EFFECT OF TETRAMETHYL LEAD ON FRESHWATER GREEN ALGAE B. A. SILVERBERG,P. T. S. WONGand Y. K. CHAU Canada Centrefor Inland Waters,Department of the Environment POB 5050,Burlington L7R4A6,Ontario, Canada
The toxicity of tetramethyl lead (Me4Pb)towards freshwater algae was studied by bubbling biologically generated Me4Pb from one flask containing 5 mg of Pb I - ' as MeaPbOAe into the culture medium in another flask where a test alga Scenedesmus quadricauda was grown. As Me4Pb is not soluble in water and is volatile, the exposure of an alga to thi.s lead compound was only momentary. It was estimated that less than 0.5 mg of Pb (Me4Pb) had passed through the culture medium. The primary productivity and cell growth (determined by dry weight), however, decreased by 85% and 32% respectively, as compared with the controis without exposure to Me4Pb. Furthermore, cells exposed to Me4Pb tended to clump together and striking alterations in cell fine-structure were observed. An electron microscopic analysis by an energy dispersive spectrometer revealed that Pb ions had penetrated the cell and were deposited within concretion bodies. Similar results were obtained with the green algae Ankistrodesmus falcatus and Chlorella pyrenoidosa. Environmental concern has drawn much public attention to inorganic lead as a potentially hazardous contaminant, based upon its long-term persistence in the various trophic levels of our environment and rapid uptake and accumulation of toxic concentrations by plants and animals. Lead in aquatic environments and its toxicity to algae has been the subject of a number of recent reviews (Cairns et al. 1972, Malanchuk and Grnendling 1973, Whitton 1970). Nothing, however, has been known about the existence o f organic forms of lead in the environment as a result of biotransformation 1. It has been well documented that both the chemical behaviour and the toxicity of metals such as mercury and arsenic are further complicated by the ability of microorganisms to methylate inorganic metals (Wood 1974), which is probably responsible for increasing the toxicity and geochemical mobility of the metals in aquatic environments. O f particular concern is evidence from this laboratory for the transformation of some inorganic and organic lead salts to tetramethyl lead (Me4Pb) by microbes in lake sediments (Wong et al. 1975). We conclude from the results of 50 experiments that incubation of some lead-containing sediments generates Me4Pb; that Me3Pb + salts are readily converted to Me4Pb by microorganisms in lake water or nutrient medium, with or without the sediment, and in the presence or the absence of light; that conversion of inorganic lead such as lead nitrate or lead chloride to Me4Pb occurred on several occasions in the presence of tLead. Airborne lead in perspective, 18 (Natn. Acad. Sci., Washington, D.C., 1972). Archives of Environmental Contamination and Toxicology Vol. 5,305-313 (1977) o 1977 by Springer-VerlagNew YorkInc. 305
306
B.A. Silverberg et al.
certain sediments; and that the conversion is purely a biological process. Little is known, however, about the harmful effects of volatile Me4Pb to higher organisms. Further, there exists a dearth of information on the cytotoxic effects of Me4Pb to algae, despite their importance as primary producers in the food chain. In the present study the authors have attempted to discern the effect of Me4Pb on the structure and physiology of freshwater algae. The resultant toxicity of Me4Pb on growth and primary productivity with the concomitant morphological changes are outlined herein.
Materials and Methods Preparation of bacterial and algal inocula. Aeromonas sp. isolated from Lake Ontario water was grown in 300 ml of glucose (0.1%), nutrient broth, (Difco, 0.5%) and yeast extract (0. I%) for 18 hr in a rotary shaker at 20~ Axenic cultures of Chlorella pyrenoidosa, Scendesmus quadricauda and Ankistrodesmus falcatus were each grown in 500 ml of CHU-10 mineral salt medium (Chu 1942) under continuous light (5000 lux) at 200C for 7 to 9 days. Generation and bioassay of volatile tetramethyl lead. Tetramethyl lead (Me4Pb) was generated from trimethyl lead acetate (Me3PbOAc) either by Aeromonas sp. or indigenous microorganisms in Hamilton Harbour water and sediment. Trimethyl lead acetate was used because it could be readily converted to Me4Pb by microorganisms in lake water or nutrient medium, with or without the sediment (Wong et al. 1975). To generate the Me4Pb, 150 ml of the bacterial inoculum was added into 1350 ml of the fresh nutrient medium with and without ME3PbOAc (final concentration 5 mg 1-1 as Pb) in a 4-L culture flask. When sediment was used, 500 gm of sediment and 1 L of lake water with and without Me3PbOAc were incubated with glucose (0.1%) to stimulate microbial growth. After one week incubation, the gas phase in the incubation flask was withdrawn through the side-arm by means of a peristaltic pump and transferred to a U-tube containing 3% QV-1 at -70"C (dry ice methanol bath). The U-tube was warmed to 50~ and the trapped air sample was swept into a gas chromatograph-atomic absorption spectrophotometer system for the separation and analysis of the volatile lead compound (Chau et al. 1975). When Me4Pb was detected in the air sample with reference to retention time of a synthetic compound and was further confirmed by gas chromatography and mass spectrometry, the incubation flask was connected to the testing algal flask with a glass tubing (Fig. 1). The algal flask was prepared by innoculating 100 ml of algae into 1.4 L of fresh CHU-10 medium in the 4-L culture flask. Me4Pb was continuously withdrawn from the generating flask and bubbled through the medium in the algal flask with a peristaltic pump. Daily observations of the algae were made. Measurement of algal growth. The amount of algal growth was estimated by both cell dry weight and microscopic cell count. Aliquots of 40 ml of algal suspensions were filtered through tared Sartorious membrane filters (0.45/z pore size). To correct for loss of weight of filters during drying, an equal aliquot of CHU-10 was filtered through the mem-
Effect of Tetramethyl Lead on Green Algae
307
Fig. 1. Experimental design for testing the toxicity of biologically generated Me4Pb to freshwater green algae.
brane filter. The filters were dried until constant weight at a 700C oven. Cell number was determined with a Fuchs-Rosenthal counting chamber. All cell count data were based upon 4 separate counts of each sample.
Measurement of primary productivity. The primary productivity of the algae was tested with the procedures of Malanchuk and Gruendling (1973). Algae (2 ml) with and without exposure to Me4Pb were inoculated into 12.2 ml of CHU-10 medium in 25 ml micro-fernbach flasks and placed upon a rotary shaker under 5000 lux fluorescent lights. Then 0.8 ml of Na~14CO3 (1/~Ci ml-1; 58.8/~Ci/zmole -1 Amersham Co.) was added to each flask. The flasks were incubated for 4 hr at 20~ Neutralized formalin (0.1 mi) was added to "fix" the algae which were then filtered through 0.45/x Sartorious membrane filters. The filters were placed in scintillation vials and dried in a desiccator. The method of Lind and Campbell (1969) was used to prepare for liquid scintillation counting. Electron microscopy and X-ray microanalysis. After 7 days ceils were collected by gentle centrifugation and fixed for 2 to 24 hr at cold temperatures either in 2.5% glutaraldehyde in 0.1 M phosphate buffer, in veronal acetate buffer, or in sodium cacodylate buffer at pH 7.2. After thorough washing, cells were postfixed in 1% osmium tetroxide at 4~ in the respective buffers. Dehydration was in a graded series of ethyl alcohols, and acetone ~vas used as a solvent for Spun" embedment (Spurr 1969). To avoid confusion with the cellular Pb deposits, sections were viewed both unstained and counterstained with heavy metals. Unstained, 900 to 2400 ,~ thick sections mounted on Formvar-coated nylon grids, with carbon coating, were used for electron probe analysis. Analyses were done with a Philips 300 electron microscope operated at 80 kV with a total beam current of 50/zA and equipped with Edax energy dispersive microanalyzing system (Edax International, Prairie View, Ill.) operated at 20 eV per channel. Usually, but not invariably, the spot sizes ranged from 0.32 to 0.2 ~ m in diameter.
308
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For scanning electron microscopy glutaraldehyde-fixed cells were dehydrated in ethanol, following standard procedures used before embedding material in plastic for ultramicrotomy. When the cells were thoroughly infiltrated with 100% ethanol, the alcohol was replaced by 100% amyl acetate by passing the material through a graded series of mixtures of the 2 liquids. The cells were dried by the critical-point method of Anderson (1951) using liquid carbon dioxide as the transitional fluid. The specimens were mounted on specimen stubs with silver-conducting paint, coated with 200 to 400 ,~ of gold vaporized onto them in a Varian VE 10 vacuum evaporator, and observed with an AMR 1000 stereoscan electron microscope. Results
The effect of tetramethyl lead on growth and photosynthesis was different for the 3 species of algae tested (Table 1). As Me4Pb is not soluble in water and is volatile, the exposure of an alga to this lead compound was only momentary. We estimated that less than 0.5 mg of Pb (Me4Pb) had passed through the culture medium. We found that the primary productivity and cell growth (determined by dry weight), however, decreased as compared with the controls without exposure to Me4Pb.
Table 1. Toxicity o f tetramethyl lead upon three green algal species % decrease as compared to control Species
Growth a
Photosynthesis/~
Chlorella pyrenoidosa c Scenedesmus quadricauda a Ankistrodesmus falcatus c
74 32 32
83 85 49
aGrowth was determined by d.ry weight as well as by cell counts. 100% gave approximately 50 mg 1-1 and 6.4 x 106 ceils ml-l bPhotosynthesis was determined by 14 C-carbonate method Me4 Pb was generated by A eromonas sp. in a chemically defined medium rime4 Pb was generated by microorganisms in water-sediment system
A significant change in pigment was observed accompanying the loss of culture turbidity. Microscopic examination of Me4Pb-exposed cultures revealed a loss of the bright green colour, turning tO a semitransparent yellow with time. The effect of Me4Pb was manifested as the formation of palmelloid-like colonies (Fig. 2), which are gelatinous masses reminiscent of senescent phase. Contents of these enlarged cells appeared abnormal and deformed. Since there was no obvious difference in the morphological alterations between the treated cultures of the three species of algae, they will be discussed collectively using Scenedesmus quadricauda for illustrative purposes. From electron microscopic examination, the Me4Pb-exposed cells were observed in a process of degeneration, resulting in a heterogeneous population of cells. Among the vari-
Effect of Tetramethyl Lead on Green Algae
309
Fig. 2. Scanning electron micrographs of Scenedesmus quadricauda. (a) A eoenobium ofS. quadricauda containing 4 individual ceils arranged in a single file. Characteristic of this species is the presence of 4 conspicuous terminal spines ( x 18,000). (b) The effect of Me4Pb exposure is the formation of palmelloids which are gelatinous masses consisting of many cell generations ( x 10,800).
310
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ous organelles of the cell the chloroplast exhibited the most pronounced morphological changes upon being exposed to Me4Pb. The cell had one large parietal chloroplast lying along the cell wall (Fig. 3a). At its middle a pyrenoid was bulging inwards the cell center. The thylakoids were aggregated in bundles of three and traversed the whole length of the chloroplast. The pyrenoid region was free from thylakoids and generally surrounded by starch plates. Found dispersed throughout the stroma were occasional osmiophilic globules or plastoglobuli. Chloroplasts of lead-exposed cells (Fig. 3b) were observed fragmented. Thylakoid membranes were often disrupted and frequently the thylakoidal lamellae formed compressed multilayered stacks. Most noticeably, the starch grains increased in size and number causing a disarrangement of the thylakoids. The pyrenoid remained very prominent but lacked a distinct starch sheath. In addition to the modified lameUar organization, exposure to Me4Pb affected the plastoglobuli. These lipid inclusions appeared to be smaller in size and greater in number than in chloroplasts of control cells. Me4Pb also caused a general degeneration of the cytoplasm. The nucleus seemed to be subjected to compressive forces becoming irregularly convoluted among groups of concretion bodies and starch grains. In Chlorella pyrenoid6sa the nuclear membrane was greatly dilated. The other organelles such as Golgi bodies and endoplasmic reticulum appeared normal and showed the same sort of associations as in control cells. Many of the cells contained vesicles that were bounded by double membranes and are considered to be mitochondria. Although no change in the form or size of this organelle was evident, a definite decrease in the staining affinity of the miticbondrial cristae was observed. The decrease in membrane staining may be a result of decreased oxidative and phosphorylative abilities (Goyer and Krall 1969). Dense cytoplasmic concretions, spherical or ovoid in shape, and varied in size, with diameters up to 0.5 urn, were prominent. The concretions were clearly surrounded by a single membrane and were found only in Me4Pb-exposed cells. These bodies consisted of a densely packed matrix impregnated with electron-opaque, coarsely granular substance. An electron microscopic analysis of these bodies was made by means of an energy dispersive spectrometer (Edax) using X-ray energies greater than I0 KeV, and revealed peaks at L~ and L/3 lines and also a small peak at the L0 line for Pb (Fig. 4). The characteristic peaks for Pb in this energy area are found at 10.550 (La line), 12.612 (L/3 line), and 14.762 (LO line).
Discussion The results of this investigation clearly illustrate the toxic nature of tetramethyl lead in the growth medium of freshwater green algae. To obtain similar inhibition of primary productivity and cell growth, twice as much lead, in the form of Me3PbOAc, and 20 times as much lead nitrate would be required. These numbers were calculated from our studies on the toxicity of various forms of lead compounds on algae. From the present findings it is concluded that Me4Pb-exposure results in Pb ions penetrating the cell and being deposited within concretion bodies which are similar to polypbosphate inclusions commonly found in the algae. Polyphosphate may be .suspected as one of the substances contributing to binding of lead ions. Me4Pb toxicity manifests itself by loss of the algal's physiological and structural integrity.
Effect of Tetramethyl Lead on Green Algae
311
b
Fig. 3. Transmission electronmicrographs ofScenedesraus quadricauda. (a) Control cell. The chloroplast occupies the periphery of the cell and is closely appressed to the cell wall. A prominent pyrenoid with surrounding starch plates is located within the chloroplast. There is a relatively small amount of cytoplasm containing mitochondria, endoplasmic reticulum and Golgi bodies ( x 20,600). (b) Various kinds of aberrations can be observed in a Me4Pb-exposed cell. Arrows point to concretion bodies within the cytoplasm, which are presumed to be polyphosphate in nature ( • 20,000).
312
B.A. Silverberg et al.
I
I L8
I L=
Fig. 4. X-ray spectra for energies greater than 10 KeV for concretion bodies showing 3 peaks for lead: the I.a-line at 10.550 KeV, the L/3-1ineat 12.612 KeV and the LO-line at 14.762 KeV. Counting time 200 see at 80 kV.
There are two factors which at least partially explain the fine-structural alterations in the chloroplast. First, photosynthetic activity is impaired in ceils exposed to Me4Pb, and it would be expected that inhibited photosynthesis would be accompanied by changes in thylakoid appearance. Since thylakoid organization is necessary for the integrated functioning of photosystems I and II (Arntzen et al. 1972, Miller and Nobel 1972), it is not surprising that disorganization of the lamellar system was observed. Secondly, the marked increase in osmiophilic plastoglobuli may result from accumulated lipids that would otherwise have been used in thylakoid development. This accumulation may arise from inhibited structural protein formation as a result of blocked photosynthesis and ATP synthesis. In view of the very volatile and transient nature of Me4Pb in solution, a comparison between the effects of the biologically generated Me4Pb and commercially available MeaPb on freshwater algae was sought. Our attempts to measure the effects of laboratory Me4Pb have been frustrated by the extreme and rapid escape of Me4Pb by volatilization. Since algae are at the base of the food chain in aquatic environments, the importance of their response to the presence of Me4Pb is obvious. The potential danger of algae greatly concentrating Pb ions in the food chain warrants future studies to determine the penetration into and accumulation of the metal by other aquatic organisms. This is of importance in explaining the mechanism of action of tetramethyl lead, and, in developing conditions under which it may be cytotoxic.
Effect of TetramethylLeadon Green Algae
313
References Anderson, T.F.: Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci. Ser. 11, 13, 13 (1951). Arntzen, C.J., R.A. Dilley, G.A. Peters, and E.R. Shaw: Photochemical activity and structural studies of photosystems derived from chloroplast grana and stroma lamellae. Biochim. Biophys. Acta 256, 85 (1972). Cairns, J., G.R. Lanza, and B.C. Parker: Pollution related structural and functional changes in aquatic communities with emphasis on freshwater algae and protozoa. Proceed. Acad. Nat. Sci. (Philadel.) 124, 79 (1972). Chau, Y.K., P.T.S. Wong, and H. Saitoh.: Determination or tetramethyl lead compounds in the atmosphere. J. Chromatogr. Sci. In press (1975). Chu, S.P.: The influence of the mineral composition of the medium on the growth of planktonic algae. I. Methods and culture media. J. Ecol. 30, 284 (1942). Goyer, R.A., and R. Krall: Ultrastructural transformation in mitochondria isolated from kidneys of normal and lead intoxicated rats. J. Cell. Biol. 41,393 (1969). Lind, O.T., and R.S. Campbell: Comments on the use of liquid scintillation for routine determination of ~4C activity in production studies. Limnol. Oceanogr. 14, 787 (1969). Malanchuk, J.L., and G.K. Gruendling: Toxicity of lead nitrate to algae. Water, Air, and Soil Pollution 2, 181 (1973). Miller, M.M., and P.S. Nobel: Light-induced changes in the ultrastructure of pea chloroplasts in vivo. Relationship to development and photosynthesis. PI. Physiol. 49, 535 (1972). Spurt, A.R.: A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31 (1969). Whitton, B.A.: Toxicity of heavy metals to freshwater algae: a review. Phykos 9, 116 (1970). Wong, P.T.S., Y.K. Chau, and P.L. Luxon: Methylation of lead in the environment. Nature 253, 263 (1975). Wood, J.M.: Biological cycles for toxic elements in the environment. Science 183, 1049 (1974).
Manuscript received July 21, 1975; accepted January 1, 1976.
The toxicity of tetramethyl lead (Me4Pb)towards freshwater algae was studied by bubbling biologically generated Me4Pb from one flask containing 5 mg of Pb I - ' as MeaPbOAe into the culture medium in another flask where a test alga Scenedesmus quadricauda was grown. As Me4Pb is not soluble in water and is volatile, the exposure of an alga to thi.s lead compound was only momentary. It was estimated that less than 0.5 mg of Pb (Me4Pb) had passed through the culture medium. The primary productivity and cell growth (determined by dry weight), however, decreased by 85% and 32% respectively, as compared with the controis without exposure to Me4Pb. Furthermore, cells exposed to Me4Pb tended to clump together and striking alterations in cell fine-structure were observed. An electron microscopic analysis by an energy dispersive spectrometer revealed that Pb ions had penetrated the cell and were deposited within concretion bodies. Similar results were obtained with the green algae Ankistrodesmus falcatus and Chlorella pyrenoidosa. Environmental concern has drawn much public attention to inorganic lead as a potentially hazardous contaminant, based upon its long-term persistence in the various trophic levels of our environment and rapid uptake and accumulation of toxic concentrations by plants and animals. Lead in aquatic environments and its toxicity to algae has been the subject of a number of recent reviews (Cairns et al. 1972, Malanchuk and Grnendling 1973, Whitton 1970). Nothing, however, has been known about the existence o f organic forms of lead in the environment as a result of biotransformation 1. It has been well documented that both the chemical behaviour and the toxicity of metals such as mercury and arsenic are further complicated by the ability of microorganisms to methylate inorganic metals (Wood 1974), which is probably responsible for increasing the toxicity and geochemical mobility of the metals in aquatic environments. O f particular concern is evidence from this laboratory for the transformation of some inorganic and organic lead salts to tetramethyl lead (Me4Pb) by microbes in lake sediments (Wong et al. 1975). We conclude from the results of 50 experiments that incubation of some lead-containing sediments generates Me4Pb; that Me3Pb + salts are readily converted to Me4Pb by microorganisms in lake water or nutrient medium, with or without the sediment, and in the presence or the absence of light; that conversion of inorganic lead such as lead nitrate or lead chloride to Me4Pb occurred on several occasions in the presence of tLead. Airborne lead in perspective, 18 (Natn. Acad. Sci., Washington, D.C., 1972). Archives of Environmental Contamination and Toxicology Vol. 5,305-313 (1977) o 1977 by Springer-VerlagNew YorkInc. 305
306
B.A. Silverberg et al.
certain sediments; and that the conversion is purely a biological process. Little is known, however, about the harmful effects of volatile Me4Pb to higher organisms. Further, there exists a dearth of information on the cytotoxic effects of Me4Pb to algae, despite their importance as primary producers in the food chain. In the present study the authors have attempted to discern the effect of Me4Pb on the structure and physiology of freshwater algae. The resultant toxicity of Me4Pb on growth and primary productivity with the concomitant morphological changes are outlined herein.
Materials and Methods Preparation of bacterial and algal inocula. Aeromonas sp. isolated from Lake Ontario water was grown in 300 ml of glucose (0.1%), nutrient broth, (Difco, 0.5%) and yeast extract (0. I%) for 18 hr in a rotary shaker at 20~ Axenic cultures of Chlorella pyrenoidosa, Scendesmus quadricauda and Ankistrodesmus falcatus were each grown in 500 ml of CHU-10 mineral salt medium (Chu 1942) under continuous light (5000 lux) at 200C for 7 to 9 days. Generation and bioassay of volatile tetramethyl lead. Tetramethyl lead (Me4Pb) was generated from trimethyl lead acetate (Me3PbOAc) either by Aeromonas sp. or indigenous microorganisms in Hamilton Harbour water and sediment. Trimethyl lead acetate was used because it could be readily converted to Me4Pb by microorganisms in lake water or nutrient medium, with or without the sediment (Wong et al. 1975). To generate the Me4Pb, 150 ml of the bacterial inoculum was added into 1350 ml of the fresh nutrient medium with and without ME3PbOAc (final concentration 5 mg 1-1 as Pb) in a 4-L culture flask. When sediment was used, 500 gm of sediment and 1 L of lake water with and without Me3PbOAc were incubated with glucose (0.1%) to stimulate microbial growth. After one week incubation, the gas phase in the incubation flask was withdrawn through the side-arm by means of a peristaltic pump and transferred to a U-tube containing 3% QV-1 at -70"C (dry ice methanol bath). The U-tube was warmed to 50~ and the trapped air sample was swept into a gas chromatograph-atomic absorption spectrophotometer system for the separation and analysis of the volatile lead compound (Chau et al. 1975). When Me4Pb was detected in the air sample with reference to retention time of a synthetic compound and was further confirmed by gas chromatography and mass spectrometry, the incubation flask was connected to the testing algal flask with a glass tubing (Fig. 1). The algal flask was prepared by innoculating 100 ml of algae into 1.4 L of fresh CHU-10 medium in the 4-L culture flask. Me4Pb was continuously withdrawn from the generating flask and bubbled through the medium in the algal flask with a peristaltic pump. Daily observations of the algae were made. Measurement of algal growth. The amount of algal growth was estimated by both cell dry weight and microscopic cell count. Aliquots of 40 ml of algal suspensions were filtered through tared Sartorious membrane filters (0.45/z pore size). To correct for loss of weight of filters during drying, an equal aliquot of CHU-10 was filtered through the mem-
Effect of Tetramethyl Lead on Green Algae
307
Fig. 1. Experimental design for testing the toxicity of biologically generated Me4Pb to freshwater green algae.
brane filter. The filters were dried until constant weight at a 700C oven. Cell number was determined with a Fuchs-Rosenthal counting chamber. All cell count data were based upon 4 separate counts of each sample.
Measurement of primary productivity. The primary productivity of the algae was tested with the procedures of Malanchuk and Gruendling (1973). Algae (2 ml) with and without exposure to Me4Pb were inoculated into 12.2 ml of CHU-10 medium in 25 ml micro-fernbach flasks and placed upon a rotary shaker under 5000 lux fluorescent lights. Then 0.8 ml of Na~14CO3 (1/~Ci ml-1; 58.8/~Ci/zmole -1 Amersham Co.) was added to each flask. The flasks were incubated for 4 hr at 20~ Neutralized formalin (0.1 mi) was added to "fix" the algae which were then filtered through 0.45/x Sartorious membrane filters. The filters were placed in scintillation vials and dried in a desiccator. The method of Lind and Campbell (1969) was used to prepare for liquid scintillation counting. Electron microscopy and X-ray microanalysis. After 7 days ceils were collected by gentle centrifugation and fixed for 2 to 24 hr at cold temperatures either in 2.5% glutaraldehyde in 0.1 M phosphate buffer, in veronal acetate buffer, or in sodium cacodylate buffer at pH 7.2. After thorough washing, cells were postfixed in 1% osmium tetroxide at 4~ in the respective buffers. Dehydration was in a graded series of ethyl alcohols, and acetone ~vas used as a solvent for Spun" embedment (Spurr 1969). To avoid confusion with the cellular Pb deposits, sections were viewed both unstained and counterstained with heavy metals. Unstained, 900 to 2400 ,~ thick sections mounted on Formvar-coated nylon grids, with carbon coating, were used for electron probe analysis. Analyses were done with a Philips 300 electron microscope operated at 80 kV with a total beam current of 50/zA and equipped with Edax energy dispersive microanalyzing system (Edax International, Prairie View, Ill.) operated at 20 eV per channel. Usually, but not invariably, the spot sizes ranged from 0.32 to 0.2 ~ m in diameter.
308
B.A. Silverberg et al.
For scanning electron microscopy glutaraldehyde-fixed cells were dehydrated in ethanol, following standard procedures used before embedding material in plastic for ultramicrotomy. When the cells were thoroughly infiltrated with 100% ethanol, the alcohol was replaced by 100% amyl acetate by passing the material through a graded series of mixtures of the 2 liquids. The cells were dried by the critical-point method of Anderson (1951) using liquid carbon dioxide as the transitional fluid. The specimens were mounted on specimen stubs with silver-conducting paint, coated with 200 to 400 ,~ of gold vaporized onto them in a Varian VE 10 vacuum evaporator, and observed with an AMR 1000 stereoscan electron microscope. Results
The effect of tetramethyl lead on growth and photosynthesis was different for the 3 species of algae tested (Table 1). As Me4Pb is not soluble in water and is volatile, the exposure of an alga to this lead compound was only momentary. We estimated that less than 0.5 mg of Pb (Me4Pb) had passed through the culture medium. We found that the primary productivity and cell growth (determined by dry weight), however, decreased as compared with the controls without exposure to Me4Pb.
Table 1. Toxicity o f tetramethyl lead upon three green algal species % decrease as compared to control Species
Growth a
Photosynthesis/~
Chlorella pyrenoidosa c Scenedesmus quadricauda a Ankistrodesmus falcatus c
74 32 32
83 85 49
aGrowth was determined by d.ry weight as well as by cell counts. 100% gave approximately 50 mg 1-1 and 6.4 x 106 ceils ml-l bPhotosynthesis was determined by 14 C-carbonate method Me4 Pb was generated by A eromonas sp. in a chemically defined medium rime4 Pb was generated by microorganisms in water-sediment system
A significant change in pigment was observed accompanying the loss of culture turbidity. Microscopic examination of Me4Pb-exposed cultures revealed a loss of the bright green colour, turning tO a semitransparent yellow with time. The effect of Me4Pb was manifested as the formation of palmelloid-like colonies (Fig. 2), which are gelatinous masses reminiscent of senescent phase. Contents of these enlarged cells appeared abnormal and deformed. Since there was no obvious difference in the morphological alterations between the treated cultures of the three species of algae, they will be discussed collectively using Scenedesmus quadricauda for illustrative purposes. From electron microscopic examination, the Me4Pb-exposed cells were observed in a process of degeneration, resulting in a heterogeneous population of cells. Among the vari-
Effect of Tetramethyl Lead on Green Algae
309
Fig. 2. Scanning electron micrographs of Scenedesmus quadricauda. (a) A eoenobium ofS. quadricauda containing 4 individual ceils arranged in a single file. Characteristic of this species is the presence of 4 conspicuous terminal spines ( x 18,000). (b) The effect of Me4Pb exposure is the formation of palmelloids which are gelatinous masses consisting of many cell generations ( x 10,800).
310
B.A. Silverberg et al.
ous organelles of the cell the chloroplast exhibited the most pronounced morphological changes upon being exposed to Me4Pb. The cell had one large parietal chloroplast lying along the cell wall (Fig. 3a). At its middle a pyrenoid was bulging inwards the cell center. The thylakoids were aggregated in bundles of three and traversed the whole length of the chloroplast. The pyrenoid region was free from thylakoids and generally surrounded by starch plates. Found dispersed throughout the stroma were occasional osmiophilic globules or plastoglobuli. Chloroplasts of lead-exposed cells (Fig. 3b) were observed fragmented. Thylakoid membranes were often disrupted and frequently the thylakoidal lamellae formed compressed multilayered stacks. Most noticeably, the starch grains increased in size and number causing a disarrangement of the thylakoids. The pyrenoid remained very prominent but lacked a distinct starch sheath. In addition to the modified lameUar organization, exposure to Me4Pb affected the plastoglobuli. These lipid inclusions appeared to be smaller in size and greater in number than in chloroplasts of control cells. Me4Pb also caused a general degeneration of the cytoplasm. The nucleus seemed to be subjected to compressive forces becoming irregularly convoluted among groups of concretion bodies and starch grains. In Chlorella pyrenoid6sa the nuclear membrane was greatly dilated. The other organelles such as Golgi bodies and endoplasmic reticulum appeared normal and showed the same sort of associations as in control cells. Many of the cells contained vesicles that were bounded by double membranes and are considered to be mitochondria. Although no change in the form or size of this organelle was evident, a definite decrease in the staining affinity of the miticbondrial cristae was observed. The decrease in membrane staining may be a result of decreased oxidative and phosphorylative abilities (Goyer and Krall 1969). Dense cytoplasmic concretions, spherical or ovoid in shape, and varied in size, with diameters up to 0.5 urn, were prominent. The concretions were clearly surrounded by a single membrane and were found only in Me4Pb-exposed cells. These bodies consisted of a densely packed matrix impregnated with electron-opaque, coarsely granular substance. An electron microscopic analysis of these bodies was made by means of an energy dispersive spectrometer (Edax) using X-ray energies greater than I0 KeV, and revealed peaks at L~ and L/3 lines and also a small peak at the L0 line for Pb (Fig. 4). The characteristic peaks for Pb in this energy area are found at 10.550 (La line), 12.612 (L/3 line), and 14.762 (LO line).
Discussion The results of this investigation clearly illustrate the toxic nature of tetramethyl lead in the growth medium of freshwater green algae. To obtain similar inhibition of primary productivity and cell growth, twice as much lead, in the form of Me3PbOAc, and 20 times as much lead nitrate would be required. These numbers were calculated from our studies on the toxicity of various forms of lead compounds on algae. From the present findings it is concluded that Me4Pb-exposure results in Pb ions penetrating the cell and being deposited within concretion bodies which are similar to polypbosphate inclusions commonly found in the algae. Polyphosphate may be .suspected as one of the substances contributing to binding of lead ions. Me4Pb toxicity manifests itself by loss of the algal's physiological and structural integrity.
Effect of Tetramethyl Lead on Green Algae
311
b
Fig. 3. Transmission electronmicrographs ofScenedesraus quadricauda. (a) Control cell. The chloroplast occupies the periphery of the cell and is closely appressed to the cell wall. A prominent pyrenoid with surrounding starch plates is located within the chloroplast. There is a relatively small amount of cytoplasm containing mitochondria, endoplasmic reticulum and Golgi bodies ( x 20,600). (b) Various kinds of aberrations can be observed in a Me4Pb-exposed cell. Arrows point to concretion bodies within the cytoplasm, which are presumed to be polyphosphate in nature ( • 20,000).
312
B.A. Silverberg et al.
I
I L8
I L=
Fig. 4. X-ray spectra for energies greater than 10 KeV for concretion bodies showing 3 peaks for lead: the I.a-line at 10.550 KeV, the L/3-1ineat 12.612 KeV and the LO-line at 14.762 KeV. Counting time 200 see at 80 kV.
There are two factors which at least partially explain the fine-structural alterations in the chloroplast. First, photosynthetic activity is impaired in ceils exposed to Me4Pb, and it would be expected that inhibited photosynthesis would be accompanied by changes in thylakoid appearance. Since thylakoid organization is necessary for the integrated functioning of photosystems I and II (Arntzen et al. 1972, Miller and Nobel 1972), it is not surprising that disorganization of the lamellar system was observed. Secondly, the marked increase in osmiophilic plastoglobuli may result from accumulated lipids that would otherwise have been used in thylakoid development. This accumulation may arise from inhibited structural protein formation as a result of blocked photosynthesis and ATP synthesis. In view of the very volatile and transient nature of Me4Pb in solution, a comparison between the effects of the biologically generated Me4Pb and commercially available MeaPb on freshwater algae was sought. Our attempts to measure the effects of laboratory Me4Pb have been frustrated by the extreme and rapid escape of Me4Pb by volatilization. Since algae are at the base of the food chain in aquatic environments, the importance of their response to the presence of Me4Pb is obvious. The potential danger of algae greatly concentrating Pb ions in the food chain warrants future studies to determine the penetration into and accumulation of the metal by other aquatic organisms. This is of importance in explaining the mechanism of action of tetramethyl lead, and, in developing conditions under which it may be cytotoxic.
Effect of TetramethylLeadon Green Algae
313
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Manuscript received July 21, 1975; accepted January 1, 1976.
