Interactive Toxic Effect and Distribution of Heavy Metals in Phytoplankton

Transcription

1 Interactive Toxic Effect and Distribution of Heavy Metals in Phytoplankton Hideo Okamura and lsao Aoyama* Division of Ecological Chemistry, Research Institute for Bioresources, Okayama University, 2-2-1, Chu-o, Kurashiki, 71, Japan Assessing the interactive toxic effect of chemicals in the environment is becoming a matter of increasing public focus on and concern with ecotoxicological aspects. The purpose of this study is to find the relationship between an interactive toxic effect and distribution of heavy metals in algal cells. The green alga Chlorella ellipsoidea Gernec(lAMC-27) was cultured for 6 days in the presence of cadmium and/or chromium. Algal cells were divided into 4 fractions by centrifugation after the cells were disrupted using a French press. The amounts of the metals in each fraction we re determined. The interaction effect between the two metals on algal growth was investigated. The amount of one metal taken up in the cells and the growth inhibition rate increased with the concentration of metals in the medium. The amount of one metal in the cells was increased due to the presence of the other metal. Accordingly, the growth inhibition rate also increased. The amounts of Cd accumulated in the soluble fraction and in the membrane fraction of algal cells were 5 and 2%, respectively, of the total amount in the cells. The presence of Cr changed the Cd concentration in both fractions to 4%. The amount of Cr accumulated in each fraction was almost the same in the absence of Cd. The amount of Cr accumulated in the cell wall fraction rose to 9% after 3 days of exposure and it stayed as high as 5% even at the end of the six-day study period in the presence of Cd. It was assumed that the presence of one metal influenced the distribution of the other metal in the cells, which resulted in the synergistic toxic effect by John Wiley & Sons, Inc. Keywords: heavy metals. cadmium. chromium * green alga. Chlorella ellipsoidea. interactive effect. growth inhibition. bioaccumulation * synergistic effect. INTRODUCTION Organisms in an ecosystem are seldom exposed to only single chemicals, and in nearly all cases the stress of pollution on ecosystems is attributable to the combined effects of many chemicals. It is, therefore, necessary and important to assess the effects of chemical mixtures on microorganisms, plants and animal populations, communities, and ecosystems, in contrast to the usual practice of assessing the effects of single chemicals on individuals. We have been conducting the re- *To whom correspondence should be addressed. search on the combined toxicity of chemicals, such as heavy metals and herbicides, using a primary producer phytoplankton from the aspect of ecotoxicology (Aoyama and Okamur 1984; Aoyama et al., 1987). As the result of the interaction effect between heavy metals, it was found that cadmium stimulated the uptake of chromium by algal cells, but chromium showed no effect on the uptake of cadmium by the cells using continuous algal culture technique (Aoyama and Okamur 1993). After uptake into a cell, chemicals may remain unchanged in solution, they may be deposited, or they may become reversibly or irreversibly bound to endogenous cellular constituents of low or high molecular Environmental Toxicology and Water Quality: An International Journal, Vol. 9 (1994) by John Wiley & Sons, Inc. CCC l94lO17-9 7

2 8 OKAMURA AND AOYAMA weight. Relatively little is known about the factors that influence the partitioning of chemicals among the various cell compartments (organelles), although it appears that lipophilicity favors accumulation within the endoplasmic reticulum and those several carboxylic acids and inorganic ions are actively taken up by, for example, mitochondria. Reaction with a cellular component such as glutathione may result in a rapid clearance of the complex from the cell, while the buildup of particular inorganic ions, for example Cd2+, Hg2+, or Cu2+ (alone or in combination), can trigger the synthesis of specific binding proteins, such as metallothionein in certain cells (SCOPE, 1987). There is no evidence to indicate whether or not the presence of one metal changes the cellular distribution of the other metal to cause an interactive toxic effect. This information may be important in clarifying the presence of some mechanism that can lead to a better understanding of the interactive effect between metals and cellular distribution of metals. The purpose of this study is to find the relationship between interactive toxic effects and distribution of heavy metals in algal cells. MATERIALS AND METHODS Culture of Algae The freshwater green algae, Chlorella ellipsoidea Gernec(1AMC-27), provided by the Institute of Applied Microbiology, Tokyo University, was used as a test organism. As a culture medium, Meyers 1N-A5 solution, whose composition was modified from FeSO, to Fe-citrate, was diluted to 1 times with distilled water before using (Okamura et al., 1989). Algal density was determined with the Cell Counter (Nippon Koden Co., Ltd.) and expressed as cells per milliliter of algal suspension. Cadmium (Cd) and Chromium (Cr) were chosen as test chemicals. The chemical formulas of the salts used were Cd(NO,), and K2Cr4. The heavy metal ions dissolved in deionized-distilled water were stored at 4 C until used. The following 4 concentrations of each heavy metal were prepared to examine the interaction effect. The experiment was performed with 16 variations for the combinations. Cd:, 2, 1, 2 meq/l of medium Cr:, 1, 2, 5 meq/l of medium The cultures were maintained at 25 C with continuous illumination of 6 lux at the surface of the medium vessel. Each vessel, containing 1 ml of the medium with and without heavy metal, was prepared and Chlorella were added to a concentration of lo4 cells/ml from an exponential growth phase algal culture. The algae were cultured for 6 days in a rotary orbital incubator after inoculation. Algal density and the concentration of heavy metals both in algal cells and in the medium were determined every 24 h. Algal Cell Fractionation Disruption of algal cells is needed as the first step for fractionation of cell component. A French press is the most useful tool for disruption of Chlorellu (Taked and Hirokaw 1984). The procedure of cell fractionation is shown in Scheme 1. Algal cells harvested by centrifugation at 3 x g for 1 min were were washed twice with 5 mm Tris HCl buffer (ph 7.5). The algal cells were disrupted using a French press at 15 kgfl cm2 three times. The homogenate was centrifuged at 3 x g for 2 min; the pellet was allowed to stand in 1 ml of.5% sodium n-dodecyl sulfate (SDS) for 3 min. The homogenate with SDS was centrifuged at 1, x g for 2 min to remove the soluble component, and then the pellet was boiled in 8% ethanol for 2 min. The cell wall fraction was prepared by centrifuging the ethanol boiled pellet at 1, x g for 2 min. The miscellaneous fraction was prepared as the mixed supernatant after the treatment of SDS and ethanol. The homogenate after disruption was centrifuged at 2,~ g for 3 min. The membrane fraction was prepared as the pellet and the soluble fraction was prepared as the supernatant. The amounts of heavy metals in each fraction after acid digestion were determined using a flameless atomic absorption spectrophotometer and were compensated by the ratio to the total amount in the homogenate of intact cells. Heavy Metal Concentration Determination The algal suspension containing test heavy metals was filtered using.45 p membrane filter. The algal cells containing the metals on the filter were digested with nitric acid : perchloric acid (3 : 1) at 2 C overnight, and the medium containing the metals was not pretreated. The amount of heavy metals both in algal cells and in the medium was determined using a flameless atomic absorption spectrophotometer (AA-82, Nippon Jarrel-Ash Co., Ltd.). RESULTS AND DISCUSSION Interactive Toxic Effect Between Heavy Metals on Algal Growth Figure 1 shows the growth curves of Chlorellu that were grown in the medium containing test metals of 16 different concentration combinations. Algal growth

3 INTERACTIVE TOXIC EFFECT AND DISTRIBUTION 9 sup. (medium) algal cells at 39 for lmin. sup.:supernatant ppt.:precipitation PPt. added 5mM Tris-HCI buffer(ph 7.5) centrifuged at 39 for lmin. i disrupted using a French press at 15 kgf/cm2 centrifuged at 39 for 2min. centrifuged at 29 for centrifuged at 1 OOOOg for 2Omin. tboiled in 8% EtOH for 2Omin. fraction Scheme 1. Procedure for the fractionation of algal cell. decreased slightly when each metal was administered alone. However, cultures grown in the medium with the highest concentrations of both metals showed a marked growth inhibition after 24 h incubation. Relative growth rates were calculated, using initial algal cell density and the density at 24 h after inoculation, to evaluate the interaction effect between the metals. Table I shows the interaction modes evaluated, employing the method modified by Stratton (1983), from the inhibition rates calculated using the relative growth rates. It seems to be more practical to evaluate the interaction effect at metal concentrations in which the inhibition rates obtained under the single administration of test chemicals was around 5%, because the inhibition rate obtained under low metal concentration was not significantly different from the control. Although an additive effect was observed when the concentration of Cd was the lowest, it was found that the interaction effect between Cd and Cr was synergistic for the relative growth rate of Chlorella. This result was different from that previously reported by Aoyama et al. (1987), i.e., the interaction effect between Cd and Cr for the growth of C. ellipsoidea was antagonistic. It is believed that the inconsis- tency of these results was due to the differences of the culture condition, the concentrations tested, and the evaluation index for interactive effect. The weight of algal cells was calculated from cell density (cells/ml) using the following equation: Y = x lo-* C'.557 (r2 =.995) where Y is the dry weight/ml (mg/ml), C is the cell number/ml (cells/ml), and r is the regression coefficient. This equation was valid when cell density is in the range 3 x lo5 to 3 x lo6 cells/ml. The average weight of an algal cell was estimated to be 7 pg (p = from the above equation. Accumulation of Cd in Algae The changes of Cd concentration in the medium are shown in Fig. 2. The concentration of Cd in the medium gradually decreased when Cr was not present. On the other hand, the concentration of Cd increased after 3 days of exposure, in the presence of Cr when the Cd concentration was high. The increase in Cd concentra-

4 1 OKAMURA AND AOYAMA -- - O '- [.. I TABLE 1. Interaction effect between Cd and Cr on the relative growth rate of C. ellipsoidea a a Syn: synergistic effect; Add: additive effect. tion in the medium suggests that Cd taken up in the cells from the medium was excreted back to the medium. The changes in Cd concentration within the algal cells (fg/cell: f = are shown in Fig. 3. Cd was not detected in the inoculated cells and in the control cells. When Cd was administered alone, algal cells accumulated Cd up to 14 fg/cell (ca. 2.% w/w) after 1 day of exposure and decreased the Cd concentration to less than 2 fg/cell in the stationary growth phase. On the other hand, the amount of Cd taken up in the cells increased with the concentration of Cr in the medium, whereas in this experiment the accumulation of Cd in cells was inhibited when the concentration of Cd and Cr was the highest. Fig. 1. Growth curves of C. ellipsoidea in the medium containing Cd and Cr. Accumulation of Cr in Algae Figure 4 shows the changes in Cr concentrations in the medium. The concentration of Cr in the medium did not always decrease gradually, unlike the changes in Cd concentrations in the medium when Cd was administered alone. When Cr alone was administered to the medium, an increase in Cr concentration in the medium was observed at a low concentration addition of Cr. The increase in Cr concentration suggests that Cr, which had been taken up from the medium by the cells, was excreted back to the medium. Even in the presence of Cd, the concentration of Cr in the medium did not decrease drastically and remained at the initial level at the end of the culture period. It was found from Figs. 2 and 4 that the changes in the concentration of one heavy metal in the medium was influenced by the presence of the other metal. The changes in metal concentration in the medium were related to the changes in the uptake and excretion of the metal by algal cells. Figure 5 shows the changes in the amount of Cr in the cells. Even when Cr was not administered to the medium, Cr in the medium was accumulated in the cells and the concentration was as high as 1 fg/cell, after 1 day of exposure. These results indicated that the test medium contained a trace of Cr that could not be detected by the chemical detection method used.

5 INTERACTIVE TOXIC EFFECT AND DISTRIBUTION 11 IWl Cd. Cr I B b.- 18 I e ,8 E 4, - P Q. --w I Ina(dolo1 I y g ll I I Ins1da)nt I Ilmlday~) I Imo(dap1 Fig. 2. Changes in Cd concentration of the medium Fig. 3. Changes in the amount of Cd in algal cells.

6 ~~ 12 OKAMURA AND AOYAMA a--- 9, a *--, $B ---z, ie b-- 2, s --- 2,58 a--- 1, te e--- 19,a 19, 58 *-- a--- 1e --- a *-- 58 I I nm Idom) Fig. 4. Changes in Cr concentration of the medium. Fig. 5. Changes in the amount of Cr in algal cells.

7 INTERACTIVE TOXIC EFFECT AND DISTRIBUTION 13 r 1 t 1 I n - Q) 2 1 CI) r Y CI C J E Q 1. 2 A 1 2 L Cd concentration (peq/l) Cr concentration(peq/l) Fig. 6. Relationships between the initial metal concentration in the medium and the amount in algal cells at the end of the study period. The cells accumulated Cr up to 3 fg/cell after 1 day of exposure and the amount of Cr decreased to 1 fg/ cell after 3 days of exposure when the highest concentration of Cr was added to the medium. The amount of Cr in the cells grown in the presence of Cd was higher than those in the absence of Cd. In the presence of the highest concentration of Cd in the medium, the amount of Cr in the cells reached 14 fg/cell (ca. 2.% w/w) after 1 day of exposure. When the highest concentrations of each metal were administered to Chlorellu, the amount of Cr in cells decreased after 4 days of exposure as shown in Fig. 5 and the concentration of Cr in the medium slightly increased as shown in Fig. 4. At the same time, there were no changes in cell density as shown in Fig. 1. Therefore it was confirmed that Cr taken up into the cells was excreted to the medium due to the presence of Cd. Relationships Between Metal Concentration in the Medium and Amount of Metal in Algal Cells Figure 6A shows the relationship between the initial concentration of Cd in the medium and the amount in the cells at the end of the study period. When only Cd was administered, the amount of Cd in the cells was likely to reach a saturation point at higher concentrations of Cd than 1 yeqll in the medium. At low concentration additions of Cr, the amount of Cd in the cells increased with increasing concentrations of Cd. When the highest concentration of Cd and Cr was present in the medium, the amount of Cd in the cells was lower than that obtained when the concentration of Cr was lower. The copresence of Cr in the medium had a tendency to stimulate the uptake of Cd. The relationship between the initial concentration of Cr in the medium and the amount in the cells at the end of the culture are shown in Fig. 6B. The amount of Cr in the cells tended to increase with the concentration of Cr in the medium when only Cr was administered. The amount of Cr in the cells of the control, in which Cr was not added, increased with the concentration of Cd in the medium. As a whole the amount of Cr tended to increase with increasing concentrations of Cd in the medium, although the accumulation of Cr in cells was inhibited when the highest concentration of each metal was administered to the medium. The amount of one metal in the cells at the end of the study period tended to be higher with higher concentration of the other metal in the medium, except for the case when the highest concentration of each test metal was administered. Distribution of Heavy Metals in Algae The interaction effect of Cd and Cr on the distribution of each metal in algal cells was investigated. The concentrations tested were 2 peqll for Cd and 5 yeql L for Cr. Algal cells grown in the medium containing

8 14 OKAMURA AND AOYAMA n i 2 3 m 6 n X u) % m E 3 '.- I - P Cd amount(fglcel1) 2' (A) I cell wall a! membrane soluble the others lnltlal Cd:2)req/l Initial Cr:Ouea/l H cell wall membrane soluble the others 8. The amount of Cr accumulated in each fraction was almost the same in the absence of Cd after 6 days, although the cells accumulated 4% of Cr in the soluble fraction after 3 days of exposure. In the presence of Cd, the amount of Cr accumulated in the cells increased 4 times in comparison to the amount in the absence of Cd. The amount of Cr in the cell wall fraction rose to 9% after 3 days of exposure and it stayed as high as 5% even at the end of the culture in the presence of Cd. Thus, the presence of Cd in the medium drastically influenced the distribution of Cr in the cells. It was assumed that the presence of one metal increased the amount of the other metal in algal cells as well as influencing the distribution of the other metal within the cells. This relationship resulted in a synergistic toxic effect. CONCLUSIONS The following conclusions may be made: Fig. 7. Distribution of Cd in algal cells grown in the medium containing (A) Cd only and (B) Cd and Cr. these heavy metals were harvested at the middle (3rd day) and the end (6th day) of the study period. The cells were fractionated into 4 fractions: cell wall fraction, membrane fraction, soluble fraction, and the other (miscellaneous) fraction after the cells were disrupted using a French press. The amount of metal in whole cells and in each cell fraction was determined. The amount of Cd in each fraction is shown in Fig. 7. The total amount of Cd in the cells after 6 days of exposure was higher than that obtained after 3 days of exposure when only Cd was administered alone. The amount of Cd accumulated in the soluble fraction and in the membrane fraction of algal cells after 3 days of exposure was found to be 35% and 35% of the total amount in whole cells. After 6 days of exposure, each of these two fractions was found to contain Cd of 5% and 2%, respectively. This result suggests the cells tend to accumulate Cd in the soluble fraction that contains the protein metallothionein. When Cr was present in the medium, the amount of Cd in the soluble fraction increased to 6% after 3 days of exposure. Therefore, it was suggested that Cr stimulated the induction of Cd-binding protein. After 6 days of exposure, the amount of Cd in the soluble fraction decreased to 4% and that in the membrane fraction increased to 4%. Thus, the distribution of Cd in the cells was influenced by the presence of Cr in the medium. The amount of Cr in each fraction is shown in Fig. 1. The interactive toxic effect between Cd and Cr on the relative growth rate of Chlorella was synergistic. 2. Cd in the medium was accumulated in algal cells with increasing Cd concentration. The presence of Cr stimulated the accumulation of Cd in algal cells. 3. Cr in the medium was also accumulated in algal cells m % m 5 3 '.- ti P L 3 m 6 P x L 3 a 6 x Cr arnount(fglcel1) Cr amount(fglce1l) H cell wall membrane soluble the olhers I nit ial Cd :Opeq/l lnltlal Cr:SOpeqll P (B ) Initial Cd:2peq/l, I lnltlal Cr:5peq/l Fig. 8. Distribution of Cr in algal cells grown in the medium containing (A) Cr only and (B) Cd and Cr.

9 INTERACTIVE TOXIC EFFECT AND DISTRIBUTION 15 with increasing of the concentration. The accumulation of Cr in the cells was stimulated due to the presence of Cd. In the presence of the other metal in the medium, algal cells tend to excrete excessive amount of Cd and/or Cr which was taken up in cells. Cd tended to be accumulated in the soluble fraction of algal cells in the absence of Cr. The distribution of Cd in the cells was influenced due to the presence of Cr. Cr was equally accumulated in each fraction of the cells in the absence of Cd. Most of the Cr was accumulated in the cell wall fraction in the presence of Cd. The distribution of Cr in the cells was influenced due to the presence of Cd. It was assumed that synergistic toxic effect between the two metals occurred because one metal stimulated the taking up of the other metal in the medium and influenced the cellular distribution of the other metal. REFERENCES Aoyam I., and H. Okamura Toxicity evaluation of heavy metals in phytoplankton, P In Toxicity Screening Procedures Using Bacterial Systems. Edited by Dickson Liu and Bernard J. Dutka. Marcel Dekker, New York. Aoyama. I., H. Okamur and M. Yagi The interaction effects of toxic chemical combinations on Chlorella ellipsoidea. Toxic Assess. 2: Aoyam I., and H. Okamura Interactive toxic effect and bioconcentration between Cd and Cr using continuous algal culture. Environ. Toxicol. Water Qual. 8: Okamur H., I. Aoyam and M. Yagi Effect of culture conditions on algal growth and toxicity evaluation of heavy metals. Suishitu Odaku Kenkyu l2: Scientific Committee on Problems of the Environment (SCOPE) Methods for Assessing the Effects of Mixtures of Chemicals. John Wiley & Sons, New York, 894. Stratton, G. W Interaction effect of permethrin and atrazine combinations towards several non-target microorganisms. Bull. Environ. Contam. Toxicol Taked H., and T. Hirokawa Studies on the cell wall The authors gratefully acknowledge the financial support of Chlorella 5. Comparison of the cell wall chemical compofor this work provided by The Nippon Life Insurance Foun- sitions in the strains of Chlorella ellipsoidea. Plant Cell dation. Physiol