DDE inhibition of marine algal cell division and photosynthesis per cell

PESTICIDE

BIOCHEMISTRY

DDE Inhibition

AND

PHYSIOLOGY

of Marine

10,306

-312 (1979)

Algal Cell Division Cell’

C.DONALDPOWERS,CHARLES

and Photosynthesis

per

F. WURSTER,ANDRALPHG.ROWLAND

Marine Sciences Research Center, State University of New York, Stony Brook, New York 11794 Received

February 21, 1978; accepted July 20, 1978

Exuviella baltica, a marine dinoflagellate, was exposed to DDE, the major metabolite of DDT, at a concentration of 25 &liter of medium. At intervals of 1 hr and 1, 2, 3, and 4 days, cells were withdrawn from the culture, washed, and reseeded in DDE-free medium, and their growth (cell division) and photosynthesis were monitored for 14 days. No increase in cell numbers occurred until cells were removed from DDE, and lag phases, proportional to the duration of DDE exposure and lasting up to 5 days, preceded exponential growth. Cell densities comparable to controls were eventually reached in all treated cultures. A similar pattern of “‘C uptake per milliliter of culture and per cell was observed. A I-hr exposure to DDE resulted in a maximum reduction of 45% in carbon fixed per cell, while longer exposures caused reductions as great as 91%, relative to controls. INTRODUCTION

The suppression of phytoplankton growth by organochlorines is well documented (l-12), but questions remain regarding the effect of these compounds on the process of photosynthesis. While inhibition of algal photosynthesis has been reported on the basis of both 14C uptake per unit volume of culture (1,2,4,9, 13-19) and per cell (9, 12, 19, 20) and, in some instances, a mechanism has been described (21-23), it is not universally agreed that decreased carbon fixation in a treated culture is necessarily the result of an interference with photosynthesis. Fisher (24), observing no decrease in 14C uptake per cell in Thalassiosira pseudonana, suggested that reduced photosynthesis in treated (growthinhibited) cultures was due only to the presence of fewer photosynthesizing cells, implying that the primary impact of organochlorines was on cell division rather than photosynthesis. The above studies were all short term with cells continuously exposed to the toxicant. Algae in nature, however, may encounter a given pollutant only intermitr Contribution 236 of the Marine Sciences Research Center, State University of New York at Stony Brook.

Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

MATERIALS

AND

METHODS

The marine dinoflagellate Exuviefla baltica was cultured axenically and 600 ml of exponentially growing cells was dispensed, as previously described (8), into each of two 800-ml Blake bottles (Fig. 1). DDE,* prepared as described earlier (8) to yield a final concentration in the test cultures of 25 pg/liter, was injected into one bottle while the other received an identical volume of methanol. Both bottles were shaken vigorously and incubated at 21°C under 5400 L/m* illumination for 1 hr, after which 80-ml aliquots were removed from each and centrifuged. Supematants were discarded and, after resuspension of each pellet in 20 ml of DDE-free medium, both samples ZDDE, 1,l - Dichloro - 2.2 - his@-chlorophenybethylene.

306 0048-3575/79/030306-07$02.00/O

tently. It may therefore be important to study cells exposed to a pollutant for a limited time, after which they are incubated free of the toxicant for several days. One can then observe the effect of temporary exposure to a pollutant on both growth rate and photosynthesis per cell and, further, determine whether such briefly exposed cells regain normal functions or are irreversibly damaged.

DDE

INHIBITION

OF ALGAL

CELL

DIVISION

Exponentially

AND

307

PHOTOSYNTHESIS

growing Cells

Blake

Blake

bottle

Incubate

I-

bottle DDE

methanol Incubate

1 hr 80 ml centrifuged

I same as TREATED series 20

A

Pellet resuspended ml DDE-free

in medium

Pellet resuspended in 10 ml DDE-free medium: transferred to 180 ml DDE-free Incubate

30

-I

Supernatant (discarded)

Incubate

EO-ml processed on each of

c Centrifuged I

1 hr

I aliquots as at left next 4 days

Sutxrnatant (discarded) medium

min.

Incubate

FIG.

l&ml

aliquotk

as above day (for

on as

1. Procedure

for

each many

washing

processed successive as 14 days)

cells and monitoring

were recentrifuged. Supernatants were again discarded and each pellet was resuspended in 10 ml of DDE-free medium. These washed cells were then transferred to 180 ml of DDE-free f/2 medium in each of two 200-ml prescription bottles. Both bottles (one containing washed DDE-treated cells, the other washed control cells) were then incubated for 30 mitt, after which 16 ml was withdrawn from each, 10 for cell density determination by Coulter count and 6 for assay of photosynthetic activity. Washed subcultures were then incubated along with the unwashed (original Blake bottle) cultures. On each of the next 4 days, additional

their

growth

und photosynthesis.

BO-ml aliquots were withdrawn from the unwashed (Blake bottle) cultures and the cells were washed and reseeded in DDEfree medium as described above. Likewise, 16 ml was withdrawn from all subcultures from each previous day for cell density and photosynthesis analyses, thereby providing a daily index of growth and photosynthetic activity in untreated cells and in cells exposed to DDE for periods ranging from 1 hr to 4 days. Subcultures were monitored in this fashion for the next 11 days, or until cells entered the stationary phase of growth. For Coulter counts, aliquots were fixed with 4% filtered formaldehyde. For photo-

308

POWERS,

WURSTER,

AND

ROWLAND

CONTROLS @

-----m

1052

IO4

f

IO3

0.04 1.0

-2 -3 -4

DAY (I HOUR) ” DAYS 11 ,I

cr, to6 DDE- TREATED

5

6

T/ME

7

8

9

I4

(DA YSJ

FIG. 2. Growth

of E. baltica, untreated (A) or treated with 25 t.~g ofDDElliter of culture (B). Curves originate at the time of washing and reseeding of cells, as indicated by the legend. A broken line joining two points indicates that data for the day between the points were not available.

synthesis determinations, each 6-ml aliquot , previously incubated for 20 min in a 12-ml glass vial sealed with Parafilm, received 0.1 ml (0.1 PCi) of i4C-labeled sodium bicarbonate (New England Nuclear Corp., Boston, Mass.), as did a blank consisting of 6 ml of cell-free f/2 medium. To ensure mixing, vials were shaken initially and at 30-min intervals throughout the 2-hr labeling period. No dark uptake vials were included, as other experiments had demonstrated negligible assimilation of 14C in the dark by this axenic culture. To terminate photosynthesis, 1 ml of 0.5 N HCl was added to each vial, the contents

were Millipore-filtered, and the filters were dried under a heat lamp. Filters were then placed in scintillation vials containing 5 ml of fluor (0.3% PPO, 0.01% POPOP in toluene), and radioactivity was determined by liquid scintillation counting (Mark II, Searle Analytic, Inc.). RESU

L-l-S

Untreated cells, whether those originally inoculated into the Blake bottle or daily aliquots (washed and reseeded) thereof, grew at the same steady rate and to the same final density (Fig. 2A). DDE-Treated cells, however, did not increase in number

DDE

INHIBITION

OF ALGAL

CELL

IO’

DIVISION

AND

309

PHOTOSYNTHESIS

-

0.04

-

1.0

DAY (1 HOUR)

”

-2 n-u3 -4

DAYS II II

lo0

-0

I

2345676

9

IO

II

12

13

14

FIG. 3. Incorporation (per ml of culture) of NaH14C03 by E. baltica, untreated (A) or treated with 25 pg of DDElliter of culture (B). Curves originate at the time of washing and reseeding qfcells. a.~ indicated by the legend. A broken line joining two points indicates that data for the day between the points were not available.

until removed from DDE, and those exposed for 1 day (D-l), 2 days (D-2), 3 days (D-3), and 4 days (D-4) took as many as 5 additional days to achieve consistent exponential growth (Fig. 2B). Even cells exposed to DDE for only 1 hr (D-0.04) grew at a slower rate for the first 4 days than the corresponding controls (1.062 vs 1.332 divisions per day,3 respectively). “Growth rates expressed as divisions per day were calculated according to the expression p = (35) (In nl In n,,)lt - t,,) where t - t,, represents elapsed time in hours and nl and n,,; are the population densities at times t and to, respectively (25).

Erratic changes in cell numbers between Days 4 and 6 occurred in cultures D- 1, D-2, D-3, and D-4. Such fluctuations in growth were not observed in control or D-0.04 cultures. D-l, D-2, D-3, and D-4 cultures then displayed lag phases proportional to their respective durations of DDE exposure, but all treated cultures eventually reached cell densities comparable to controls. A similar pattern of r4C uptake was observed. Whereas carbon assimilation proceeded at approximately the same rate in all untreated cultures (Fig. 3A), inhibition occurred in those treated with DDE (Fig. 3B),

310

POWERS,

WURSTER,

AND

TABLE Incorporation

ROWLAND

1

(per Cell) of NaHW03 by E. baltica, Untreated (C Series) or Treated with 25 pg of DDElliter of Culture (D Series) Days after start of experiment

Sample

0

1

2

3

4

5

6

7

C-0.04” D-0.04 C-l D-l c-2 D-2 c-3 D-3 c-4 D-4

1.8* 1.1

7.6 4.2 6.4 1.4

11.3 6.9 7.1 1.5 7.8 1.9

11.3 8.1 8.6 1.6 6.4 1.4 4.0 1.7

12.9 9.5 10.4 2.7 9.4 1.4 5.1 1.5 2.6 0.9

8.2 7.5 8.9 1.8 5.1 1.9 7.2 0.7 3.4 0.5

2.1 1.7 2.9 6.6 2.3 2.6 2.2 1.5 3.9 2.6

r ( c d p 3.7 1 2.4 cl 1.0

8

9

11

12

13

14

4.2

3.7

0.9

c

6.2

5.9

7.9

4.3

1.5

r

1.3 1.2 1.0

4.9 0.7 0.6

11.0 0.2 9.4

6.9 o 7.3

7.1

2.3

6.6

5.1

B Time (in days) of washing and reseeding the cells is indicated by the number following C or D. b Counts per minute per cell (x lOma). c Stationary phase; no samples taken. rl Cell counts for calculating cpm per cell not available.

the duration being proportional to duration of DDE exposure. Three of the treated cultures (D-2, D-3, and D-4) required 4 to 5 days following removal from DDE to register a consistent increase in photosynthetic activity, though all eventually attained levels of activity comparable to those in control cultures. Erratic changes in carbon fixation in these three treated cultures on Days 5 and 6 corresponded to the fluctuations in cell numbers observed on the same days (Fig. 2B). Increases were not proportional in all cases, however, as cell numbers rose more than 14C uptake in cultures D-l, D-3, and D-4. Carbon fixation per cell was inhibited by DDE in all treated cultures (Table 1). A I-hr pulse of DDE (D-0.04 culture) immediately reduced photosynthetic activity per cell to 61% of controls (C-0.04). Twenty-four hours later, these cells exhibited only 55% of the r4C uptake of control cells, rising to 74% of controls on Day 4. Inhibition per cell was still more pronounced in cultures D-l, D-2, D-3, and D-4, reaching a low of 9% of control values. Though maximum values of photosynthesis per cell in control cultures decreased from the C-O.04 to C-4 series, concomitant inhibition per treated

cell was nevertheless clearly evident for 3 to 5 days after removal from DDE. As in D-0.04, recovery from DDE was only partial in D-l and D-2 cultures in that incorporation per cell never equaled the maximum values registered by their respective controls. D-3 and D-4 values eventually exceeded the highest values attained by their controls but none of the treated cultures achieved more than 85% of the maximum photosynthetic activity recorded in the experiment (12.9 x 10e3 cpmcell in C-O.04 on Day 4). Direct comparison of cultures D-l, D-2, D-3, and D-4 with control cultures was complicated by the fact that control cells, not having been inhibited, were 1 to 4 days ahead of DDE-treated cells in growth and were declining metabolically as the treated cells recovered. To clarify this, data were normalized for cell density, allowing comparisons between treated and untreated cultures (based upon the same amount of radioactive carbon available to each cell), regardless of actual differences in their growth cycles on a given day (Table 2). DDE-Induced inhibition of r4C incorporation per cell was again evident, at least until the stationary phase of growth was ap-

DDE

INHIBITION

OF

ALGAL

CELL

DIVISION

TABLE Incorporution

Cell density (cells/ml) 5 x 103 1 x IO” 5 x 10’ 1 x lo” 2 x 105

(per

Cell) of NaH’4C03

by DDElliter

C-O.04

D-0.04

1.3b (9.0)’ 1.9 (11.0) 3.5 (12.0) 4.2 (12.0) 5.0 (8.2)

1.4 (3.4) 2.4 (7.7) 4.1

(9.2) 4.9

(8.5) 5.7 (2.9)

AND

311

PHOTOSYNTHESIS

2

E. baltica, Untreated of Culture

(C Series) (D Series)O

or Treated

with 25 E;LK0.f’

C-l

D-l

C-2

D-2

c-3

D-3

c-4

1.1

3.3 (1.9) 4.2 (2.6) 6.8 (5.0) 7.5 (5.0) 8.5 (4.2)

r,

I. I (3.6) 8.0 (6.2) 9.6 (6.0) 10.3 (7.0) 11.0 (7.9)

d

8.7 (3.2) 9.8

rl

(6.0) 1.8 (7.0) 3.7 (10.0) 4.4 (9.0) 5.4 (6.0)

” 3.7 (8.6) 4.4

(8.6) 4.8

(6.0)

d

(4.6) d

(6.5) 3.8

(6.0) 4.6 (7.0) 5.4 (4.8)

11.3 (7.0) 11.5 (4.7) 12.5 (5.5)

D-4 -.--10.5 11.3

(8.7) ” 5.0 (3.4) 5.9 (3.9)

12.5 (6.8) 13.0 (6.6) 13.8 (6.0)

n Data are normalized for cell density. b Day (during sustained exponential growth) on which indicated cell density was reached. c 14C uptake per cell (X IO-? in parentheses. ” Cell density exceeded that specified in column I at the time of washing and reseeding cells.

proached. Likewise, the exposure timedependent lag in recovery of treated cells was evident. DISCUSSION

These results indicate that both cell division and photosynthesis were inhibited by DDE in this algal species, even with an exposure time as brief as 1 hr. Inhibition of photosynthesis was not merely a reflection of fewer photosynthesizing cells in treated cultures but involved suppression of 14C assimilation per cell. Increasing the periods of exposure by 24-hr increments lengthened the lag periods preceding resumption of normal rates of cell division and carbon fixation. All treated cultures eventually recovered, but only after removal from DDE. This study differed from others dealing with photosynthesis per cell in organochlorine-treated cultures in that cells were removed from their toxic environment prior to analysis, and metabolic processes were monitored for 2 weeks. In contrast, MacFarlane er al. (19) utilized a single assay after 24 hr, Fisher (24) a single assay after 48 hr, Magnani et a/. (12) three assays over 5 days, and Powers et al. (9) three assays over 4 days. Removing the cells from the presence of the inhibitor and monitoring

cell division and photosynthesis daily over an extended period allows one to determine whether damage done by the pollutant to either or both processes is reversible or permanent. Bowes (22) and Adamich and Towle (26) reported difficulty in separating algal cells from DDT by means of low-speed centrifugation. Our results suggest, however, that we liberated the cells from much of the unincorporated DDE since growth, almost completely inhibited in this organism by just 10 lug of DDE/liter (8), resumed at a near normal rate in the culture exposed for 24 hr soon after washing and reseeding in DDE-free medium. ACKNOWLEDGMENTS

This work was supported by grants from the U.S. National Science Foundation (BMS74-1966.5). the Rockefeller Foundation (RF 76018), the MESA New York Bight Project (ERL-NOAA), and the New York Sea Grant Institute. REFERENCES

1. C. F. Wurster, Jr., DDT reduces photosynthesis by marine phytoplankton, Science 159, 1474 (1968). 2. D. W. Menzel, J. Anderson, and A. Randtke, Marine phytoplankton vary in their response to chlorinated hydrocarbons, Science 167, 1724 (1970).

312

POWERS,

WURSTER,

3. J. C. Batterton, G. M. Boush, and F. Matsumura, Growth response of blue-green algae to aldrin, dieldrin, endrin and their metabolites, Bull. Environ. Contam. Toxicol. 6,589 (1971). 4. L. Stadnyk, R. S. Campbell, and B. T. Johnson, Pesticide effect on growth and 14C assimilation in a freshwater alga, Bull. Environ. Contam. Toxicol. 6, 1 (1971). 5. J. L. Mosser, N. S. Fisher, T.-C. Teng, and C. F. Wurster, Polychlorinated biphenyls: Toxicity to certain phytoplankters, Science 175, 191 (1972). 6. J. L. Mosser, N. S. Fisher, and C. F. Wurster, Polychlorinated biphenyls and DDT alter species composition in mixed cultures of algae, Science 176, 533 (1972). 7. N. S. Fisher, E. J. Carpenter, C. C. Remsen, and C. F. Wurster, Effects of PCB on interspecific competition in natural and gnotobiotic phytoplankton communities in continuous and batch cultures, Microbial Ecol. 1, 39 (1974). 8. C. D. Powers, R. G. Rowland, R. A. Michaels, N. S. Fisher, and C. F. Wurster, The toxicity of DDE to a marine dinoflagellate, Environ. Pollut. 9, 253 (1975). 9. C. D. Powers, R. G. Rowland, H. B. O’Connors, Jr., and C. F. Wurster, Response to PCB of marine phytoplankton isolates cultured under natural conditions, Appl. Environ. Microbial. 34, 760 (1977). 10. C. D. Powers, R. G. Rowland, and C. F. Wurster, Dieldrin-induced destruction of marine algal cells with concomitant decrease in size of survivors and their progeny, Environ. Pollut. 12, 17 (1977). 11. G. E. Walsh, K. A. Ainsworth, and L. Faas, Effects and uptake of chlorinated naphthalenes in marine unicellular algae, Bull. Environ. Contam. Toxicol. 18, 297 (1977). 12. B. Magnani, C. D. Powers, C. F. Wurster, and H. B. O’Connors, Jr., Effects of chlordane and heptachlor on the marine dinoflagellate, Exuviella baltica, Lohmann, Bull. Environ. Contam. Toxicol. 20, 1 (1978). 13. S. A. Moore, Jr. and R. C. Harriss, Effects of polychlorinated biphenyl on marine phytoplankton communities, Nature (London) 240, 356 (1972). 14. E. J. Luard, Sensitivity of Dunaliella and Scenedesmus (Chlorophyceae) to chlorinated hydrocarbons, Phycologia 12,29 (1973).

AND

ROWLAND

15. D. R. Cole and F. W. Plapp, Jr., Inhibition of growth and photosynthesis in Chlorella pyrenoidosa by a polychlorinated biphenyl and several insecticides, Environ. Entomol. 3, 217 (1974). 16. V. Glooschenko and W. Glooschenko, Effect of polychlorinated biphenyl compounds on growth of Great Lakes phytoplankton, Canad. J. Bot. 53, 653 (1975). 17. L. W. Harding, Jr., Polychlorinated biphenyl inhibition of marine phytoplankton photosynthesis in the northern Adriatic Sea, BUN. Environ. Contam. Toxicoi. 16, 559 (1976). 18. J. C. Kricher and C. L. Bayer, Depression of primary productivity by Aroclor 1232 in an interspecific lentic algal assemblage, BUN. Environ. Contam. Toxicol. 18, 14 (1977). 19. R. B. MacFarlane, W. A. Glooschenko, and R. C. Harriss, The interaction of light intensity and DDT concentration upon the marine diatom, Nitzschia delicatissitna Cleve, Hydrobiologia 39, 373 (1972). 20. M. L. Hawes, J. C. Kricher, and J. C. Urey, The effects of various Aroclor fractions on the productivity of Chlorella pyrenoidosa, Bull. Environ. Contam. Toxical. 15, 588 (1976). 21. G. W. Bowes and R. W. Gee, Inhibition of photosynthetic electron transport by DDT and DDE, Bioenergetics 2, 47 (1971). 22. G. W. Bowes, Uptake and metabolism of 2,2-bis@-chlorophenyl)- 1,l ,l-trichlorethane (DDT) by marine phytoplankton and its effect on growth and chloroplast electron transport, Plant Physiol. 49, 172 (1972). 23. S. S. Lee, S. C. Fang, and V. H. Freed, Effect of DDT on photosynthesis of Selanastrum capricormutum, Pesiic. Biochem. Physiol. 6, 46 (1976). 24. N. S. Fisher, Chlorinated hydrocarbon pollutants and photosynthesis of marine phytoplankton: A reassessment, Science 189, 463 (1975). 25. R. W. Eppley and J. D. H. Strickland, Kinetics of marine phytoplankton growth, in “Advances in Microbiology of the Sea” (M. R. Droop and E. J. F. Wood, Eds.), Vol. 1, pp. 23-62, Academic Press, London and New York, 1%8. 26. M. Adamich and A. Towle, Ficoll density gradient separation of extracellular DDT from Chlorella, Bull. Environ. Contam. Toxicof. 12, 562 (1974).