc on the Growth of a Freshwater Alga, Scenedesmus quadricauda : The Significance of Chemical Speciation Richard Petersen Biology Department, Portland State University, Portland, Oregon 97201
rn Scenedesmus quadricauda was grown in a defined medium with a wide range of copper and zinc concentrations. The growth rate of the alga was influenced by the concentration of each of the two metal ions, Cua? and Znaq2+.The growth rate of the alga can be predicted (eq 5) from the concentrations of the two metal ions. These data indicate that zinc is an essential trace metal for Scenedesmus quadricauda and that copper and zinc are toxic when present in excess. The results suggest also that the free metal ion is the chemical form of each metal that is significant in controlling biological effects (both nutrition and toxicity). The expression (4) used to describe these results is a special case of a general expression that may be used to describe the combined effect on algal growth of any number of metals in solution, including a consideration of the chemical speciation of each metal. Introduction The toxicities of copper and zinc to aquatic organisms have been examined extensively. I t has been shown that each of these metals is toxic to a wide variety of aquatic organisms including phytoplankton (1-3), zooplankton (4-6), and fish (7,8).There have also been studies of the effect of having elevated concentrations of both metals present at the same time (9-12) as well as efforts to synthesize this information in a systematic fashion (13,14). In general, the joint action of any two or more metals has usually been described as “additive” with respect to the total concentration of the metals (14). That is, toxicity is commonly observed when the sum of the total concentrations of all of the toxic metals reaches a certain value. Because of large differences in the chemical composition of the natural water or nutrient medium used in such studies, it has not been poesible to directly compare results. However, it has recently been shown that the chemical speciation of a single toxic metal is important in determining toxic effects and that a consideration of speciation allows more direct comparison of the results from different natural waters or nutrient media (15-18). The applicability of the concept of metal speciation to studies of the joint action of two or more metals has not been examined. The work reported here has been designed to address this question over a wide range of copper and zinc concentrations. A freshwater alga, Scenedesmus quadricauda, was grown in a defined nutrient medium, to which copper sulfate, zinc sulfate, or both were added. The growth rate of the alga was determined as a function of the amount of each of these metals present in the medium. 00 13-938X/82/09 16-0443$0 1.2510
Growth data were analyzed in terms of total concentration of the metals as well as in terms of the calculated chemical speciation of the metals. A general model is proposed that succinctly accounts for the observed biological effects as a function of the chemical speciation of both metals. The model would allow for the comparison of the combined action of two or more metals in different natural waters or culture media and suggests why two metals would often appear to be ”additive” in their effect, if only the total concentrations of the metals are considered. Methods Inoculum Preparation. Scenedesmus quadricauda (culture 11, Freshwater Institute algal culture collection, Freshwater Institute, Environment Canada, Winnipeg, Canada) was used as a test organism. One drop of culture medium containing cells from a stationary phase culture was inoculated into 50 mL of the nutrient medium Fraquil (19),in a 250-mL glass Erlenmeyer flask. After 7 days, 1.00 mL of this culture was inoculated into 100 mL of nutrient medium and incubated for 4 days. Each flask in a bioassay experiment was inoculated with 1.00 mL from this 4day-old culture, giving an initial cell density of 2-8 X 1Q2 cells/mL. This procedure ensured that the inoculum used in all the bioassay experiments was in log phase growth, and no “lag phase” was evident in any of the bioassay experiments. Culture Conditions. Each bioassay culture was grown for 5 days. Cell densities were determined immediately after inoculation and at the same time of day on each of the following 4 days with a Coulter Counter Model B particle counter with a 1Wpm aperture tube. The cultures were grown in a Psycrotherm incubator (New Brunswick Scientific Co.) at 20 “C at a light intensity of approximately 90 peinsteins/ (m2s) (continuous illumination) and were shaken at 100 rpm. Culture Medium. The medium used in these experiments was based on Fraquil (19). The chemical composition of the medium is presented in Table I. Departures from standard Fraquil include the addition of 1.00 X lo-’ M H3B03,the addition of 1.8 X 10” M KC1, the removal of all
[email protected] (1.25 X 10” M in standard Fraquil), and a 10-fold reduction in the concentration of K2HP04 (from 10” to 10* M). The latter two changes were necessary to avoid the precipitation of zinc minerals. KCl was added to replace the potassium in K2HP0,, and H3B03was added because it is thought that Scenedesmus requires boron.
0 1982 American Chemical Society
Environ. Scl. Technol., Vol. 16, No. 8, 1982
443
Table I. Modified Fraquil Culture Medium chemical chemical compoconcn, compoconcn, nenta mol/L nent mol/L Caz+ 2.5 X C0:1.5 x 10-4 1.5 x 10-4 Mg2+ 1.5 x K+ 2.0 x 10-5 ci5.2 x 10-4 Na+ 2.6 x P O : 1.0 x lo-$ B(OH),1.0x 10-7 Fe3+ 4.5 x Mnz+ 2.3x lo-* MOO:1.5 x 10-9 CO~+ 2 . 5 ~10-9 NO,1.0x 10-4 HtC 1 . 6 ~ EDTA4-d 5.0 x CIA'+,Zn2+ variable 1.3 x NH, a Chemical components are defined as in ref 20. The components represent the total stoichiometric amount of each element or ion added to the medium in the form of reagent grade salt, not the actual or estimated concentration of the chemical species in the final medium. The list of components and their concentrations uniquely defines the chemical speciation to be expected in the medium, Cu2+and Znz were added to the medium as sulfate salts. Accordingly, the amount of SO-: was slightly higher in culture flasks receiving Cuz+ or Znz+ treatments. Observed pH ranged from 6.7 to 7.3. The calculated pH EDTA = ethylenedia(according t o MINEQL)was 7.3. minetetraacetate. +
The major ion solutions and macronutrient solutions were passed through a column of Chelex 100 (Bio-Rad Laboratories) to remove trace-metal contaminants (19). All experimental solutions were allowed 48 h to reach chemical equilibrium before being inoculated with Scenedesmus. The water used throughout these experiments was obtained fresh from a Millipore Super Q system. Chemical Speciation. The chemical speciation of the medium, including each of the experimental manipulations, was estimated with the computer program MINEQL (20). Chemical Treatments. Each bioassay employed a fixed amount of metal buffering ligand (5 X lo* M EDTA). Eight different bioassays were carried out in which there was a fixed amount of either ZnSO, or CuSO1. With one of the two metals fixed, the concentration of the second was varied over a range of concentrations of interest. Three additional bioassays were carried out in which both CuSO, and ZnSO, were varied. For all bioassays, each treatment was run in duplicate. Seven of the bioassays were carried out in 250-mL Teflon Erlenmeyer flasks, and four of the bioassays were carried out in 250-mL glass Erlenmeyer flasks. The apparent total concentrations of copper and zinc were measured by flameless atomic absorption spectroscopy before and after each experiment. Estimation of Growth Rates. The number of cells of Scenedesmus quadricauda in each culture flask increased exponentially during the course of the bioassays. The rate of increase, or growth rate, was estimated for each culture by fitting a least-squares linear regression to the data. The growth rate of each culture serves as a measure of the impact of the added metals on the growth of the alga. Results and Discussion Effect of Added Copper. Five separate bioassays were carried out in which the total amount of ZnS04 added was the same in all flasks but in which the total amount of CuSO, was varied. The data from these bioassays clearly indicate that the growth rate of the alga is slowed when excess CuSO, is added to the medium and that the amount of CuSO, required to slow the growth rate is to some extent 444
Environ. Sci. Technot., Vol. 16, No. 8, 1982
w
l
EC-50
I
13
\
12
I I
18
8
9
7
, 6
PCU Figure 1. The growth rate of Scenedesmus qua&icaUds over a range of concentrations of free copper ion, Cu A M concentration of Cu reduces the growth rate bylalf (Le., EC-50 = M Cu,/f,
'+
'+.
Table 11. Concentration of Copper and Zinc Which in Combination Produces a 50% Reduction in Growth Rate (Le., EC,) bio- metal as- held say con- total Cu, total Zn, M PCU M PZn no, stant 8.82 4.0 X lom9 9.88 T-3 Zn 4.5 X lo-' 5.13 T-5 Cu 1.0 X 12.12 10.5 X T-6 Zn 2.1 X lo-' 3.0 X 6.17 8.47 9.46 8.6 x lo-' 5.48 T-8 Cu 1.0 x G-1 Zn 4.5 X 0.5 X 6.94 8.08 G-2 Zn 2.8 X lo-' 9.06 2.0 X lo-' 7.01 G-3 Z n 1.9 X 4.0 X 5.73 8.25 G-4 CU 2.0 X lo-' 9.20 3.1 X lo-' 6.81
related to the amount of ZnSO, already present in the medium. The results of one of these bioassays is presented in Figure 1. For this bioassay, the total amount of zinc sulfate was 4 X lo4 M, and the amount of copper added varied from 0 to 5.5 X lo* M. The concentration of cupric ion, CuqZ', present in each culture was estimated by using the computer program MINEQL(20),which calculated the expected chemical speciation of all the chemical components of the medium. The growth rates are presented as a function of these calculated concentrations of cupric ion. The abscissa of Figure 1is presented in units of pCu (15), defined by p c u = -log [CU,2+]
(1)
A least-squares regression line has been fitted to all the values which show reduced growth rate due to metal toxicity. The pCu value that corresponds to a growth rate equal to half the maximum growth rate is indicated in the figure and labeled as EC-50. Total copper, total zinc, pCu, and pZn corresponding to a 50% reduction in growth rate for each bioassay are presented in Table 11, for the five bioassays in which the total amount of zinc was held constant and the three bioassays for which total copper was held constant. Effect of Added Zinc. The results of one of the three bioassays in which the total copper was held constant and the total zinc varied are presented in Figure 2. For this bioassay, total copper was l0-g M, and zinc was varied from 0 to 4 X 10" M. The results of added zinc are similar to added copper. When the concentration of ionic zinc is sufficiently high, the growth rate is reduced. Joint Action of Copper and Zinc. The combination of copper and zinc that reduces growth is tabulated in Table 11. A complication in interpreting the results of
I
Q
EC-50
1 II
3 7
8
1 8 9
5
6
4
PZn Flgure 2. The growth rate of Scsnedesmus quadricat& over a range M concentration of concentrations of free zinc ion, ha:+.A reduces the growth rate by half ( l a , EC-50 = M of Zn Zna:8).
+:
,
1 2 1 1
1 8 9
,
8
7
.
,
6
5
I 4
PZn Flgure 3. The combinations of copper ion and zinc ion that reduce growth by half. Each point represents the concentrations of copper and zinc ions at which growth rate was reduced by half for a particular bioassay.
these bioassays results from the competition of the two metals for the complexing ligand. Although the total concentration of one of the metals is held constant, the concentration of the ionic species of that metal (and for that matter, all the other trace metals in the medium) increases as the second metal is added. That is, when total zinc is held constant and copper is added
+
m l
’\
piin5.1
Cu2+ ZnEDTA2- a Zn2++ CuEDTA2(2) so that both Cu,? and Zna? increase when CuSOl is added to the growth medium. Nonetheless, for some of the bioassays, the calculated concentrations of one of the metals is so low that the depression in growth rate may be safely assumed to be solely the result of the other metal. For bioassay T-3 (Table 11), total zinc was 4 X lo* M, and the highest zinc ion concentration was lo*.@ M, much less than the amount of ionic zinc required to inhibit growth when the total copper in the medium is very low. Based on this bioassay, then, the concentration of cupric ion that reduces the growth rate by half is [Cu!?] = 10-8.8 M, or pCu = 8.8. Similarly, the data from bioassay T-5 can be used to estimate the zinc ion concentration that by itself will reduce growth rate by half. For this bioassay, the maximum concentration of cupric ion was 10-11.62M, less than the amount required to produce a toxic effect due to copper alone. Based on bioassay T-5,the concentration of zinc required to reduce growth rate by half is [Znaq2+] = lo”.’ M, or pZn = 5.1. The results of the remaining six bioassays can be compared to these two, as is shown in Figure 3. It appears, based on this analysis, that there is no clear evidence of interaction between copper ion and zinc ion in producing toxicity. Under most circumstances, copper ion appears to be responsible for producing toxicity. This is in spite
0
5
0
. m 10
15
MICROMOLES ZINC Flgure 4. The combinations of added copper and zinc that reduce growth by half. The axes represent the total amount of copper and zinc added without regard to the chemical speciation in the culture medium.
of the greater affinity of the complexing ligand for copper and is a reflection of the much greater sensitivity of the alga to cupric ion, (EC-50 = 10-8.8M), than to zinc ion (EC-50 = lo”.’ M). It is particularly of interest that bioassay T-8 indicates that the observed reduction in growth may be related to the increase in the cupric ion resulting from displacement of Cusp from CuEDTA%by the addition of ZnS04. Toxicity as a Function of Total Copper and Zinc. The data presented in Table I1 can also be used to present the apparent pattern of toxicity as a function of total copper and total zinc (Figure 4), as is usually done in trace-metal bioassays involving aquatic organisms and more than one metal. As is evident, when the data are presented in this fashion, it appears that the two metals are not acting independently but “additively” (14). A combination of total zinc and total copper that adds to 5 pM will reduce the growth rate by 50%. However, this more traditional view does not explain why more than 5 pM total zinc is required to slow the growth of the alga if copper is absent from the medium. If the chemical speciation of the medium is considered, it is evident that the apparent additivity of the two metals is probably related to the competition between the two metal ions for the complexing ligands and not to a direct biological effect. An Alternate Model. Because of the ambiguity involved in attempts to attribute apparent toxicity to only the metal added, whereas in fact the concentration of each metal ion is changing, a model is here proposed that simultaneously makes use of all the data from all the bioassays. The model proposed (21) is given in eq 3, where
= growth rate, pmax= maximum growth rate, [Zna92+] = concentration of zinc ion, KuptskeZn= a half-saturation constant for the uptake of zinc as a required micronutrient, Lt = the total concentration of a hypothetical cell-bound “ligand” which is essential to the growth of the organism and which may be inactivated by copper or zinc ions when these are present in excess, and Lf = the “free” ligand, or the amount of the hypothetical ligand not complexed by toxic metal. Furthermore, we may suppose that the tendency of the two metals to inactivate the ligand may be described as a reversible complexation reaction, that is p
CU~? + L Zn,,Z+
CUL
[CUL] = Khx,c”[c~a,Z+][L]
+ L a ZnL
[ZnL] = Kbx,z”[Znaq2+I[L]
F?
where KbxiPis the formation constant for the complex formed between copper and the hypothetical ligand, and Environ. Sci. Technoi., Vol. 18, No. 8, 1982
445
Table 111. Estimated Values of the Parameters of the Proposed Model Pmax
Teflon only glass only
KuptakeZn
1.00 6.87 x 1.30 1.26 x
lo-''
KtoxicZn
ot.338
m
KtoxicCU
0+.241
9.47 x lo4 9.73 x l o 8 1.00 x l o 6 1.00 X lo9
0 t%;0078
0+.007 0+.047
0+.%?3
Kbicz" is the formation constant for the complex formed between zinc and the ligand. Thus W L t = (Lf)/(Lf + [CuLl + [ZnLI)
L f / L , = (1 + Ktori?[Cuaq2+]
By substitution, the model becomes
(1+ KbicCu[Cuaq2+]
1 .
+ Kbxi?[Znaq2+])-l
7
5
6
PZn Flgure 5. The difference between the observed growth rate and the growth rate predicted by the suggested model for cultures employing concentrations of copper ion C ()U ':, and zinc ion (Zn,?') approximately equal to the amounts expected to reduce growth rate by half.
+ Kbxi?[Znaq2+])-l (4)
This model assumes that both metal ions can contribute
to toxicity when present in excess. In this study, there was evidence of growth limitation due to zinc deficiency, when the concentration of zinc was very low, and therefore the model also includes the assumption that some zinc is required for growth. There was no evidence, in this study, of growth limitation due to a lack of copper or any other trace metal. Data are available for a total of 159 cultures where p, [ZnaF],and [Cu,?] are known so that the remaining parameters p-, Kupezn, KtoLic zn, and Ktoxiccu may be estimated by a nonlinear least-squares regression (nonlinear regression, SAS Institute, Inc., Raleigh, NC). Results of the Model. The data were analyzed with this model separately according to whether the cultures were carried out in Teflon or glass Erlenmeyer flasks. (There was some evidence that p.- was lower in the Teflon flasks, probably due to light limitation, as the Teflon flasks were not as transparent as the glass flasks.) The results of the analysis are presented in Table 111. The data from the bioassays carried out in glass containers are not very useful for determining the value of KuptakF because the treatments all employed relatively high concentrations of added ZnS04. The data from the bioassays carried out in Teflon containers are based on a wider range of added copper and zinc and are discussed further here. The toxicity results from the glass containers do not differ significantly. The results of analyzing the data with the proposed model suggest that growth-rate limitation due to insufficient zinc may be expected when [Zn,?] lo* M and that growth-rate inhibition due to excess zinc may be expected when [ZnaF] M. If M, then any trace-metal toxicity would [Cuaq2+] stem primarily from excess zinc, and if [Zna,2+]
