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Effects of cadmium on microorganisms
ECOTOXICOLOGY
AND
ENVIRONMENTAL
Effects
SAFETY
6, 157-165
of Cadmium
(1982)
on Microorganisms
BRIGITTE BISCHOFF Universitdt
Oldenburg,
Fachbereich Received
May
XII,
Oldenburg,
Wesr Germany
20, 1981
A short review was made of current knowledge, particularly of experimental and laboratory studies, relating to cadmium toxicity on microorganisms. The survey includes several aspects of cadmium toxicology, such as growth inhibition, lethality, physiological alterations, microbial resistarme to cadmium, uptake, and bioaccumulation. The spectrum of heavy metal toxicity on microorganisms involves initial growth inhibition up to the total elimination of a microbial population, and includes alterations of morphology, e.g., mitochondria, Golgi, and ribosomes as probable sites of damage, of metabolic pathways or enzymatic activities. Heavy metals may cause cytostatic or mutagenic effects, as described for Salmonella spp. and Escherichia coli. Microorganisms have the potential to accumulate and deposit heavy metals without showing adverse effects, to develop mechanisms of resistance, and to adapt to increasing concentrations of heavy metals. Cadmium is found to be virtually ubiquitous (18, 31). It is not only deposited and accumulated in various body tissues, but is also found in varying concentrations throughout all environmental compartments, since there has been an increasing introduction of cadmium into the environment as a result of mining operations, industrial pollution, and agricultural runoff ((19) cp. also (8, 25)). The toxicity of cadmium has been studied with regard to several aspects (7, 17-l 9, 30, 32, 43). Since cadmium is known to be a hazardous toxicant and enters into food webs as a result of various processes, it becomes important to study the toxic effects on the microb,iota to acquire a broad knowledge of the toxic potential of this metal. INVOLVEMENT OF MICROORGANISMS IN BIOLOGICAL PROCESSES AND CONTAMINATION Soil microorganisms are also involved in biogeochemical and decomposition processes necessary for nutrient cycling (39) and maintenance of soil fertility and in numerous interactions with other microorganisms, plants, and animals. (5). An essential part of microbiological activities is concerned with decomposition of organic matter like plant litter and mineralization of organically bound elements like nitroglen. An interruption or reduction in organic matter decomposition and mineralization might eventually result in a deficiency of essential nutrients for plants and., as a consequence, in a limitation of plant growth. On the other hand, microorganisms themselves are involved in pollution in multiple ways (4): ( 1) microorganisms are a primary natural biotic source of substantial quantities of gaseous pollutants (emission of inorganic and organic gases and volatiles),
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(2) they serve as sinks for a variety of pollutants, and (3) they are capable of reactivating contaminants from terrestrial and aqueous environments by transforming decomposed and deposited pollutants into more or less toxic forms (cp. (I, 27)). Contaminants in the form of air pollutants or as components of sewage or agricultural runoff can affect the microbiota on different levels of microbial activities and relations. The proven detrimental effect on the microbial reproductive potential (e.g., reduction in growth rates, inhibition of fungal sporulation) could influence not only microbial establishment, population dynamics, and interactions between microorganisms, but also the general ecology of the microbiota in natural habitats (7) and, as a consequence, can cause profound alterations in the contaminated environment. Another aspect of the ecological effects of heavy metal pollution is the problem of deficient or absent vegetation in a contaminated area in regard to colonization with microorganisms. This aspect has been described as the “rhizophere effect” (1.5, 28). INFLUENCE
OF VARIOUS INHIBITION
CONDITIONS BY CADMIUM
ON GROWTH
The toxicity of a contaminant is dependent on the physicochemical characteristics of the environment into which it is deposited, resulting in a final decrease or increase of the toxicity of the contaminant (cp. (5)). The amount of available cadmium is partially dependent on the cation-exchange capacity (CEC) of the environment, which, in turn, is a function of the types and concentrations of organic matter and clay minerals (6). Clay minerals in particular evolve a protective effect against cadmium toxicity, just as Babich and Stotzky proved for fungi (5) as well as for bacteria, actinomycetes, and filamentous fungi (6). The protective effect of the clay minerals studied, montmorillonite and kaolinite, is correlated to their CEC (cp. also (13)); increasing protection is apparently related to higher CEC and the capacity to absorb greater quantities of exogenous cadmium. In the absence of cadmium, in montmorillonite-amended agar, bacterial growth was stimulated, and fungal respiration was inhibited, but was unaffected by kaolinite (6), whereas in the presence of cadmium, montmorillonite and, to a lesser extent, kaolinite decreased the inhibitory effects of cadmium to bacteria, actinomycetes, and fungi. Cadmium is strongly bound to organic matter, and the degree of binding of cadmium ions in different types of soils follows the sequence organic soil > heavy clay soil > sandy and silt loam soil > sandy soil (6). In pure culture studies actinomycetes showed more tolerance to cadmium than eubacteria, and gram-negative more than gram-positive eubacteria. Fungal sporulation was inhibited to a higher extent than mycelial growth (7). The toxicity of cadmium to eubacteria, actinomycetes, and fungi turned out to be pH dependent, and toxicity was potentiated at pH 8 or 9 in the cases of Bacillus cereus, Alcaligenes faecalis, Agrobacterium tumefaciens, Nocardia parajjinae, Aspergillus niger, Trichoderma viride, and Rhizopus stolonifer; the exception was Streptomyces olivaceus in which the toxicity of cadmium was independent of pH. It was not
EFFECTS
OF CADMIUM
ON
MICROORGANISMS
159
clarified whether the increased toxicity was a reflection of the formation of complex ionic and molecular species of hydroxylated cadmium (7). On the other hand, growth inhibition of Chlorella pyrenoidosa seemed more pronounced at pH 7 than pH 8 (23) suggesting that removal of cadmium from the culture medium and its simultaneous deposition within the cells might be a pHdependent process (heavy metals associate with particles that are formed to a larger size under alkaline conditions in the medium). Inhibition of growth rates of Aspergillus niger was more pronounced by 100 or 250 pg of Cd/g in soil adjusted to pH 7.2 than in the same soil at its natural pH of 5.1, but there were no differences in growth rates at both pH 5.1 and 7.2 in the case of A. fischerii (5). The reaction of A. niger was supposed to be due to either an inability of the fungus to tolerate an additional heavy metal stress at alkaline levels or a synergistic interaction between cadmium and the higher pH level. At pH 6-7 cadmium is present in the divalent ionic form and is completely soluble in the absence of precipitating anions, such as phosphate and sulfide, resulting in availability for adsorption on suspended mineral colloids and complexation with organic matter, and cadmium may move in these forms (21). To determine heavy metal distribution in the environment, chlorides, under certain circumstances, may also be of great significance (21). In areas where organic matter accumulation takes place, organo-metal ion complexes are formed which may exist in soluble or colloidal form and, therefore, differ in degree of mobility. Results indicated that the hydroxy and chloride complexes may contribute to the mobilization of heavy metal ions in the environment (21). Yet, indlependent of environmental circumstances, the toxicity of cadmium to several microbial organisms might be graduated. Studies of organisms isolated from cadmium-amended and -unamended soils indicated that each organism had its own characteristic tolerance level to added cadmium (47). Regarding cadmium toxicity on algal growth in aqueous environments, effects of copper, zinc, and cadmium on Selanastrum capricornutum were investigated by Bartlett et al. (9). They found that combinations of copper, zinc, and cadmium were similar in toxicity to equal concentrations of zinc. Combinations of copper and cadmium resulted in greater growth than equal concentrations of copper, suggesting that cadmium inhibits copper toxicity. The algicidal concentration of cadmium was 0.65 mg/liter. Investigations with extremely Cd-sensitive and Cd-tolerant bacteria and extremely Pb-sensitive and Pb-tolerant bacteria isolated from brackish estuarine water showed that cadmium was more inhibitory to growth of Pb-sensitive bacteria than lead was to growth of Cd-sensitive strains (40). In general, neither cadmium nor lead had a pronounced specific effect of growth inhibition on single species of bacteria: Cd-sensitive bacteria were, too, more sensitive to lead than Cd-tolerant strains. The exalmination of 259 strains of marine origin pointed out that gram-negative bacteria w’ere more resistant to cadmium than gram-positive bacteria (for similar observations, cp. (7)). Moreover, a pronounced sensitivity or resistance to cadmium and lead was dependent on the genus, as well as on the geographical origin, of the marine bacteria studied (40).
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PHYSIOLOGICAL ALTERATIONS BY CADMIUM BY COz FIXATION, RESPIRATION, NITROGEN TRANSFORMATION
EXEMPLIFIED AND
Under conditions which promote accumulation of cadmium, Chlorella pyrenoidosa was still fixing CO*, albeit at reduced rates (at 1.OO mg of Cd/liter 39% fixation rate of the control), whereas O2 evolution was inhibited to a lesser extent (at 1.00 mg of Cd/liter 80%) (23). The reduction of a microbial culture as a result of a lethal cadmium effect obviously is connected with decreased simultaneous CO2 evolution. In a culture medium of Escherichia coli 6 mg of Cd/liter (as CdC12) and more caused decreased CO* production, correlated with a decrease in the number of cells surviving (48). Low levels of zinc and cadmium seemed to have a slightly stimulating effect on litter decomposition by a mixed population of soil and litter microorganisms, whereas high concentrations were shown to decrease respiration rates (13). The toxic effect of high concentrations of Cd2+ and Zn2+ on the activity of soil and litter microorganisms was suggested to be due to their ability to compete with essential elements (Mn, Fe, Mg) for the active sites (-SH, -NH2, -NH) of enzymes, but a mechanism for the stimulatory effect was stated to be unknown (13). Cadmium, as CdC12 (mean concentrations of 0.01 and 10.0 ppm), introduced into soil/litter microcosms markedly altered soil respiration without a concomitant decrease in organisms. This observation was suggested to indicate a slowdown of organismal activity, presumably to a level more conducive to long-term survival at low nutrient levels (IO). Varying levels of cadmium in sandy (50 to 1000 ppm) and alluvial (50 to 100 ppm) soil were registered to have an apparently stimulatory effect on nitrogen fixation (38), but only during the first 3 months of cadmium incubation; after 8 to 12 months there was a remarkable decrease in nitrogen fixation. In sandy soil, consisting of no organic matter, cadmium had the most deleterious effect on nitrogen fixation. Nitrate utilization by organisms in cadmium-amended soils was not influenced by levels of cadmium up to 100 pg/g of soil, but seemed to be inhibited by cadmium contents greater than 100 pg/g of soil (47). At 1000 pg Cd/g of fine sandy loam soil, the rates of ammonium utilization and nitrate production were reduced; however, the inhibition was not evident. Both nitrogen transformations were completely blocked at 10,000 pg Cd/g. It would appear that inhibition at this rate was related to an unfavorable pH in conjunction with metal toxicity. The results indicate that nitrification in soil is relatively resistant to the toxic action of heavy metals (36). Inhibition of nitrification by Ag(I), Hg(II), and Cd(I1) in various soils causes accumulation of NH4-N (33), as long as the ammonification rate is not reduced at the same time, as reported by Pancholy et al. (37). Furthermore, low activities of urease and dehydrogenase determined in soil from a denuded and contaminated area indicated the reduction of various other physiological activities (37). UPTAKE
AND
BIOACCUMULATION
OF CADMIUM
From consideration of the increasing cadmium concentrations in rivers, lakes, coastal waters, and sediments the ability of microorganisms to accumulate cadmium at a high rate appears to be possible. As reported by Maclean et al. (cited by (16)),
EFFECTS
OF CADMIUM
ON
MICROORGANISMS
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E. coli, A,uacystis midulans, and Chlorella spp. grown in a culture medium containing lo9CdC12 (lo-’ M) took up 9, 98, and 80% of the available cadmium, respectively, paralleled to growth. The ability of microorganisms, particularly E. coli and B. cereus, to immobilize large amounts of cadmium from the broth and to grow in the presence of relatively high concentrations of cadmium might indicate, according to Doyle et a/. (16), the possession of specific inherited resistance to inorganic ions by these organisms. Experimental studies investigating toxicity and bioaccumulation of cadmium in C. pyrenoidosa (23) pointed out that the amount of cadmium accumulated by the organisms was directly proportional to the concentration of metal initially present in the medium. There was no accumulation in the dark, at 4”C, or by dead cells. In this experiment, cadmium accumulation was not affected by concentrations of Ca, Mg, MO, Cu, Zn, or Co in the growth medium, but results concerning this problem are very different. Antagonistic or synergistic effects are possibilities, when combinations of different metals are present (44). Only manganese, at a level equal to or greater than 0.20 mg/liter, completely blocked cadmium accumulation (23). Iron might also play a role in regulating cadmium accumulation, since Fe-EDTA complex o’r additional iron as a supplement (FeCl,) restored the growth rate of C. pyrenoidosa to normal. Manganese was more effective as a blocking agent than iron; competitive inhibition was presumed as a most likely mechanism for the action of manganese on this system (23). The toxicity of Hg, Cd, and Pb not only may be modified by other elements such as Se but also may interfere with normal metabolism of essential trace elements. In animal dietary studies it was found that cadmium interferes with both copper and zinc metabolism, apparently because Cd*+ has the same chemical parameters as Zn*+ and Cu*+ (26). Further studies suggest that cadmium obviously shares an uptake system with one or more cations that are normally required for growth and there is evidence for the possibility that cadmium and manganese are transported by a common uptake system (22): -manganese inhibits cadmium uptake in a concentration-dependent manner and vice versa, --‘09Cd efflux from cells was brought about to the same extent by manganese as by “*Cd, -kinetic studies showed that cadmium and manganese appear to compete for the same binding sites and manganese might be expected to show a higher affinity than cadmium for cell binding sites (cp. also 12). Mang and Thromballa (35) suspected the zinc transport system of being the carrier mediating cadmium uptake, based on kinetic studies comparing the uptake of cadmium and zinc ions and examining their mutual inhibition. The experiments conducted under different conditions of energy supply showed, besides strong, fast, and for the most part reversible binding by absorption, very effective energy-dependent uptake, obviously by intake into the interior of the cell. Mang and Thromballa assurned active transport, as is the case with zinc. MECHANISMS
OF RESISTANCE
Several :investigators confirmed microbial manifestations of resistance to heavy metals (3, 1 I, 14, 20, 4.5, 46, 48). Babich and Stotzky (5) stated that A. niger and
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A. fischeri& isolated from an area contaminated with heavy metals near a Japanese smelter (28 pg of Cd/g of soil, pH 5.7), turned out to be relatively resistant to cadmium, since the growth obtained in the contaminated soil was the same as growth some distance away (4 pg of Cd/g of soil, pH 6.1). The toxicity of cadmium to A. niger was mitigated by addition of zinc; high concentrations of zinc (1.460 pg/g) had also been determined in the soil near the Japanese smelter. In this context, Babich and Stotzky (5) emphasized the significance of the physicochemical characteristics of the soil regarding toxicity of cadmium on microorganisms. Fusarium oxysporium isolated from a contaminated area had more tolerance to heavy metals than the same species isolated from the control area, suggesting that fungi isolated from contaminated soils have a greater than normal tolerance to heavy metals (24). To explain the resistance of Staphylococcus aureus to cadmium (and also mercury) ions, Chopra (I 4) took into consideration penicillinase plasmids and some related extrachromosomal elements. The resistance of some strains of S. aureus has been related to other physiological characteristics: resistance against tetracycline and several other antibiotics in connection with high penicillinase production. Mercury-resistant staphylococci are usually, though not invariably, tetracycline resistant, whereas tetracycline resistance in mercury-sensitive strains occurs only sporadically (46). Extrachromosomal determinants of penicillin resistance in organisms are linked to those of cadmium resistance (42). Coresistance of bacteria against heavy metals and several antibiotics is considered to be a current problem of hospitals (34, 46). Outside hospital environments metal and antibiotic coincidence has been detected in aquatic habitats, e.g., in guts of fish living in heavy metal- or sewage-polluted rivers and in sediments with high concentrations of heavy metals (34). Cadmium, added to a mixed population litter system (Douglas fir needle litter microcosm), benefited bacteria that were both resistant to cadmium inhibition and more or less resistant to antibiotic inhibition, depending on the antibiotic. These cadmiumtreated microcosms had facilitated cadmium and gentamicin resistance and streptomycin and chloramphenicol sensitivity (34). Chopra (14) investigated the uptake of ‘I5 mCdC12 by a sensitive and a resistant strain of S. aureus and found that the total cadmium uptake by the sensitive cells was about 15 times higher than the uptake by the resistant cells, and that 40% of the radioactive cadmium ions accumulated in the sensitive cells were nonexchangeable, obviously bound strongly at intracellular structures rather than absorbed adventitiously to the surface. The increased cadmium uptake by sensitive cells proved by Chopra might refer to the hypothesis that resistance of organisms against inorganic ions is based on impermeability of the cell wall for these ions, as formulated by Tornabene and Edwards (41) who postulated the important role of the cell wall as a protection mechanism for the microbial cell, in connection with studies concerning the uptake of lead by the lead-resistant bacteria Micrococcus luteus and Azotobacter sp. In the case of C. pyrenoidosa the ability to synthesize a metallothionein-like protein is suggested by Hart et al. (22) on the basis of preliminary experiments. Metallothionein has a high metal and sulfur content, cysteinyl residues accounting for 30-35s of the total amino acid composition, and it has the highest cysteine
EFFECTS
OF CADMIUM
ON
MICROORGANISMS
163
and, hence, -SH content of any protein known (29). Instead of S-S bridges each three cysteines bind one atom of cadmium (or other metal). A cadmium- and zinc-binding macromolecule of molecular weight 10,000 to 12,000 hais been reported by Maclean et al. (cited by (29)) in a blue-green alga, A. nidufans, grown in a medium containing CdClz and ZnCl,. Because of the high amount of metal-binding sites and increased biosynthesis by administration of cadmium, copper, or zinc (29), the hypothesis of detoxification by metallothionein gains support. Ashworth and Amin (3) postulated a pool of intracellular SH that is free of protein and protects enzyme systems by forming complexes with the toxicant, in order to explain the tolerance of mycelium of A. niger to mercury. Yet, according to Chopra (14), the biochemical basis of microbial resistance to cadmium is not totally clarifed. Resistance against heavy metals or other toxic substances may be generated or increased Iduring continuous influence of the toxic substance. In that case, it is a question of “adaptation” or “training” of sensitive organisms to the toxicant, a process that is not based on genetic determination (cp. (46)). Two basic measures for the development of resistance are: (a) the concentration of the toxicant has to be increased to accomplish the same deleterious effect as before, and (b) the same concentration of the toxicant causes a less deleterious effect than before (2). REFERENCES I. 2.
3. 4. 5.
6. 7.
8. 9. 10.
II. 12. 13.
The breakdown of pesticides in soils. In Agriculture and the Qualify of Our Environment (N. C. Brady, Ed.), Vol. 85, pp. 331-342. ASHIDA, J. (1965). Adaptation of fungi to metal toxicants. Annu. Rev. Phytopathol. 3, 153-175. ASHWORTH, L. J., AND AMIN, J. V. (1964). A mechanism for mercury tolerance in fungi. Phytopafhology 54, 1459-1463. BABICH, H., AND STOTZKY, G. (1974). Air pollution and microbial ecology. Crit. Rev. Environ. Control 4, 353-421. BABICH, H., AND STOTZKY. G. (1977). Effect of cadmium on fungi and on interactions between fungi and bacteria in soil: Influence of clay minerals and pH. Appl. Environ. Microbial. 33(5), 1059~1066. BABICH. H., AND STOTZKY, G. (1977). Reductions in the toxicity of cadmium to microorganisms by clay minerals. Appl. Environ. Microbial. 33( 3), 696-705. BABICH, H., AND STOTZKY, G. (1977). Sensitivity of various bacteria, including actinomycetes, and fungi to cadmium and the influence of pH on sensitivity. Appl. Environ. Microbial. 33(3), 681695. BANAT, I<.. F~RSTNER, U., AND MUELLER, G. (1972). Schwermetalle in Sedimenten von Donau, Rhein, Ems, Weser und Elbe im Bereich der BRD. Naturwissenschuften 59, 525-528. BARTLETT, L., RABE, F. W.. AND FUNK, W. H. (1974). Effects of copper, zinc, and cadmium on Selanastrum capricornutum. Water Rex 8. 179- 185. BOND, H,, LIGHTHART, R., SHIMABUKU, R., AND RUSSELL, L. (1976). Some effects of cadmium on coniferous forest soil and litter microcosms. Soil Sci. 121(5), 278-287. BOOTH, G. H., AND MERCER, S. J. (1963). Resistance to copper of some oxidizing and reducing bacteria. Nature (London) 199, 622. BROWN, ‘T. A., AND SMITH, D. G. (1977). Cytochemical localization of mercury in Cryptococcus albidus grown in the presence of mercuric chloride. .I. Gen. Microbial. 99, 435-439. CHANEY, W. R.. KELLY, J. M., AND STRICKLAND, R. C. (1978). Influence of cadmium and zinc on carbon dioxide evolution from litter and soil from a black oak forest. J. Environ. Qual. 7(l), 115-119. ALEXANDER,
M.
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14. CHOPRA, I. (1971). Decreased uptake of cadmium by a resistant strain of Staphylococcus aureus. J. Gen. Microbial. 63, 265-267. 15. DE LAVAL, J., AND DEMONTY, J. (1972). l?volution de la microflore du sol en fonction de sa concentration en Zn et en Pb. Rev. Ecol. Biol. Sol. 9, 491-504. 16. DOYLE, J. J., MARSHALL, R. T., AND PFANDER, W. H. (1975). Effects of cadmium on the growth and uptake of cadmium by microorganisms. Appl. Microbial. 29, 562-564. 17. ERNST, W. (1972). Zink- und Cadmiumimmissionen auf B&ien und Pflazen in der Umgebung einer Zinkhiitte. Ber. Dtsch. Bof. Ges. 85, 295-300. 18. FLICK, D. F., KRAYBILL, H. F., AND DIMITROFF, J. M. (1971). Toxic effects of cadmium: A review. Environ. Rex 4, 7 l-85. 19. FRIBERG, L., PISCATOR, M., NORDBERG, G. F., AND KJELLSTROM, T. (1974). Cadmium in the Environment. CRC Press, Cleveland. 20. FURUKAWA, K., SUZUKI, T., AND TONOMURA, K. (1969). Decomposition of organic mercurial compounds by mercury-resistant bacteria. Agr. Biol. Chem. 33, 128- 130. 21. HAHNE, H. C. H., AND KROONTJE,W. (1973). Significance of pH and chloride concentration on behaiovr of heavy metal pollutants: Mercury (II), cadmium (II), zinc (II). and lead (II). J. Environ. Qual. 2, 444-450. 22. HART, B. A., BERTRAM, P. E., AND SCAIFE, B. D. (1979). Cadmium transport by Chlorella pyrenoidosa. Environ, Rex 8, 327-335. 23. HART, B. A., AND SCAIFE, B. D. (1977). Toxicity and bioaccumulation of cadmium in Chlorella pyrenoidosa. Environ. Res. 14, 40 l-4 13. 24. HARTMANN, L. M. (1974). A preliminary report: Fungal flora of the soil as conditioned by varying concentrations of heavy metals. Amer. J. Bat. 61(Suppl. 5), 23. 25. HERRMANN, R. (1976). Modellvorstellung zur rgumlichen Verteilung von Spurenmetallverunreinigungen in der BRD, angezeigt durch den Metallgehalt in epiphytischen Moosen. Erdkunde 30(4), 241-253. 26. HUISINGH, D. (1974). Heavy metals: Implications for agriculture. Annu. Rev. Phyfopathol. 12, 375-388. 27. JERNEL~V, A. (1969). Conversion of mercury compounds. In Chemical Fallout (M. W. Miller, and G. G. Berg, Eds.), Chap. 4. C. C Thomas, Springfield, Ill. 28. JORDAN, M. J., AND LECHEVALIER, M. P. (1975). Effects of zinc smelter emissions on forest soil microflora. Canad. J. Microbial. 2l( 1 1 ), 1855- 1865. 29. K~GI, J. H. R., AND NORDBERG, M. (1979). Metallothionein. In Proceedings, First, International Meeting on Metallofhionein and Orher Low Molecular Weight Metal-Binding Proteins, Ziirich. July 17-22, 1978. Birkhiiser, Basel/Boston/Stuttgart. 30. KLOKE, A. (1972). Zur Anreicherung von Cadmium in Bidden und Pflanzen. Landwirtsch Forsch. 27, 200-206. 31. LAGERWERFF, J. V. (1972). Lead, mercury, and cadmium as environmental contaminants. In Micronutrienfs in Agriculture (J. J. Mortvedt, Ed.). Amer. Sot. of Agronomy, Madison, Wise. 32. LEPP, N. W., AND ROBERTS, M. J. (1977). Some effects of cadmium on growth of bryophytes. Bryologist 80(3), 533-536. 33. LIANG, C. N., AND TABATABAI, M. A. (1977). Effects of trace elements on nitrogen mineralisation in soils. Environ. Pollut. 12, 141-147. 34. LIGHTHART, B. (1979). Enrichment of cadmium-mediated antibiotic resistant bacteria in a DouglasFir (Pseudotsuga menziesii) litter microcosm. Appt. Environ. MicrobioI. 37(5), 859-861. 35. MANG, S., AND THROMBALLA, H. W. (1978). Aufnahme von Cadmium durch Chlorella fusca. Z. Pflanzenphysiol. 90, 293-302. 36. MORRISSEY, R. F., DUGAN, E. P., AND KOTHS, J. S. (1974). Inhibition of nitrification by the incorporation of select heavy metals in soil. Absrr. Annu. Meet. Amer. Sot. Microbial. 74, 2. 37. PANCHOLY, S. K., ELROY, L. R., AND TURNER, J. A. (1975). Soil factors preventing revegetation of a denuded area near an abandoned zinc smelter in Oklahoma. J. Appl. Ecol. 12, 337-342. 38. PFEIFFER-MALISZEWSKA, W. (1977). Effects of boron and cadmium on nitrogen fixation in soil. Acta Microbial. Pal. 26(3), 295-300. 39. STARK, N. (1972). Nutrient cycling pathways and litter fungi. Bioscience 22(6), 355-360. 40. THORMANN, D., AND WEYLAND, H. (1979). Beziehungen zwischen verschiedenen Brackwasserund Meeresbakterien und der wachstumshemmenden Wirkung von Cadmium und Blei. Veroeff: Inst. Meeresforsch. Bremerhaven 17, 163- 188.
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41. TORNABENE, T. G., AND EDWARDS, H. W. (1972). Microbial uptake of lead. Science 176, 1334. 42. VALLEE, B. L., AND ULMER, D. D. (1972). Biochemical effects of mercury, cadmium and lead. Annu. Rev. Biochem. 41, 91-128. 43. WACHS. B. (1978). Kontamination der Oberflachengewasser durch Cadmium. In Mtinchener Beitriige zur Abwasser-, Fischerei-, und Flussbiologie, Band 30, ed. by Bayerische Landesanstalt fur Wasserforschung. Munchen. 44. WEBB, M. (Ed.). (1979). The Chemistry, Biochemistry and Biology of Cadmium. Elsevier/NorthHolland Biochemical Press, Amsterdam/New York/Oxford. 45. WILLIAIMS, J. I., AND PUGH, G. J. F. (1975). Resistance of Chrysosporium pannorum to an organ0 mercury fungicide. Trans. Brif. Mycol. Sot. 64(2), 255-264. 46. WILLIAMS, R. F. (1967). Mercury resistance and tetracycline resistance in Staphylococcus aureus. J. Hjg. 65, 299-309. 47. WOLLUI~~, A. G. (1973). Effects on the agricultural ecosystem immobilization of cadmium by soil microorganisms. Environ. Health Perspect. 4, 103. 48. ZWARUN, A. A. (1973). Tolerance of Escherichia coli to cadmium. J. Environ. Quaf. 2(3), 353355.
AND
ENVIRONMENTAL
Effects
SAFETY
6, 157-165
of Cadmium
(1982)
on Microorganisms
BRIGITTE BISCHOFF Universitdt
Oldenburg,
Fachbereich Received
May
XII,
Oldenburg,
Wesr Germany
20, 1981
A short review was made of current knowledge, particularly of experimental and laboratory studies, relating to cadmium toxicity on microorganisms. The survey includes several aspects of cadmium toxicology, such as growth inhibition, lethality, physiological alterations, microbial resistarme to cadmium, uptake, and bioaccumulation. The spectrum of heavy metal toxicity on microorganisms involves initial growth inhibition up to the total elimination of a microbial population, and includes alterations of morphology, e.g., mitochondria, Golgi, and ribosomes as probable sites of damage, of metabolic pathways or enzymatic activities. Heavy metals may cause cytostatic or mutagenic effects, as described for Salmonella spp. and Escherichia coli. Microorganisms have the potential to accumulate and deposit heavy metals without showing adverse effects, to develop mechanisms of resistance, and to adapt to increasing concentrations of heavy metals. Cadmium is found to be virtually ubiquitous (18, 31). It is not only deposited and accumulated in various body tissues, but is also found in varying concentrations throughout all environmental compartments, since there has been an increasing introduction of cadmium into the environment as a result of mining operations, industrial pollution, and agricultural runoff ((19) cp. also (8, 25)). The toxicity of cadmium has been studied with regard to several aspects (7, 17-l 9, 30, 32, 43). Since cadmium is known to be a hazardous toxicant and enters into food webs as a result of various processes, it becomes important to study the toxic effects on the microb,iota to acquire a broad knowledge of the toxic potential of this metal. INVOLVEMENT OF MICROORGANISMS IN BIOLOGICAL PROCESSES AND CONTAMINATION Soil microorganisms are also involved in biogeochemical and decomposition processes necessary for nutrient cycling (39) and maintenance of soil fertility and in numerous interactions with other microorganisms, plants, and animals. (5). An essential part of microbiological activities is concerned with decomposition of organic matter like plant litter and mineralization of organically bound elements like nitroglen. An interruption or reduction in organic matter decomposition and mineralization might eventually result in a deficiency of essential nutrients for plants and., as a consequence, in a limitation of plant growth. On the other hand, microorganisms themselves are involved in pollution in multiple ways (4): ( 1) microorganisms are a primary natural biotic source of substantial quantities of gaseous pollutants (emission of inorganic and organic gases and volatiles),
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(2) they serve as sinks for a variety of pollutants, and (3) they are capable of reactivating contaminants from terrestrial and aqueous environments by transforming decomposed and deposited pollutants into more or less toxic forms (cp. (I, 27)). Contaminants in the form of air pollutants or as components of sewage or agricultural runoff can affect the microbiota on different levels of microbial activities and relations. The proven detrimental effect on the microbial reproductive potential (e.g., reduction in growth rates, inhibition of fungal sporulation) could influence not only microbial establishment, population dynamics, and interactions between microorganisms, but also the general ecology of the microbiota in natural habitats (7) and, as a consequence, can cause profound alterations in the contaminated environment. Another aspect of the ecological effects of heavy metal pollution is the problem of deficient or absent vegetation in a contaminated area in regard to colonization with microorganisms. This aspect has been described as the “rhizophere effect” (1.5, 28). INFLUENCE
OF VARIOUS INHIBITION
CONDITIONS BY CADMIUM
ON GROWTH
The toxicity of a contaminant is dependent on the physicochemical characteristics of the environment into which it is deposited, resulting in a final decrease or increase of the toxicity of the contaminant (cp. (5)). The amount of available cadmium is partially dependent on the cation-exchange capacity (CEC) of the environment, which, in turn, is a function of the types and concentrations of organic matter and clay minerals (6). Clay minerals in particular evolve a protective effect against cadmium toxicity, just as Babich and Stotzky proved for fungi (5) as well as for bacteria, actinomycetes, and filamentous fungi (6). The protective effect of the clay minerals studied, montmorillonite and kaolinite, is correlated to their CEC (cp. also (13)); increasing protection is apparently related to higher CEC and the capacity to absorb greater quantities of exogenous cadmium. In the absence of cadmium, in montmorillonite-amended agar, bacterial growth was stimulated, and fungal respiration was inhibited, but was unaffected by kaolinite (6), whereas in the presence of cadmium, montmorillonite and, to a lesser extent, kaolinite decreased the inhibitory effects of cadmium to bacteria, actinomycetes, and fungi. Cadmium is strongly bound to organic matter, and the degree of binding of cadmium ions in different types of soils follows the sequence organic soil > heavy clay soil > sandy and silt loam soil > sandy soil (6). In pure culture studies actinomycetes showed more tolerance to cadmium than eubacteria, and gram-negative more than gram-positive eubacteria. Fungal sporulation was inhibited to a higher extent than mycelial growth (7). The toxicity of cadmium to eubacteria, actinomycetes, and fungi turned out to be pH dependent, and toxicity was potentiated at pH 8 or 9 in the cases of Bacillus cereus, Alcaligenes faecalis, Agrobacterium tumefaciens, Nocardia parajjinae, Aspergillus niger, Trichoderma viride, and Rhizopus stolonifer; the exception was Streptomyces olivaceus in which the toxicity of cadmium was independent of pH. It was not
EFFECTS
OF CADMIUM
ON
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clarified whether the increased toxicity was a reflection of the formation of complex ionic and molecular species of hydroxylated cadmium (7). On the other hand, growth inhibition of Chlorella pyrenoidosa seemed more pronounced at pH 7 than pH 8 (23) suggesting that removal of cadmium from the culture medium and its simultaneous deposition within the cells might be a pHdependent process (heavy metals associate with particles that are formed to a larger size under alkaline conditions in the medium). Inhibition of growth rates of Aspergillus niger was more pronounced by 100 or 250 pg of Cd/g in soil adjusted to pH 7.2 than in the same soil at its natural pH of 5.1, but there were no differences in growth rates at both pH 5.1 and 7.2 in the case of A. fischerii (5). The reaction of A. niger was supposed to be due to either an inability of the fungus to tolerate an additional heavy metal stress at alkaline levels or a synergistic interaction between cadmium and the higher pH level. At pH 6-7 cadmium is present in the divalent ionic form and is completely soluble in the absence of precipitating anions, such as phosphate and sulfide, resulting in availability for adsorption on suspended mineral colloids and complexation with organic matter, and cadmium may move in these forms (21). To determine heavy metal distribution in the environment, chlorides, under certain circumstances, may also be of great significance (21). In areas where organic matter accumulation takes place, organo-metal ion complexes are formed which may exist in soluble or colloidal form and, therefore, differ in degree of mobility. Results indicated that the hydroxy and chloride complexes may contribute to the mobilization of heavy metal ions in the environment (21). Yet, indlependent of environmental circumstances, the toxicity of cadmium to several microbial organisms might be graduated. Studies of organisms isolated from cadmium-amended and -unamended soils indicated that each organism had its own characteristic tolerance level to added cadmium (47). Regarding cadmium toxicity on algal growth in aqueous environments, effects of copper, zinc, and cadmium on Selanastrum capricornutum were investigated by Bartlett et al. (9). They found that combinations of copper, zinc, and cadmium were similar in toxicity to equal concentrations of zinc. Combinations of copper and cadmium resulted in greater growth than equal concentrations of copper, suggesting that cadmium inhibits copper toxicity. The algicidal concentration of cadmium was 0.65 mg/liter. Investigations with extremely Cd-sensitive and Cd-tolerant bacteria and extremely Pb-sensitive and Pb-tolerant bacteria isolated from brackish estuarine water showed that cadmium was more inhibitory to growth of Pb-sensitive bacteria than lead was to growth of Cd-sensitive strains (40). In general, neither cadmium nor lead had a pronounced specific effect of growth inhibition on single species of bacteria: Cd-sensitive bacteria were, too, more sensitive to lead than Cd-tolerant strains. The exalmination of 259 strains of marine origin pointed out that gram-negative bacteria w’ere more resistant to cadmium than gram-positive bacteria (for similar observations, cp. (7)). Moreover, a pronounced sensitivity or resistance to cadmium and lead was dependent on the genus, as well as on the geographical origin, of the marine bacteria studied (40).
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PHYSIOLOGICAL ALTERATIONS BY CADMIUM BY COz FIXATION, RESPIRATION, NITROGEN TRANSFORMATION
EXEMPLIFIED AND
Under conditions which promote accumulation of cadmium, Chlorella pyrenoidosa was still fixing CO*, albeit at reduced rates (at 1.OO mg of Cd/liter 39% fixation rate of the control), whereas O2 evolution was inhibited to a lesser extent (at 1.00 mg of Cd/liter 80%) (23). The reduction of a microbial culture as a result of a lethal cadmium effect obviously is connected with decreased simultaneous CO2 evolution. In a culture medium of Escherichia coli 6 mg of Cd/liter (as CdC12) and more caused decreased CO* production, correlated with a decrease in the number of cells surviving (48). Low levels of zinc and cadmium seemed to have a slightly stimulating effect on litter decomposition by a mixed population of soil and litter microorganisms, whereas high concentrations were shown to decrease respiration rates (13). The toxic effect of high concentrations of Cd2+ and Zn2+ on the activity of soil and litter microorganisms was suggested to be due to their ability to compete with essential elements (Mn, Fe, Mg) for the active sites (-SH, -NH2, -NH) of enzymes, but a mechanism for the stimulatory effect was stated to be unknown (13). Cadmium, as CdC12 (mean concentrations of 0.01 and 10.0 ppm), introduced into soil/litter microcosms markedly altered soil respiration without a concomitant decrease in organisms. This observation was suggested to indicate a slowdown of organismal activity, presumably to a level more conducive to long-term survival at low nutrient levels (IO). Varying levels of cadmium in sandy (50 to 1000 ppm) and alluvial (50 to 100 ppm) soil were registered to have an apparently stimulatory effect on nitrogen fixation (38), but only during the first 3 months of cadmium incubation; after 8 to 12 months there was a remarkable decrease in nitrogen fixation. In sandy soil, consisting of no organic matter, cadmium had the most deleterious effect on nitrogen fixation. Nitrate utilization by organisms in cadmium-amended soils was not influenced by levels of cadmium up to 100 pg/g of soil, but seemed to be inhibited by cadmium contents greater than 100 pg/g of soil (47). At 1000 pg Cd/g of fine sandy loam soil, the rates of ammonium utilization and nitrate production were reduced; however, the inhibition was not evident. Both nitrogen transformations were completely blocked at 10,000 pg Cd/g. It would appear that inhibition at this rate was related to an unfavorable pH in conjunction with metal toxicity. The results indicate that nitrification in soil is relatively resistant to the toxic action of heavy metals (36). Inhibition of nitrification by Ag(I), Hg(II), and Cd(I1) in various soils causes accumulation of NH4-N (33), as long as the ammonification rate is not reduced at the same time, as reported by Pancholy et al. (37). Furthermore, low activities of urease and dehydrogenase determined in soil from a denuded and contaminated area indicated the reduction of various other physiological activities (37). UPTAKE
AND
BIOACCUMULATION
OF CADMIUM
From consideration of the increasing cadmium concentrations in rivers, lakes, coastal waters, and sediments the ability of microorganisms to accumulate cadmium at a high rate appears to be possible. As reported by Maclean et al. (cited by (16)),
EFFECTS
OF CADMIUM
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E. coli, A,uacystis midulans, and Chlorella spp. grown in a culture medium containing lo9CdC12 (lo-’ M) took up 9, 98, and 80% of the available cadmium, respectively, paralleled to growth. The ability of microorganisms, particularly E. coli and B. cereus, to immobilize large amounts of cadmium from the broth and to grow in the presence of relatively high concentrations of cadmium might indicate, according to Doyle et a/. (16), the possession of specific inherited resistance to inorganic ions by these organisms. Experimental studies investigating toxicity and bioaccumulation of cadmium in C. pyrenoidosa (23) pointed out that the amount of cadmium accumulated by the organisms was directly proportional to the concentration of metal initially present in the medium. There was no accumulation in the dark, at 4”C, or by dead cells. In this experiment, cadmium accumulation was not affected by concentrations of Ca, Mg, MO, Cu, Zn, or Co in the growth medium, but results concerning this problem are very different. Antagonistic or synergistic effects are possibilities, when combinations of different metals are present (44). Only manganese, at a level equal to or greater than 0.20 mg/liter, completely blocked cadmium accumulation (23). Iron might also play a role in regulating cadmium accumulation, since Fe-EDTA complex o’r additional iron as a supplement (FeCl,) restored the growth rate of C. pyrenoidosa to normal. Manganese was more effective as a blocking agent than iron; competitive inhibition was presumed as a most likely mechanism for the action of manganese on this system (23). The toxicity of Hg, Cd, and Pb not only may be modified by other elements such as Se but also may interfere with normal metabolism of essential trace elements. In animal dietary studies it was found that cadmium interferes with both copper and zinc metabolism, apparently because Cd*+ has the same chemical parameters as Zn*+ and Cu*+ (26). Further studies suggest that cadmium obviously shares an uptake system with one or more cations that are normally required for growth and there is evidence for the possibility that cadmium and manganese are transported by a common uptake system (22): -manganese inhibits cadmium uptake in a concentration-dependent manner and vice versa, --‘09Cd efflux from cells was brought about to the same extent by manganese as by “*Cd, -kinetic studies showed that cadmium and manganese appear to compete for the same binding sites and manganese might be expected to show a higher affinity than cadmium for cell binding sites (cp. also 12). Mang and Thromballa (35) suspected the zinc transport system of being the carrier mediating cadmium uptake, based on kinetic studies comparing the uptake of cadmium and zinc ions and examining their mutual inhibition. The experiments conducted under different conditions of energy supply showed, besides strong, fast, and for the most part reversible binding by absorption, very effective energy-dependent uptake, obviously by intake into the interior of the cell. Mang and Thromballa assurned active transport, as is the case with zinc. MECHANISMS
OF RESISTANCE
Several :investigators confirmed microbial manifestations of resistance to heavy metals (3, 1 I, 14, 20, 4.5, 46, 48). Babich and Stotzky (5) stated that A. niger and
162
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A. fischeri& isolated from an area contaminated with heavy metals near a Japanese smelter (28 pg of Cd/g of soil, pH 5.7), turned out to be relatively resistant to cadmium, since the growth obtained in the contaminated soil was the same as growth some distance away (4 pg of Cd/g of soil, pH 6.1). The toxicity of cadmium to A. niger was mitigated by addition of zinc; high concentrations of zinc (1.460 pg/g) had also been determined in the soil near the Japanese smelter. In this context, Babich and Stotzky (5) emphasized the significance of the physicochemical characteristics of the soil regarding toxicity of cadmium on microorganisms. Fusarium oxysporium isolated from a contaminated area had more tolerance to heavy metals than the same species isolated from the control area, suggesting that fungi isolated from contaminated soils have a greater than normal tolerance to heavy metals (24). To explain the resistance of Staphylococcus aureus to cadmium (and also mercury) ions, Chopra (I 4) took into consideration penicillinase plasmids and some related extrachromosomal elements. The resistance of some strains of S. aureus has been related to other physiological characteristics: resistance against tetracycline and several other antibiotics in connection with high penicillinase production. Mercury-resistant staphylococci are usually, though not invariably, tetracycline resistant, whereas tetracycline resistance in mercury-sensitive strains occurs only sporadically (46). Extrachromosomal determinants of penicillin resistance in organisms are linked to those of cadmium resistance (42). Coresistance of bacteria against heavy metals and several antibiotics is considered to be a current problem of hospitals (34, 46). Outside hospital environments metal and antibiotic coincidence has been detected in aquatic habitats, e.g., in guts of fish living in heavy metal- or sewage-polluted rivers and in sediments with high concentrations of heavy metals (34). Cadmium, added to a mixed population litter system (Douglas fir needle litter microcosm), benefited bacteria that were both resistant to cadmium inhibition and more or less resistant to antibiotic inhibition, depending on the antibiotic. These cadmiumtreated microcosms had facilitated cadmium and gentamicin resistance and streptomycin and chloramphenicol sensitivity (34). Chopra (14) investigated the uptake of ‘I5 mCdC12 by a sensitive and a resistant strain of S. aureus and found that the total cadmium uptake by the sensitive cells was about 15 times higher than the uptake by the resistant cells, and that 40% of the radioactive cadmium ions accumulated in the sensitive cells were nonexchangeable, obviously bound strongly at intracellular structures rather than absorbed adventitiously to the surface. The increased cadmium uptake by sensitive cells proved by Chopra might refer to the hypothesis that resistance of organisms against inorganic ions is based on impermeability of the cell wall for these ions, as formulated by Tornabene and Edwards (41) who postulated the important role of the cell wall as a protection mechanism for the microbial cell, in connection with studies concerning the uptake of lead by the lead-resistant bacteria Micrococcus luteus and Azotobacter sp. In the case of C. pyrenoidosa the ability to synthesize a metallothionein-like protein is suggested by Hart et al. (22) on the basis of preliminary experiments. Metallothionein has a high metal and sulfur content, cysteinyl residues accounting for 30-35s of the total amino acid composition, and it has the highest cysteine
EFFECTS
OF CADMIUM
ON
MICROORGANISMS
163
and, hence, -SH content of any protein known (29). Instead of S-S bridges each three cysteines bind one atom of cadmium (or other metal). A cadmium- and zinc-binding macromolecule of molecular weight 10,000 to 12,000 hais been reported by Maclean et al. (cited by (29)) in a blue-green alga, A. nidufans, grown in a medium containing CdClz and ZnCl,. Because of the high amount of metal-binding sites and increased biosynthesis by administration of cadmium, copper, or zinc (29), the hypothesis of detoxification by metallothionein gains support. Ashworth and Amin (3) postulated a pool of intracellular SH that is free of protein and protects enzyme systems by forming complexes with the toxicant, in order to explain the tolerance of mycelium of A. niger to mercury. Yet, according to Chopra (14), the biochemical basis of microbial resistance to cadmium is not totally clarifed. Resistance against heavy metals or other toxic substances may be generated or increased Iduring continuous influence of the toxic substance. In that case, it is a question of “adaptation” or “training” of sensitive organisms to the toxicant, a process that is not based on genetic determination (cp. (46)). Two basic measures for the development of resistance are: (a) the concentration of the toxicant has to be increased to accomplish the same deleterious effect as before, and (b) the same concentration of the toxicant causes a less deleterious effect than before (2). REFERENCES I. 2.
3. 4. 5.
6. 7.
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The breakdown of pesticides in soils. In Agriculture and the Qualify of Our Environment (N. C. Brady, Ed.), Vol. 85, pp. 331-342. ASHIDA, J. (1965). Adaptation of fungi to metal toxicants. Annu. Rev. Phytopathol. 3, 153-175. ASHWORTH, L. J., AND AMIN, J. V. (1964). A mechanism for mercury tolerance in fungi. Phytopafhology 54, 1459-1463. BABICH, H., AND STOTZKY, G. (1974). Air pollution and microbial ecology. Crit. Rev. Environ. Control 4, 353-421. BABICH, H., AND STOTZKY. G. (1977). Effect of cadmium on fungi and on interactions between fungi and bacteria in soil: Influence of clay minerals and pH. Appl. Environ. Microbial. 33(5), 1059~1066. BABICH. H., AND STOTZKY, G. (1977). Reductions in the toxicity of cadmium to microorganisms by clay minerals. Appl. Environ. Microbial. 33( 3), 696-705. BABICH, H., AND STOTZKY, G. (1977). Sensitivity of various bacteria, including actinomycetes, and fungi to cadmium and the influence of pH on sensitivity. Appl. Environ. Microbial. 33(3), 681695. BANAT, I<.. F~RSTNER, U., AND MUELLER, G. (1972). Schwermetalle in Sedimenten von Donau, Rhein, Ems, Weser und Elbe im Bereich der BRD. Naturwissenschuften 59, 525-528. BARTLETT, L., RABE, F. W.. AND FUNK, W. H. (1974). Effects of copper, zinc, and cadmium on Selanastrum capricornutum. Water Rex 8. 179- 185. BOND, H,, LIGHTHART, R., SHIMABUKU, R., AND RUSSELL, L. (1976). Some effects of cadmium on coniferous forest soil and litter microcosms. Soil Sci. 121(5), 278-287. BOOTH, G. H., AND MERCER, S. J. (1963). Resistance to copper of some oxidizing and reducing bacteria. Nature (London) 199, 622. BROWN, ‘T. A., AND SMITH, D. G. (1977). Cytochemical localization of mercury in Cryptococcus albidus grown in the presence of mercuric chloride. .I. Gen. Microbial. 99, 435-439. CHANEY, W. R.. KELLY, J. M., AND STRICKLAND, R. C. (1978). Influence of cadmium and zinc on carbon dioxide evolution from litter and soil from a black oak forest. J. Environ. Qual. 7(l), 115-119. ALEXANDER,
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41. TORNABENE, T. G., AND EDWARDS, H. W. (1972). Microbial uptake of lead. Science 176, 1334. 42. VALLEE, B. L., AND ULMER, D. D. (1972). Biochemical effects of mercury, cadmium and lead. Annu. Rev. Biochem. 41, 91-128. 43. WACHS. B. (1978). Kontamination der Oberflachengewasser durch Cadmium. In Mtinchener Beitriige zur Abwasser-, Fischerei-, und Flussbiologie, Band 30, ed. by Bayerische Landesanstalt fur Wasserforschung. Munchen. 44. WEBB, M. (Ed.). (1979). The Chemistry, Biochemistry and Biology of Cadmium. Elsevier/NorthHolland Biochemical Press, Amsterdam/New York/Oxford. 45. WILLIAIMS, J. I., AND PUGH, G. J. F. (1975). Resistance of Chrysosporium pannorum to an organ0 mercury fungicide. Trans. Brif. Mycol. Sot. 64(2), 255-264. 46. WILLIAMS, R. F. (1967). Mercury resistance and tetracycline resistance in Staphylococcus aureus. J. Hjg. 65, 299-309. 47. WOLLUI~~, A. G. (1973). Effects on the agricultural ecosystem immobilization of cadmium by soil microorganisms. Environ. Health Perspect. 4, 103. 48. ZWARUN, A. A. (1973). Tolerance of Escherichia coli to cadmium. J. Environ. Quaf. 2(3), 353355.