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The effect of cadmium ion on the growth, photosynthesis, and nitrogenase activity of Anabaena inaequalis
Chemosphere No. 5, PP 277 - 282. © P e r g a m o n Press Ltd. 1979. Printed
0045-6555/79/0501-0277~02.00/0 in Great Britain.
THE EFFECT OF CADMIUM ION ON THE GROWTH, PHOTOSYNTHESIS, NITROGENASE ACTIVITY OF ANABAENA INAEQUALIS Glenn W. Stratton Department
AND
and Charles T. Corke
of Environmental Biology, Guelph, Ontario, Canada.
University NIG 2WI.
of Guelph,
T~e growth of Anabaena inaequalis was significantly inhibited by Cd 2 concentrations greater than 0.02 ppm (~g/ml) and completely inhibited at 0.06 ppm (Day 12). Cadmium had no significant effect upon the lag phase of growth or the culture doublin~ time, but caused the retardation phase to arrive sooner. One ppm Cd ~+ significantly inhibited the ra~es of both photosynthesis and acetylene reduction, by ~. inaequalis, with complete inhibition at 4 and 20 ppm respectively. Cell sensitivity increased directly with exposure time. Cadmium caused some cell lysis of A. inaequalis and induced an increase in filament length, heterocyst frequency, and a loss of cellular contents from filament apical cells. The cellular abnormalities observed and the fact that toxicity increased with longer exposure times, suggested that metal toxicity resulted from effects of Cd 2 taken up by cells rather than Cd 2~ at the cell surface. INTRODUCTION Cadmium is a potentially hazardous pollutant in the biosphere since it is toxic to many life forms, 1 , 4 , 5 , 9 , 1 7 ~ 2 6 is a frequent contaminant in effluents 2 , 6, ~, 10, 17, 20, 26 and is extremely persistant in most environments 17, 26. It is rapidly absorbed and accumulated by plants s, 19, animals 26, and is toxic to man Iv One area of concern is the adverse effect of cadmium on phytoplankton. Much of the literature on this metal ion is concerned with its effects on the green alga Chlorella i, 12, 20, 28 and other eucaryotic algae ~' s, 9, 18, 19, while reports dealing specifically with the toxicity of cadmium towards blue-green algae are absent. Bluegreen algae are important components of the aquatic environment, since many are able to fix nitrogen, contributing up to one half of the annual nitrogen budget in some aquatic ecosystems 16. This paper describes the toxic effects of cadmium ion on the growth, photosynthetic, and nitrogen fixing abilities of a blue-green alga - Anabaena inaequalis &is part of a major investigation on the interactions of heavy metals and pesticides on biological transformations in aquatic ecosystems. MATERIALS
AND METHODS
Anabaena inaequalis was supplied by the Department logical Science, University of Guelph, Guelph, Ontario,
277
of Botany, College of BioCanada, and was maintained
27s
in a liquid, nitrogen-free medium* on a 12 hour light cycle at a temperature of 22°C and a light intensity of 7 Klux. These incubation conditions were standard for all growth, photosynthesis and nitrogen fixation experiments. All chemicals were supplied by the Fisher Scientific Co. with the exception of NaH Z~CO3, which was obtained from Amersham/Searle, Oakville, Ontario, Canada. Algal growth was assessed by measuring the optical density of the culture at 600 nm, wlth time, using a Bausch and Lomb Spectronic 20 spectrophotometer. Sidearm flasks of 500 ml capacity, containing 95 ml of medium, and I ml of an aqueous cadmium solution (as CdCI2.2½H20) were inoculated with 5 ml of a 7 day culture (standardized to give an initial algal population of 9 x i0 ~ cells/ml), and grown in still culture. Nitrogenase activity was determined using the acetylene-reduction technique z3 Tissue culture flasks with a surface area of 25 cm 2 and an internal volume of 74 ml were employed as assay chambers. Each culture flask contained 9 ml of algal culture in the logarithmic stage of growth (1.6 x 107 cells) and 1 ml of either growth medium or cadmium solution. Flasks were sealed with tight fitting rubber serum stoppers and a 10% volume of air removed and replaced with acetylene. Activity was monitored hourly for 5 hours, or at the end of 5 hours incubation. Ethylene production was assayed by the injection of i ml gas samples, taken from the sealed flasks, into a Varian Aerograph Model 1800 Gas Chromatograph, equipped with hydrogen flame ionization detectors and a Porapak N column (50/80 mesh, 1/8 in i.d. by I0 ft). The unit was operated under the following conditions: detector, 200°C; injector, 150°C; column, lO0°C; hydrogen, 30 ml/min; air, 300 ml/min; nitrogen, 48 mi/min. Ethylene peaks on recorder tracings were identified by retention time, and quantitated by comparing to standard curves. Photosynthesis was determined by following the uptake of Z~CO2 from NaHZ~C03 30 The tissue culture flasks contained 8.9 ml of algal culture in the logarithmic phase of growth (1.2 x 107 cells), 0.i ml of the labelled bicarbonate solution (to give a final activity of 0.2 ~Ci/ml) and 1 ml of elther growth medium or cadmium solution. Activity was assayed every 30 minutes for 2 hours, or after 2 hours incubation. One ml samples were filtered through 0.45 Dm membrane filters (Millipore - HAWP02500), washed successively with 5 ml of 0.i N HCI and 5 ml of water, and dried under an infrared lamp. Filter pads were placed in 20 ml scintillation vials, covered with i0 ml of a cocktail containing 5 gm PPO and 0.3 gm POPOP per litre toluene, and counted in a Beckman Mode] LS-3150T liquid scintillation counter. Counts were corrected for % counting efficiency and reduced by the background count. RESULTS AND DISCUSSION The yield (at Day 12) of Anabaena ~naequalis was significantly inhibited (based on Student's T test at e = 0.05) by ~-~2 concentrations of 0.03 ppm (~g/ml) and greater (Figure i). The~e was no significant inhibition of either growth rate or yield below 0.03 ppm Cd ~ , while 0.06 ppm was required for the complete inhibition of growth+ There was a linear relationship between percen~ inhibition of yield and logz0 Cd 2 concentration in the range 0.01 to 0.05 ppm Cd ~ (correlation coefficient, r = 0.990).
* The basal medium contained 0.039 g K2HPO~, 0.075 g MgSO~.7H20, 0.04 g CaCI2.6H20, 0.006 g citric acid monohydrate, 0.001 g EDTA, 0.02 g NaHCO3, and 0.006 g ferric citrate per litre. To this was added a micronutrient mixture, at a level of i ml/litre basal medium, which contained 2.86 g N3BO3, 1.81 g MnCI2.4H20, 0.222 g ZnSO~.TH20, 0.39 g NaMoO~.2H20, 0.079 g CuSO~.SH20 and 0.049 g CoC12.6H~O per litre. (Modified after Stanier et al. ~9.)
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279
TABLE i. The effect of Cd 2+ on acetylene reduction by Anabaena inaequalis. .-'"~ ..~*i......... b ..".~" - ~ . %:.,.."" ~ % ....... .,,~ ,,-~
0.8-
~ /•.:.'~-," , 18 o-4 ,..( d 0.2
•"."-~"
o" •
Cd z¢ concn, ~ppm) 0 1 2 4 6
~'~."
8
."~|"
I0 20
..4'-''" ~,
,i
Time
e,
I!l
lb
1~
(days)
FIGURE i. The effect of Cd 2+ on the growth of Anabaena inaequalis. (e) control; (O) 20 ppb CdZ'; (A) 30 ppb Cd2~; (4) 40 ~ b Cd2+; (m) 50 ppb Cd2+; (Q) 60 ppb Cd" . O.D. - optical density.
nmoles ethylene produced per 10 cells a ~ s.d. % Inhlb. b 2.60 0.56 i. 96 0.59 24.6 1.95 0.56 25.2 1.57 0.19 39.6 0.99 0.19 61.8 0.74 0.28 71.6 0.51 0.12 80.5 0.21 0.ii 92.0
a
b
Assayed after 5 hours incubation. Percent inhibition, based on control. All treatments differed significantly from the controls, based on a Student's T test at ~ = 0.05. The number of replicates = 11-15.
Cadmium had no significant effect upon either the lag phase of growth, which ranged from 0 to 0.8 days, or the culture doubling time, which ranged from 1.4 to 1.7 days. Cadmium did cause the retardation phase to occur sooner, indicating a delayed expression of toxicity. This is in contrast to results for Chlorella 2e, where cadmium treatment significantly increased the lag phase of growth. This difference could be related to cell concentration, as reducing the initial level of A. inaequalls to 5 from 9 x i0 ~ cells/ml also resulted in a significant increase in ~he lag phase~ from 0.5 to 2.0 days in response to 0.05 ppm Cd" . One ppm Cd 2 significantly reduced the amount of acetylene reduced over a 5 hour period, while 5 ppm was required to cause complete inhibition (Table i). +There was a linear relationship between the reduction in ethylene yield and logz0Cd 2 concentration over the range of 2 to 20 ppm for acetylene reduction (r = 0.981). When the time course of acetylene reduction was investigated, it was found that cadmium did not affect the early pattern of acetyl~ne reduction, but did cause this activity to reach zero at times dependent upon Cd 2 concentration. Control cultures demonstrated active nitrogenase activity throughout the 5 hour incubation, while activity ceased after i, 2 and 3 hours in those systems treated with I0, 5 and 1 ppm Cd 2 , respectively. Cadmium ion is removed from solution within 15 minutes by A. inaequalis cells (G.W. Stratton and C.T. Corke, unpublished data obtained using an ion specific electrode) and is known to be slowly concentrated by some algae 9, 19, and yeasts z~, 2s. The delayed toxicit~ towards growth and acetylene reduction may be related to the time required for Cd 2 to enter the cells. Cell lysis also played a role in the inhibition of acetylene reduction. Cell disruption would release the oxygen-labile nicrogenase enzyme complex into the medium, where it could undergo inactivation by oxygen i] and cadmium. The e~fect of lysis was determined by comparing the level of acetylene reduction in Cd 2 -treated flasks, containing either air or nitrogen atmospheres. The presence of a nitrogen atmosphere would reduce the amount of dissolved oxygen in the medium~ and thereby the level of 02 lltaotivation of released nitrogenase. At 5 ppm Cd 2 there was three
280
No. 5
TABLE 2. The effect of Cd 2+ on I~COz fixation by Anabaena inaequalis.
Cd zl
Counts per minute a
conch. J
s
/* oS
o
s J ~
30
eO ~o" Time (mtnulee)
lio
FIGURE 2. The effect of Cd 2+ on I~C02 fixation by Anabaena ina~qualis. (e) control; ( O ) ' - ~ p-~±Cd z ; (A) 2 ppm Cd 2 ; (~) i0 ppm Cd z~-. M c p m - i0 s counts per minute after various incubation times.
(ppm) 0 0.5 1 2 4 6 8 i0 20
X 3.18 3.21 2.09 1.00 0.12 0.24 0.13 0.ii 0.i0
s.d. 0.26 0.07 0.03 0.01 0.01 0.01 0.01 0.01 0.01
% inhib, b -0.9 c 34.3 68.6 96.1 92.3 95.9 96.3 96.8
a The number of replicates was i0. Table values are in multiples of l0 s cpm and were contained in 1.0 ml of the assay system following a 2 hour blncubation. Percent inhibition based on control C Not significantly different from the control (Student's T test at ~ = 0.05); all others were significantly different.
times more ethylene produced in the system incubated under a nitrogen atmosphere, although the level was still significantly lower than in the control. Controls evidenced the same amount of ethylene production under both air and nitrogen atmospheres. Similar results were obtained at other Cd 2 concentrations. Cadmium is known to replace Zn in enzymes 2~, to bind to nucleic acids 19, 23, and to interact with dithiol and disulphide groups in proteins 2?. That inhibition, not e~plained by cell lysis, may be due to similar interactions between (i) internal Cd 2 and nitrogenase, or (ii) between Cd 2 and enzyme systems providing the ATP and reductant pools required f~r nitrogenase activity. One ppm Cd = significantly inhibited photosynthesis, while 20 ppm was required for complete inhibition+(Table 2). There was a linear relationship between percent inhibition and log10Cd 2 concentration over the range of 0.5 to 4 ppm (r = 0.998). Cadmium ion altered the pattern of I~C02 fixation, and induced a subsequent reduction in filterable radioactivity (Figure 2). Cadmium may inhibit photosynthesis by interfeting with C02 assimilation 8 , while the loss in radioactivity can be explained by cell lysis, which would release any fixed carbon into the medium. Untreated A. inaequalls cultures containe~ from 10-50 vegetative cells and one heterocyst per ~ilament (Figure 3a), while Cd2~-treated cultures had from 60-100 vegetative cells and up to 12 heterocysts per filament (Figure 3b), often in grapelike clusters (Figure 3c). Apical cells in treated filaments were devoid of cellular contents (Figure 3d). Cadmium affects mainly cell division l, 5, 2~, 2e, probably through nonspecific enzyme inhibition le Increased heterocyst frequency may be due to an inhibition of the heterocyst spacing mechanism, which is thought to involve glutamine-derived inhibitors ~I , or localized nutrient depletion 3 . Increased heterocyst frequency may also be related to altered growth 11 Apical cells are probably more sensitive to toxicants due to their larger exposed surface area. Cadmium-treated A. inaequalis cultures yellowed with age, a response which has also been noted with Chlorella zl. In blue-green algae, pigment bleaching can be
No. 5
2+ on the morphology of _ ~ b a e n a inaequalls. FIGURE 3. The effect of Cd (A) Control filament. (B-D) Filaments treated with 0.05 ppm Cd for 7 days. (B) Increased filament length. (C) Increased heterocyst frequency. (D) Damaged apical cells. h - heterocyst; p - proheterocyst; a - apical cell. The bar represents i0 ~m. induced by CO2 deprivation 22 and nitrogen stress zz. In the latter case the cells are able to utilize their pigments as a sourse of nitrogen lZ. A. inaequalis was sensitive towards Cd 2 in the order of growth (cell yield at
The data for photosynthesis and acetylene reduction were based upon short term incubations. Preincubation of cells in the presence ~f Cd 2 for 24 hours, prior to assay, greatly increased the apparent toxiclty of Cd 2 towards Z~CO2 uptake and acetylene reduction. Under such conditions, the toxicity induced by 0.5 ppm cadmium ion was increased by 77% for acetylene reduction, and 56% for photosynthesis.+ This suggests a delayed toxicity which could be related to a gradual uptake of Cd 2 into the cells either passively, or actively. Preincubation also reversed the order of sensitivity. Cultures which had been growing for 14 days in the presence of 0.05 ppm Cd = had their acetylene reducing ability inhibited by 70%, while the level+of Z~CO2 uptake was not significantly altered. Therefore growth inhibition by Cd 2 may be related to an inability to supply sufficient fixed nitrogen. Anabaena inaequali~ is susceptible to relatively low levels of Cd 2+ in its environment. Al-~-~uough the Cd z levels studied are seldom found, at present, in unpolluted freshwater bodies 15, one would not expect to find this organism in areas evidencing cadmium pollution.
28~
282
No. 5
ACKNOWLEDGEMENTS Financial support in the form of a National Research Council of Canada Postgraduate Scholarship is gratefully acknowledged by G.W.S. Research funds were provided by the Ontario Ministry of Agriculture and Food. REFERENCES
i. 2. 3.
4. 5. 6. 7. 8. 9. i0.
ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
I.D. Anlkeeva, E.N. Vaulina, and I.G. Kogan (1975). Genetlka ii, 78. Anonymous (1977). Environ. Sci. Technol. ii, 336. M. Bahal and E.R.S. Talpasayi (1972). Control of heterocyst development in Anabaena ambi~ue Roa. In Taxonomy and Biology of Blue-Green Al~ae. ed. by T.V. Desikachary, p. 197, Bansalore Press, India. B.R. Berland, D.J. Bonln, V.I. Kapkov, S.Y. Maestrinl, and D.P. Arlhac (1976). C.R. Acad. Scie. Paris Ser. D. Scl. Natur. 282, 633. B.R. Berland, D.J. Bonln, O.J. Guerin-Ancey, V.I. Kapkov, and D.P. Arlhac (1977). Marine Biol. 42, 17. Bureau of National A/falrs, Inc. (1975). Environ. Reporter, 29 Au~., 703. K.W. Burton and E. John (1977). Water, Air, Sol1 Poll. ~, 45. A. Cedeno-Maldonado and C.I. Asencio (1976). Plant. Physiol. 57, 7 Abs. 34. H.L. Conway (1978). J. Fish Res. Bd. Can. 35, 286. W.E. Davis and Associates (1970). National inventory of sources and emissions of cadm/u~, nickel, and asbestos. Cadm/em, section i. U.S. Dept. Commerce Report PB 192250, Nat Techn. Info. Ser., Springfield, Va. G.E. Fogs, W.D.P. Steward, P. Fay, and A.E. Walsby (1973). The Blue-Green Allae. Academic Press, N.Y. S.S. Greenfield (1942). Amer. J. Bot. 29, 121. R.W.F. Hardy, R.C. Burns, and R.D. Holsten (1973). Soil. Bio. Biochem. ~, 47. R. Heldwein, H.W. Tromballa, and E. Broda (1977). Z. Allgemeine Mikrobiol. 17, 299. A. Henriksen and R.F. Wright (1978). Water Res. 12, i01. A.J. H o m e and G.R. Goldman (1974). Science 183, 409. IARC Clnternational Agency for Research on Cancer) (1976). IARC monographs on the evaluation of carclnosenic risk of chemicals to man. ii, 39. E. Klass, D.W. Rowe, and E.J. Massaro (1974). Bull. Environ. Contamin. Toxlcol. 12, 442. M.W. McLean and F.B. Willlamson (1977). Physiol. Plant. 41, 268. H.M. Malin (1971). Environ. Sci. Technol. ~, 754. S. Mang and E. Broda (1976). Naturwissenschaften 63, 295. L.S. Miller and S.C. Holt (1977). Arch. Microbiol. i15, 185. R.S. Mitra and I.A. Bernstein (1978). J. Bacteriol. 133, 75. Y. Nakano, K. Oskamoto, and K. Fuwa (1978). Agric. Biol. Chem. 42, 901. P.R. Norris and D.P. Kelly (1977). J. Gen. Microbiol. 99, 317. A.L. Page and F.T. Bingham (1973). Residue Rev. 48, i. H. Passow, A. Rothstein, and T.W. Clarkson (1961). Pharmacol. Rev. 13, 185. J.J. Rosko and J.W. Rachlin (1977). Bull. Torrey Bot. Club 104, 226. R.Y. Stanier, R. Kunisawa, M. Mandel, and G. Cohen-Bazine (1971). Bacteriol. Rev. 35, 171. W.D.P. Steward and G. Alexander (1971). Freshwater Biol. ~, 389. J. Thomas, J.C. Meeks, C.P. Wolk, P.W. Shaffer, S.M. Austin, and W.S. Chien (1977). J. Bacteriol. 129, 1545.
(Reoeived in The Netherlands 15 Ms/oh 1979)
0045-6555/79/0501-0277~02.00/0 in Great Britain.
THE EFFECT OF CADMIUM ION ON THE GROWTH, PHOTOSYNTHESIS, NITROGENASE ACTIVITY OF ANABAENA INAEQUALIS Glenn W. Stratton Department
AND
and Charles T. Corke
of Environmental Biology, Guelph, Ontario, Canada.
University NIG 2WI.
of Guelph,
T~e growth of Anabaena inaequalis was significantly inhibited by Cd 2 concentrations greater than 0.02 ppm (~g/ml) and completely inhibited at 0.06 ppm (Day 12). Cadmium had no significant effect upon the lag phase of growth or the culture doublin~ time, but caused the retardation phase to arrive sooner. One ppm Cd ~+ significantly inhibited the ra~es of both photosynthesis and acetylene reduction, by ~. inaequalis, with complete inhibition at 4 and 20 ppm respectively. Cell sensitivity increased directly with exposure time. Cadmium caused some cell lysis of A. inaequalis and induced an increase in filament length, heterocyst frequency, and a loss of cellular contents from filament apical cells. The cellular abnormalities observed and the fact that toxicity increased with longer exposure times, suggested that metal toxicity resulted from effects of Cd 2 taken up by cells rather than Cd 2~ at the cell surface. INTRODUCTION Cadmium is a potentially hazardous pollutant in the biosphere since it is toxic to many life forms, 1 , 4 , 5 , 9 , 1 7 ~ 2 6 is a frequent contaminant in effluents 2 , 6, ~, 10, 17, 20, 26 and is extremely persistant in most environments 17, 26. It is rapidly absorbed and accumulated by plants s, 19, animals 26, and is toxic to man Iv One area of concern is the adverse effect of cadmium on phytoplankton. Much of the literature on this metal ion is concerned with its effects on the green alga Chlorella i, 12, 20, 28 and other eucaryotic algae ~' s, 9, 18, 19, while reports dealing specifically with the toxicity of cadmium towards blue-green algae are absent. Bluegreen algae are important components of the aquatic environment, since many are able to fix nitrogen, contributing up to one half of the annual nitrogen budget in some aquatic ecosystems 16. This paper describes the toxic effects of cadmium ion on the growth, photosynthetic, and nitrogen fixing abilities of a blue-green alga - Anabaena inaequalis &is part of a major investigation on the interactions of heavy metals and pesticides on biological transformations in aquatic ecosystems. MATERIALS
AND METHODS
Anabaena inaequalis was supplied by the Department logical Science, University of Guelph, Guelph, Ontario,
277
of Botany, College of BioCanada, and was maintained
27s
in a liquid, nitrogen-free medium* on a 12 hour light cycle at a temperature of 22°C and a light intensity of 7 Klux. These incubation conditions were standard for all growth, photosynthesis and nitrogen fixation experiments. All chemicals were supplied by the Fisher Scientific Co. with the exception of NaH Z~CO3, which was obtained from Amersham/Searle, Oakville, Ontario, Canada. Algal growth was assessed by measuring the optical density of the culture at 600 nm, wlth time, using a Bausch and Lomb Spectronic 20 spectrophotometer. Sidearm flasks of 500 ml capacity, containing 95 ml of medium, and I ml of an aqueous cadmium solution (as CdCI2.2½H20) were inoculated with 5 ml of a 7 day culture (standardized to give an initial algal population of 9 x i0 ~ cells/ml), and grown in still culture. Nitrogenase activity was determined using the acetylene-reduction technique z3 Tissue culture flasks with a surface area of 25 cm 2 and an internal volume of 74 ml were employed as assay chambers. Each culture flask contained 9 ml of algal culture in the logarithmic stage of growth (1.6 x 107 cells) and 1 ml of either growth medium or cadmium solution. Flasks were sealed with tight fitting rubber serum stoppers and a 10% volume of air removed and replaced with acetylene. Activity was monitored hourly for 5 hours, or at the end of 5 hours incubation. Ethylene production was assayed by the injection of i ml gas samples, taken from the sealed flasks, into a Varian Aerograph Model 1800 Gas Chromatograph, equipped with hydrogen flame ionization detectors and a Porapak N column (50/80 mesh, 1/8 in i.d. by I0 ft). The unit was operated under the following conditions: detector, 200°C; injector, 150°C; column, lO0°C; hydrogen, 30 ml/min; air, 300 ml/min; nitrogen, 48 mi/min. Ethylene peaks on recorder tracings were identified by retention time, and quantitated by comparing to standard curves. Photosynthesis was determined by following the uptake of Z~CO2 from NaHZ~C03 30 The tissue culture flasks contained 8.9 ml of algal culture in the logarithmic phase of growth (1.2 x 107 cells), 0.i ml of the labelled bicarbonate solution (to give a final activity of 0.2 ~Ci/ml) and 1 ml of elther growth medium or cadmium solution. Activity was assayed every 30 minutes for 2 hours, or after 2 hours incubation. One ml samples were filtered through 0.45 Dm membrane filters (Millipore - HAWP02500), washed successively with 5 ml of 0.i N HCI and 5 ml of water, and dried under an infrared lamp. Filter pads were placed in 20 ml scintillation vials, covered with i0 ml of a cocktail containing 5 gm PPO and 0.3 gm POPOP per litre toluene, and counted in a Beckman Mode] LS-3150T liquid scintillation counter. Counts were corrected for % counting efficiency and reduced by the background count. RESULTS AND DISCUSSION The yield (at Day 12) of Anabaena ~naequalis was significantly inhibited (based on Student's T test at e = 0.05) by ~-~2 concentrations of 0.03 ppm (~g/ml) and greater (Figure i). The~e was no significant inhibition of either growth rate or yield below 0.03 ppm Cd ~ , while 0.06 ppm was required for the complete inhibition of growth+ There was a linear relationship between percen~ inhibition of yield and logz0 Cd 2 concentration in the range 0.01 to 0.05 ppm Cd ~ (correlation coefficient, r = 0.990).
* The basal medium contained 0.039 g K2HPO~, 0.075 g MgSO~.7H20, 0.04 g CaCI2.6H20, 0.006 g citric acid monohydrate, 0.001 g EDTA, 0.02 g NaHCO3, and 0.006 g ferric citrate per litre. To this was added a micronutrient mixture, at a level of i ml/litre basal medium, which contained 2.86 g N3BO3, 1.81 g MnCI2.4H20, 0.222 g ZnSO~.TH20, 0.39 g NaMoO~.2H20, 0.079 g CuSO~.SH20 and 0.049 g CoC12.6H~O per litre. (Modified after Stanier et al. ~9.)
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279
TABLE i. The effect of Cd 2+ on acetylene reduction by Anabaena inaequalis. .-'"~ ..~*i......... b ..".~" - ~ . %:.,.."" ~ % ....... .,,~ ,,-~
0.8-
~ /•.:.'~-," , 18 o-4 ,..( d 0.2
•"."-~"
o" •
Cd z¢ concn, ~ppm) 0 1 2 4 6
~'~."
8
."~|"
I0 20
..4'-''" ~,
,i
Time
e,
I!l
lb
1~
(days)
FIGURE i. The effect of Cd 2+ on the growth of Anabaena inaequalis. (e) control; (O) 20 ppb CdZ'; (A) 30 ppb Cd2~; (4) 40 ~ b Cd2+; (m) 50 ppb Cd2+; (Q) 60 ppb Cd" . O.D. - optical density.
nmoles ethylene produced per 10 cells a ~ s.d. % Inhlb. b 2.60 0.56 i. 96 0.59 24.6 1.95 0.56 25.2 1.57 0.19 39.6 0.99 0.19 61.8 0.74 0.28 71.6 0.51 0.12 80.5 0.21 0.ii 92.0
a
b
Assayed after 5 hours incubation. Percent inhibition, based on control. All treatments differed significantly from the controls, based on a Student's T test at ~ = 0.05. The number of replicates = 11-15.
Cadmium had no significant effect upon either the lag phase of growth, which ranged from 0 to 0.8 days, or the culture doubling time, which ranged from 1.4 to 1.7 days. Cadmium did cause the retardation phase to occur sooner, indicating a delayed expression of toxicity. This is in contrast to results for Chlorella 2e, where cadmium treatment significantly increased the lag phase of growth. This difference could be related to cell concentration, as reducing the initial level of A. inaequalls to 5 from 9 x i0 ~ cells/ml also resulted in a significant increase in ~he lag phase~ from 0.5 to 2.0 days in response to 0.05 ppm Cd" . One ppm Cd 2 significantly reduced the amount of acetylene reduced over a 5 hour period, while 5 ppm was required to cause complete inhibition (Table i). +There was a linear relationship between the reduction in ethylene yield and logz0Cd 2 concentration over the range of 2 to 20 ppm for acetylene reduction (r = 0.981). When the time course of acetylene reduction was investigated, it was found that cadmium did not affect the early pattern of acetyl~ne reduction, but did cause this activity to reach zero at times dependent upon Cd 2 concentration. Control cultures demonstrated active nitrogenase activity throughout the 5 hour incubation, while activity ceased after i, 2 and 3 hours in those systems treated with I0, 5 and 1 ppm Cd 2 , respectively. Cadmium ion is removed from solution within 15 minutes by A. inaequalis cells (G.W. Stratton and C.T. Corke, unpublished data obtained using an ion specific electrode) and is known to be slowly concentrated by some algae 9, 19, and yeasts z~, 2s. The delayed toxicit~ towards growth and acetylene reduction may be related to the time required for Cd 2 to enter the cells. Cell lysis also played a role in the inhibition of acetylene reduction. Cell disruption would release the oxygen-labile nicrogenase enzyme complex into the medium, where it could undergo inactivation by oxygen i] and cadmium. The e~fect of lysis was determined by comparing the level of acetylene reduction in Cd 2 -treated flasks, containing either air or nitrogen atmospheres. The presence of a nitrogen atmosphere would reduce the amount of dissolved oxygen in the medium~ and thereby the level of 02 lltaotivation of released nitrogenase. At 5 ppm Cd 2 there was three
280
No. 5
TABLE 2. The effect of Cd 2+ on I~COz fixation by Anabaena inaequalis.
Cd zl
Counts per minute a
conch. J
s
/* oS
o
s J ~
30
eO ~o" Time (mtnulee)
lio
FIGURE 2. The effect of Cd 2+ on I~C02 fixation by Anabaena ina~qualis. (e) control; ( O ) ' - ~ p-~±Cd z ; (A) 2 ppm Cd 2 ; (~) i0 ppm Cd z~-. M c p m - i0 s counts per minute after various incubation times.
(ppm) 0 0.5 1 2 4 6 8 i0 20
X 3.18 3.21 2.09 1.00 0.12 0.24 0.13 0.ii 0.i0
s.d. 0.26 0.07 0.03 0.01 0.01 0.01 0.01 0.01 0.01
% inhib, b -0.9 c 34.3 68.6 96.1 92.3 95.9 96.3 96.8
a The number of replicates was i0. Table values are in multiples of l0 s cpm and were contained in 1.0 ml of the assay system following a 2 hour blncubation. Percent inhibition based on control C Not significantly different from the control (Student's T test at ~ = 0.05); all others were significantly different.
times more ethylene produced in the system incubated under a nitrogen atmosphere, although the level was still significantly lower than in the control. Controls evidenced the same amount of ethylene production under both air and nitrogen atmospheres. Similar results were obtained at other Cd 2 concentrations. Cadmium is known to replace Zn in enzymes 2~, to bind to nucleic acids 19, 23, and to interact with dithiol and disulphide groups in proteins 2?. That inhibition, not e~plained by cell lysis, may be due to similar interactions between (i) internal Cd 2 and nitrogenase, or (ii) between Cd 2 and enzyme systems providing the ATP and reductant pools required f~r nitrogenase activity. One ppm Cd = significantly inhibited photosynthesis, while 20 ppm was required for complete inhibition+(Table 2). There was a linear relationship between percent inhibition and log10Cd 2 concentration over the range of 0.5 to 4 ppm (r = 0.998). Cadmium ion altered the pattern of I~C02 fixation, and induced a subsequent reduction in filterable radioactivity (Figure 2). Cadmium may inhibit photosynthesis by interfeting with C02 assimilation 8 , while the loss in radioactivity can be explained by cell lysis, which would release any fixed carbon into the medium. Untreated A. inaequalls cultures containe~ from 10-50 vegetative cells and one heterocyst per ~ilament (Figure 3a), while Cd2~-treated cultures had from 60-100 vegetative cells and up to 12 heterocysts per filament (Figure 3b), often in grapelike clusters (Figure 3c). Apical cells in treated filaments were devoid of cellular contents (Figure 3d). Cadmium affects mainly cell division l, 5, 2~, 2e, probably through nonspecific enzyme inhibition le Increased heterocyst frequency may be due to an inhibition of the heterocyst spacing mechanism, which is thought to involve glutamine-derived inhibitors ~I , or localized nutrient depletion 3 . Increased heterocyst frequency may also be related to altered growth 11 Apical cells are probably more sensitive to toxicants due to their larger exposed surface area. Cadmium-treated A. inaequalis cultures yellowed with age, a response which has also been noted with Chlorella zl. In blue-green algae, pigment bleaching can be
No. 5
2+ on the morphology of _ ~ b a e n a inaequalls. FIGURE 3. The effect of Cd (A) Control filament. (B-D) Filaments treated with 0.05 ppm Cd for 7 days. (B) Increased filament length. (C) Increased heterocyst frequency. (D) Damaged apical cells. h - heterocyst; p - proheterocyst; a - apical cell. The bar represents i0 ~m. induced by CO2 deprivation 22 and nitrogen stress zz. In the latter case the cells are able to utilize their pigments as a sourse of nitrogen lZ. A. inaequalis was sensitive towards Cd 2 in the order of growth (cell yield at
The data for photosynthesis and acetylene reduction were based upon short term incubations. Preincubation of cells in the presence ~f Cd 2 for 24 hours, prior to assay, greatly increased the apparent toxiclty of Cd 2 towards Z~CO2 uptake and acetylene reduction. Under such conditions, the toxicity induced by 0.5 ppm cadmium ion was increased by 77% for acetylene reduction, and 56% for photosynthesis.+ This suggests a delayed toxicity which could be related to a gradual uptake of Cd 2 into the cells either passively, or actively. Preincubation also reversed the order of sensitivity. Cultures which had been growing for 14 days in the presence of 0.05 ppm Cd = had their acetylene reducing ability inhibited by 70%, while the level+of Z~CO2 uptake was not significantly altered. Therefore growth inhibition by Cd 2 may be related to an inability to supply sufficient fixed nitrogen. Anabaena inaequali~ is susceptible to relatively low levels of Cd 2+ in its environment. Al-~-~uough the Cd z levels studied are seldom found, at present, in unpolluted freshwater bodies 15, one would not expect to find this organism in areas evidencing cadmium pollution.
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ACKNOWLEDGEMENTS Financial support in the form of a National Research Council of Canada Postgraduate Scholarship is gratefully acknowledged by G.W.S. Research funds were provided by the Ontario Ministry of Agriculture and Food. REFERENCES
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4. 5. 6. 7. 8. 9. i0.
ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
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(Reoeived in The Netherlands 15 Ms/oh 1979)