Effects of selenium on the growth and phosphorus uptake of Scenedesmus dimorphus and Anabaena cylindrica

Eaviroa~aalEadF_zpogaeCalBolu,y, Vol. 20, pp. 207 to212 Pergamon Pre~ Ltd. 1980. Printed in Great Britain

EFFECTS OF SELENIUM ON THE GROWTH AND P H O S P H O R U S UPTAKE OF SCENEDESMUS DIMORPHUS AND A, VABAENA CTLIaVDRICA A. MOEDE. R. W. GREENE mad D. F. $~_,NOgR* Department of Biology, University of Notre Dame, Notre Dame, IN 46556, U.S.A. (Received 24 May 1979; revised 13 .Vovember 1979) MOEDE A., GREENE R. W. and SPENCER D. F. Effects of selenium on the growth and phosphorus uptake of Scenedesmus dimorphus and Anabaena cylindrica. ErCVmONMENTAL AND EXPE~M~rCrAL BOTANY20, 207--212, 1980.--Determinations of 14-day cell number and of log phase growth rate were used to assess the effects of selenite and selenate on Scenedesmus dimorphus and Anabaena qylindriea. Both salts of selenium inhibited growth of the algae when compared to controls. A previous study had suggested that selenium might interfere with mechanisms of phosphate uptake in algae, thereby inhibiting growth. This hypothesis was tested by studying phosphate uptake by P-starved cells grown in the presence of various concentrations of sodium selenate. Selenate significantly reduced phosphate uptake by both species of algae compared {o selenium-free controls.

INTRODUCTION

THE CONCENTRATIONo f trace elements in aquatic ecosystems influences the species composition of the associated algal community. (9) PATRICK et al. (1°) reported that changes in the concentration of selenium in the water of experimental streams resulted in altered abundances of the characteristic algal species. They reported that the addition of selenat¢ to the streams caused increased abundance of blue-green algae and decreased abundances of diatoms. In studies on an eutrophic lake in Indiana, SPENCER eta/. (at) found that biomass of blue-green algae was significantly correlated with the concentration of total dissolved selenium, while biomass of other groups (green algae, diatoms, and flagellates) did not show a similar relationship. PATRICK et al. (~°) also determined that the form of selenium used was important in in-

fluencing toxicity to algae. They reported that diatoms, especially Achnanthes lanceolata, appeared to be stimulated at 1.0 and 10.0rag/1. selenite, and grew well at 40 mg/1. selenite. The blue-green algae, Schizothrix calcicola and Oscillatoria sp. were not generally observed when selenite concentrations reached 10.0mg/1. or greater. In contrast, when selenate was added to the experimental streams diatom abundance decreased, while blue-green species grew well at 1.0 and 10.0mg/l. KUMAR and PRAKASH(6) obtained similar results when they compared the growth of Anacystis nidulans and Anabaena variabilis exposed to selenatc and selenite. They reported that growth of both species was inhibited more by selenite than selenate. Since information on the effects of selenium on algal growth is limited, we investigated the effect of selenium, as selenite and sclenatc, on growth and phosphorus uptake in two species of

*Present Address: Department of Biology, Indiana University-Purdue University, Indianapolis, IN 46205, U.S.A. 207

208

A. MOEDE, R. W. GREENE and D. F. SPENCER

algae commonly found in freshwater plankton communities, Anabaena cylindrica (Lemmerman) and Scenedesmus dimorphus (Kutz.). We were interested in testing the hypothesis that bluegreen algae are more tolerant of increased levels of selenium than other types of algae. METHODS

The algae used in this study were Anabaena cylindrica (UTEX Strain 693) and Scenedesmus dimorphus ( U T E X Strain 417). Cultures were maintained in full-strength Algal Assay Medium ~1) until being used for experiments. For the various studies described here, algae were transferred to modified Algal Assay Medium (see Table 1).

or Anabaena (260 filaments/ml=3600 cells/ml). Thus, test flasks were inoculated with nearly the same number of cells in each case. Flasks containing algae were placed on a rotating platform (New Brunswick Scientific-75 rev/min) in random arrangement. They were maintained in a growth chamber at 20°C on a light/dark cycle of 16L:8D, with light provided by eight Sylvania Gro-lux tubes (Fo40) at a distance of 60cm from the bottoms of the flasks. Light energy was measured with an International Light I L l 5 0 Plant Growth Photometer as 1600#W/cm 2 at 4--500rim and 4400#W/cm 2 at 6-700nm. Experiments were run for 14 days, with samples being taken each day for the first 7 days, then on the 9th, 1 l th and 14th days. One ml samples were taken

Table 1. Medium used in growth experiments: raicronutrient components deleted exceptfor maintenance of stock cultures Macmnutrients

(rag/l)

Micronutrients

NaNO 3 KzHPO , MgCI 2 MgSO4 • 7H20 CaCI 2 • 2H20 NaHCO 3

25.500 1.004 5.700 14.700 4.410 15.000

H3BO 3 MnC12 ZnCI2 CoCI2 CuC12 Na2 MoO , •2H20 FeCI3

250ml of concentrated medium (2X) was introduced into each of four 500 ml volumetric flasks. Aqueous sodium selenite or sodium selenate (Ventron) was added in appropriate quantity, then glass-distilled deionized water was added to make the total volume up to 500ml. Selenite was tested at 40mg/1, while selenate was tested at both 40 and 80 mg/l. Two concentrations of selenate were used since it had been reported to have a greater effect on algal growth. ~1°) Control flasks consisted of modified Algal Assay Medium without the addition of selenium. Each 500 ml of medium was then sub-divided into 4 samples, such that the experiments ultimately consisted of 16 flasks. The flasks were inoculated with~either Scenedesmus (3300 cell/ml)

~g/1) 183.520 264.264 32.709 0.780 0.009 7.260 96.000

from each flask and were placed into vials containing Lugol's solution, tx2~ Fixed samples were enumerated using a Sedgewick-Rafter counting cell¢4~ at 200X. Growth rates were calculated using cell counts converted to base 2 logarithms ¢'*~ as the dependent variable in a regression with time. Growth rates for S. dimorphus are based on cell counts taken on days 1-5. Cell counts on days 3-9 were used to calculate growth rates for A. cylindrica. The 14-day cell numbers were analyzed using the O N E W A Y procedure for analysis of variance in SPSS. ~s~ In addition, the selenium treatment means were analyzed by orthogonal contrasts using the Student's ttest. ~5's~ One comparison involved a test for differences between the two levels of selenate. A

EFFECTS OF SELENIUM ON S. DIMORPHUS" AND .1. C;ZI.VDRIC..I second contrast tested for differences between the two selenate levels and the selenite treatment. The second phase of the study dealt with the effect of selenium on phosphorus uptake. Algae which had been grown in P-free Algal Assay Medium for 2 weeks were added to flasks containing full-strength Algal Assay Medium and various concentrations of sodium selenate. Samples for phosphate analysis were taken at time 0, 1, 2, 4 and 6hr after inoculation. Samples were filtered through acid-washed membrane filters (0.45#m pore size), and the filtrate was analyzed for ortho-phosphate by the ascorbic acid method. (2) PO4-uptake per cell was calculated by subtracting the amount of PO,r measured in the medium from the initial P O , level of the medium, and dividing by the number of cells present.

209

Student's t-test revealed no differences either between the two levels of selenate, or between selenate and selenite.

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RESULTS AND DISCUSSION

Figure 1 demonstrates the differences between control cultures of Scenedesmus and those contalning selenium.. Contrgls showed a typical growth response for algal batch cultures. (a) All selenium-containing cultures showed a clearly negative effect on the algae. Selenium-free cultures of S. dimorphus increased at the rate of 1.195 +0.157 doublings/day, while cultures containing both selenite and selenate had negative growth rates (Table 2). The selenium-treated cultures had almost identical growth rates. The analysis of variance performed with the 14-day cell numbers yielded a significant F-statistic (F =69.93; d f = 3 ; p<0.001). Application of the

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1.195 4,0.157 0.744 4"0.112

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Fro. 1. Growth curves for S. dimorphus with and without selenium in the culture medium. Each point represents the mean of 4 replicate cultures.

Table 2. Growth rates (doublings/day)for Scenedesmus dimorphus (n =20) and Anabaena cylindrica (n=24) cultures at the tested selenium concentrations. Values given are growth rate 4" S.E..ill ,~r,,'th ra/,'~ differ significantly from zero (p < 0.001)

0

9

TIME[ (~1~1)

Selenium 40 mg/l 40 rag/1 Se03 Se04

80 rag/1 Se04

-0.264 _.+0.061 0.410 4-0.083

-0.265 4-0.063 0.347 4"0.063

-0.264 4-0.063 0.625 4"0.085

210

A. MOEDE, R. W. GREENE and D. F. SPENCER

Figure 2 shows the growth curves obtained with Anabaena in the presence and absence of selenium. Selenium-free cultures increased at cyllndrlca

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the rate of 0.744_.+0.112 doublings/day. The growth rate had decreased slightly to 0.625 +0.085 doublings/day at 40rag/1 selenate. Further decreases in growth rate were observed at 40 mg/l selenite and 80 mg/l selenate (Table 2). The analysis of variance of the 14-day filament numbers indicated that selenium in the culture medium significantly (F = 7.04; d f = 3; P <0.01) reduced the number of filaments present after 14 days growth. A priori comparisons similar to those employed for Scenedesmus showed no significant differences among the selenium treatment means (Table 3). The log-phase growth rates for S. dimorphus are clearly reduced by the test levels of selenium, whether present as selenite or selenate. In contrast, the log-phase growth rate for A. cylindrica cultures decreased only slightly at 40rag/1 selenate relative to controls, while a somewhat greater decrease occurred at 40 mg/l selenite and 80mgfl selenate. In no case, however, was the growth rate for A. cylindriea as severe as that observed for S. dimorphus cultures exposed to selenium. A similar trend occurs for the 14-day cell numbers (Table 3). After 14 days the seleniumtreated S. dimorphus cultures fell to cell densities of 5% of the controls. Similar data for A. cylindrica ranged from 12-20%. The apparently greater toxicity to A. cylindrica (compared to effects on the growth rate) based on the 14-day cell numbers can be explained by the fact that these data reflect a longer exposure period to selenium. I f selenium toxicity is related to in-

Table 3. 14-day cell numbers for Scenedcsmus dimorphus and Anabaena cylindrica. Values are ~4,S.E., n=4. Underlining shows groups not significantly different at g=0.05, based on results of orthogonal contrasts using Student's t-test (KaRK, 1968). Selenium 40 mg]l 40 mg/1 SeO 3 SeO,

80 mg/1 SeO4

101192 + 12039

5304 +_1633

3536 4" 1715

4160 "t"1176

146380 4- 45942

18460 4- 2801

29172 4"6319

17056 4-8507

0 Seenedesmus dimorphus (cells]ml) Anabaena cylindriea (filaments/ml)

EFFECTS OF SELENIUM ON S. DIMORPHUS AND A. C?'LINDRICA S, dlmorphus

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FIG. 3. Phosphorus uptake by S. dimorphus with and without selenium. Ranges are derived from 2 replicate cultures. The small (2) indicates two data points fell together.

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traceUular accumulation, then a longer exposure period should be associated with increased toxicity, and results such as reported here for the 14-day Cell numbers would be expected. The results of the a priori contrasts among the selenium treament means (Table 3) indicate that selenate and selenite are equally toxic to algal growth at the levels used. Further, the data reported here strongly support the reported differential tolerance to selenium exhibited by blue-green algae when compared to green algae and diatoms. (1°' a x) It has been suggested in the literature that interference with the mechanism of phosphate

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Fie. 4. Phosphorus uptake by A. fylindriva with and without selenium. Ranges are derived from 2 replicate cultures. The small (2) indicates two data points fell together.

uptake might explain the negative effect of selenium on algal growth. (7) The results of the phosphate uptake experiments are shown in Figs. 3 and 4. A two-way analysis of variance

Table 4. Results of 2-way analysis of variance on phosphate uptake data from axenic cultures ofAnabaena cylindrica and Scenedesmus dimorphus in response to selenate and time. SeOa

Time

SeO 4 x time

PO 4 uptake by Scenedesmus dimorphus

*

I"

NS++

PO 4 uptake by Anabaena cylindrica

*

I"

NS,+

*P<0.05. I"P<0.001. .+NS, not significant.

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212

A. MOEDE, R. W. GREENE and D. F. SPENCER

(with time and selenate as factors) was applied to these data, and the resulting F-statistics indicate that selenate significantly reduced phosphate uptake for both S. dimorphus and A. cylindrica (Table 4). Thus, the data reported here support the hypothesis that selenium may negatively affect algal growth by inhibiting the uptake of phosphorus.

5. KIRK R. E. (1968) Experimental Design: Procedures for the Behavioral Sciences. Brooks, Belmont, California 577 pp. 6. KUMAR H. D. and PRAga~sn G. (1971) Toxicity of selenium to the blue-green algae, Anacystis nidulans and Anabaena variabilis. Ann. Bot. 3kS, 697705. 7. KVUN A. (1966) The influence of photosynthetic factors and metal inhibitors on the uptake of phosphate in P-deficient Scenedesmas. Physiol. Plant. 19, 611 549. Acknowledgement--We wish to thank MARY GODDARD 8. Nm N. H., HULL C. H., JENVaNS J. G., for preparing the graphs. STEINBRENNER K. and BRENT ~). H. (1975) Statistical Package for the Social Sciences. McGrawHill, New York, 675 pp. 9. PATPaCKR. (1978) Effects of trace metals in the REFERENCES aquatic ecosystem. Am. Sci. ¢~ 185-191. 1. ANONYMOUS. (1971) Algal Assay Procedure--Bottle 10. PAT~aCKR , BOaT T. and ~ N R. (1975) The Test. U.S. Environmental Protection Agency, Role of Trace Elements in Management of Nuisance Corvallis, Oregon 82 pp. Growths. U.S. Environmental Protection Agency, 2. APHA. (1971) Standard Methods for the Corvallis, Oregon. Examination of Water and Wastewater 13th Edition. 11. SpsNCER D. F., GRSENE R. W., THExs T. L., Amer. Publ. Health Assoc., Washington, D.C. YEuNo H.-Y., Ross Q. E. and DOIZ,~E E. E. 874 pp. (1978) A study of the relationship between phyto3. EPI'LEV R. W. (1971) The growth and culture plankton abundance and trace metal concenof diatoms. Pages 24-64. in D. WARNER ed. The trations in eutrophic Lake Charles East, using Biology of Diatoms. University of California, correlation techniques. Proc. Ind. Acad. Sci. 87, Berkeley. 204-212. 4. GuxL~m~ R. R. L. (1973) Division rates. Pages 12. VOLLENWEmeR R. A. ed. (1974) A Manual on 289--311. /n J. R. STEIN ed. Handbook of Methods for Measuring Prima~.y Production in Aquatic Phycological Methods: Culture Methods and Growth Environments. Int. Biol. Programme (IBP) Measurements. Cambridge University " Press, Handbook No. 12 (2nd Ed.). Blackwells, Oxford Cambridge. 225 pp. \