Environmental Pollution (Series A) 27 (1982) 9-14
POLYCHLORINATED BIPHENYLS (PCB) INHIBIT PHOTOSYNTHESIS PER CELL IN THE MARINE DIATOM THALASSIOSIRA PSEUDO NANA*
Ropert A. MICHAELS,t R. GEORGE ROWLAND & CHARLES F. WURSTER
Marine Sciences Research Center, State University of New York, Stony Brook, NY 11794, USA
ABSTRACT
At sampling times of 1, 6, 12, 24 and 48h after inoculation, PCB at 50 parts per thousand million (10°; ptm) reduced '*C uptake per viable cell of the diatom Thalassiosira pseudonana by at least 32, 75, 72,50 and 66%, respectively. Inhibition of !*C uptake per culture was caused both by reduced photosynthesis per cell and by fewer }*C-fixing, viable cells in treated than in control cultures.
INTRODUCTION
The inhibition of photosynthesis in phytoplankton by chlorinated hydrocarbons is well documented (Wurster, 1968; Menzel et a/., 1970; Mosser et al., 1972; Biggs etal., 1978; Harding & Phillips, 1978a; O’Connors er al., 1978; Powers et al., 1979), but it is not entirely clear whether this reduction results from inhibited cell division leading to a smaller number of photosynthesising cells, to reduced photosynthesis per cell, or to both.
Fisher (1975) concluded that polychlorinated biphenyls (PCB) and DDT inhibited cell division, but not photosynthesis per cell, in three species of marine phytoplankton, including the diatom Thalassiosira pseudonana. Powers et al. (1979) and Magnani et al. (1978), however, tested a marine dinoflagellate with DDE, chlordane and heptachlor, and concluded that both cell division and photosynthesis per cell were inhibited. Powers et al. (1977) reached the same conclusion after testing two Thalassiosira species with PCB and MacFarlane eral. (1972) also reported reduced photosynthesis per cell in DDT-treated cultures of a marine diatom.
* Contribution 289 from the Marine Sciences Research Center, State University of New York at Stony
Brook. + Present address: Enviro Control, Inc., One Central Plaza, 11 300 Rockville Pike, Rockville, Md. 20852,
USA.
9 Environ. Pollut. Ser. A. 0143-1471/82/0027-0009/$02:75 © Applied Science Publishers Ltd, England, 1982 Printed in Great Britain
10 ROBERT A. MICHAELS, R. GEORGE ROWLAND, CHARLES F. WURSTER
In the present investigation we determined the effects of PCB on the rate of photosynthesis per cell of T. pseudonana. Since PCB delayed exponential growth of this organism for two to three days, samples were taken during this delay period to assure detection of any inhibition of photosynthesis per cell that might occur during this time. Only viable cells were included in the calculations.
MATERIALS AND METHODS
An estuarine isolate, clone 3H, of Thalassiosira pseudonana (formerly Cyclotella nana) from R.R. L. Guillard, Woods Hole Oceanographic Institution, Wood Hole, Mass., USA (Hasle & Heimdal, 1970) was batch cultured axenically as previously described (Powers et al., 1975).
Four prescription bottles containing 150 ml of medium were inoculated with 10* exponentially growing cells per millilitre. Into two cultures, 15 yl of PCB (Aroclor 1254) stock in methanol solution was inoculated by microsyringe to yield 50 yg litre~ + of PCB. Two control cultures received 15 yl of pure methanol. Cultures were shaken, then incubated in an environmental chamber at 24°C under continuous cool white fluorescent (Sylvania) illumination of 1:3 x 10~?cal em~? min™?.
At intervals, about 2ml of cell suspension was removed, stained with Lugol’s iodine, and counted microscopically in a counting chamber. At each counting time, triplicate 6-ml samples were exposed for 2h to 0-1 ml of ‘*C-bicarbonate solution (specific activity 1 wCim1~!), comprising two illuminated samples and one dark sample. After incubation, samples were filtered under a gentle vacuum onto 0-45-p Millipore filters, washed with medium, placed in scintillation vials with 10 ml of Aquasol (New England Nuclear Corp., Boston, Mass., USA) and counted.
The number of viable cells per PCB-treated culture was determined retrospectively by calculating the minimum number of cells dividing at the maximum growth rate necessary to generate the population counted at the next sampling time. This method assumes that cell division can occur only among viable cells. In treated cultures, therefore, the minimum number of viable cells was taken as that number of cells below which the population from which they were sampled could not possibly grow to the population size observed microscopically at the next sampling time. Since the estimate of viable cells was minimum, the estimate of photosynthesis per viable cell in PCB-treated cultures was maximum. Control cultures, by contrast, were assumed to be 100% viable, so that photosynthesis per control cell would be underestimated. Hence, any inhibitions detected by this procedure would be underestimates of actual inhibitions per cell by PCB.
RESULTS AND DISCUSSION
Control cultures of T. pseudonana grew exponentially for 48h, then tapered off to the stationary phase (Fig. 1). PCB-treated cultures, by contrast, grew slowly for about 72h, then accelerated and grew exponentially. The exponential growth of
PCBs INHIBIT DIATOM PHOTOSYNTHESIS ll
10} 7 / 7 / 7 / a 2 = , oc CONTROL w 7 a. ye 10> “ , rm / ° PCB / - 7 / ii a 7) ie im a ei Pot ‘pal it a ¥ es 104 1 1 . 1 1. 0 20 40 60 80 100 120 140 ELAPSED TIME (HRS) Fig. |. Growth of Thalassiosira pseudonana cultures. Dashed line represents cultures treated with
50 ptm of PCB, Controls received methanol solvent only. Points are mean + range of two replicates.
treated cultures, however, was reduced from a specific growth rate of 3-3day~! in controls to 1-3day~ !.
Samples taken 1, 6, 12, 24 and 48h after initial treatment with PCB exhibited inhibition of photosynthetic '*C uptake per culture, per cell and per viable cell, calculated as described (Table 1). Even when the number of viable cells was calculated as the minimum possible, giving a maximum estimate of photosynthesis per viable cell, photosynthesis per viable cell was markedly inhibited by PCB at all sampling times. Inhibition was evident as early as 1h after the addition of PCB (Table 1),
IUmay be that Fisher (1975), who sampled his PCB-treated 7, pseudonana cultures only once at 48h, measured photosynthesis per cell after exponential growth had resumed, thereby failing to detect photosynthetic inhibition per cell that had occurred earlier, During exponential growth, photosynthesis per cell would be high,
12 ROBERT A. MICHAELS, R. GEORGE ROWLAND, CHARLES F. WURSTER
TABLE | EFFECT OF 50 ptm OF PCB on '*C upTAKE BY Thalossiosira pseudonana: UPTAKE PER CULTURE, PER CELL AND PER VIABLE CELL, CALCULATED AS DESCRIBED IN THE TEXT
Sample Treatment '4C uptake per culture $C uptake per cell 14C per viable cell (A)
Mean Ve Mean % Mean vA
(CPM/6ml) Control (CPM x 10~*) Control (CPM x 10~*) Control
ol Control 179 100 29-8 100 29-8 100
PCB 122 68 20:3 68 20:3 68
6 Control 465 100 45-9 100 45-9 100
PCB 83 18 10-0 22 13) 25
12 Control 831 100 36-4 100 36-4 100
PCB 60 12) 6-0 16 10-2 28
24 Control 2555 100 31:8 100 31:8 100
PCB 59 2:3 5:2 16 15-9 50
48 Control 43096 100 56-4 100 56-4 100
PCB 97 0-2 15 13 19-0 34
CPM =Counts per minute.
even though the number of cells had not yet caught up with controls. Control cultures might at the same time have been entering the stationary phase, characterised by diminished photosynthesis per cell. A single sampling at 48 h, then, may have compared control cultures at a time in their growth cycle when photosynthesis was diminishing with PCB-treated cultures when their photosynthesis was accelerating, thereby leading to a spurious conclusion.
Several considerations make the PCB concentration of 50 ptm employed in this investigation more relevant to the PCB situation in nature than it might at first appear. Although 50 ptm is much higher than PCB concentrations found in open ocean water (Harvey efal., 1973), it is comparable with concentrations in certain coastal or estuarine situations (Duke etal., 1970; Duce etal., 1972; Huntzinger etal., 1974). Furthermore, our initial cell density of 10* cells ml~! is much higher than most natural phytoplankton population densities, thereby lowering the PCB dosage per cell.
PCB found in the water of natural marine ecosystems may be only a fraction of the total PCB available to the biota within those systems. PCB sorbed to sediments and suspended particulates can be transferred through water to the phytoplankton (Harding & Phillips, 19785; this laboratory, to be published). No such PCB reservoir was present in our experiments, the only PCB being that added directly to the water.
Our findings that a chlorinated hydrocarbon inhibited photosynthesis per cell in phytoplankton are consistent with the similar conclusions of MacFarlane etal. (1972), Powers etal. (1977), Magnani etal. (1978), Harding & Phillips (1978) and Powers etal. (1979). They are also compatible with evidence that several organochlorine compounds inhibit the photosynthetic process, possibly by
PCBs INHIBIT DIATOM PHOTOSYNTH 13
interfering with electron transport (Lawler & Rogers, 1967; Rogers etal., 1971; Bowes, 1972; Lee ez a/., 1976; Sinclair et al., 1977; Biggs et al., 1978; Harding & Phillips, 1978a). Inhibited photosynthesis by phytoplankton cultures, therefore, apparently results both from reduced photosynthesis per cell and a smaller number of photosynthesising cells, the latter quite probably being caused by the former.
ACKNOWLEDGEMENTS
We are grateful for grants in support of this research from the New York Sea Grant Institute, the MESA New York Bight Project (NOAA), the New York State Department of Environmental Conservation and the Jesse Smith Noyes Foundation.
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Publication citation
Michaels, RA; R. George Rowland, and Charles F. Wurster. Polychlorinated biphenyls (PCB) inhibit photosynthesis per cell in the diatom Thalassiosira pseudonana. Environmental Pollution (Series A), 27(1):9-14, 1982;