J. Biosci., Vol. 5, Number 1, March 1983, pp. 71 77 Printed in India. Correlation between inhibition of photosynthesis and growth of Chlorella treated with methyl parathion G. SAROJA and SALIL BOSE Department of Plant Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021 MS received 5 November 1982; revised 21 January 1983 Abstract. When methyl parathion (Ο,Ο-dimethyl O-p-nitrophenyl phosphorothioate), an organophosphorous insecticide, was added to an exponentially growing culture of Chlorella protothecoides and the effects were followed for 12 days, the following observations were made: a) In autotrophic culture the cell number and the chlorophyll content decreased as compared to the control. These changes paralleled the inhibition of the rate of net photosynthesis, suggesting that the photosynthetic apparatus was the primary target of the insecticide action. b) The inhibition of cell growth (on cell number basis) also occurred in the case of heterotrophic culture at 100μΜ insecticide concentration but the inhibition was less as compared to that of an autotrophic culture. c) The cell diameter in treated culture increased by 10-20% in both autotrophic and heterotrophic cultures. The observations, (b) and (c) suggest that apart from the photosynthetic apparatus, the insecticide has other sites of action, but the sensitivity of these sites to the insecticide is less than that of the photosynthetic apparatus. Keywords. Chlorella protothecoides; methyl parathion; photosynthesis; autotrophic; heterotrophic. Introduction Organophosphorous insecticides are widely used in modern agriculture. Very little information is available on the effects of these insecticides on non-target organisms like plants. Recently it has been shown that methyl parathion, one of the most widely used organophosphorous insecticides, inhibits growth of Chlorella, a green alga, when the insecticide is present in the growth medium (Saroja et al., 1982). Since in this study the normal cells were inoculated in a medium containing methyl parathion, it was not possible to specify whether methyl parathion affected the metabolism in the lag phase or in the exponential phase. In the present study the insecticide was added to exponentially growing cultures only to avoid greater complexity of the problem. Furthermore, based on earlier observation that methyl parathion inhibits photosynthetic electron transport when added to a suspension of chloroplast thylakoids isolated from higher plants (Anbudurai et al., 1981); in this study we have examined the correlation between inhibition of photosynthesis and inhibition of growth in Chlorella upon addition of methyl parathion to an 71
72 Saroja and Bose exponentially growing culture of Chlorella protothecoides. Materials and methods The stock culture of Chlorella protothecoides supplied from the algal culture collection of the University of Indiana (ACC No. 25) was maintained in a nutrient medium containing 1g KH 2 PO 4, 1g K 2 HPO 4, 0.3g MgSO 4, 5mg FeSO 4, 1 ml of Arnon s A5 solution and 10 μg thiamine hydrochloride per litre, plus 0.1 % NH 4 Cl as the nitrogen source (Senger et al., 1976). The medium, 140 ml in each of three 250 ml flasks was inoculated with 10 ml of cells from the stock culture and grown autotrophically for 8 days. The flasks were shaken reciprocally at 120 strokes per minute at 25 C under illumination of intensity of 3000 lux. On the 8th day, methyl parathion was added to 2 flasks to obtain concentrations of 100 μμ and 200 μμ respectively and the remaining one served as the control. A heterotrophic culture of Chlorella protothecoides was maintained in the dark in a medium similar to that for an autotrophic culture, except that the former contained 1% glucose and 0.1% urea. Ten ml of the stock culture was transferred to 140 ml of similar media but containing 100 and 200 μμ concentrations of the insecticide and the cultures were grown in the dark. Other procedures were the same as in the case of autotrophic culture. Cell number was counted with a Neubaur double haemocytometer. Cell diameter was measured microscopically using an ocular micrometer. For pigment analyses, 10 ml aliquots of the cultures were centrifuged at 2000g for 3 min, to obtain a pellet. Chlorophylls were extracted from cells with methanol, and estimated according to Holden (1967) using the extinction coefficient reported by MacKinney(1941). Photosynthesis was monitored in terms of the rate of oxygen evolution measured polarographically under saturating light intensity using a YSI 4004 Clark type oxygen electrode (Yellow Spring Instruments Co., Yellow Spring, Ohio, USA) connected to an amplifier followed by a strip chart recorder (Toshniwal Bros., India). The assay mixture contained 4 ml of the fresh nutrient medium containing 10 to 20 μg Chl/ml equivalent of cells. The temperature was maintained at 25 C by circulating water from a thermostated bath around the reaction vessel. Results Upon the addition of 100 μμ methyl parathion to an exponentially growing culture of Chlorella, increase in cell number was slightly inhibited as compared to the initial (zero day) cell number for the following 6 days (figure 1, solid circles). On 7th day onward the cell number started increasing reaching a value of about 20% more than the initial number at the 12th day. Compared to the control (without methyl parathion) culture, however, the inhibition was severe, around 80% on the 12th day. With 200 μμ methyl parathion the cell number did not increase, rather a gradual decrease in cell number occurred during the period of observation.
, Methyl parathion inhibits photosynthesis 73 Figure 1. Effect of methyl parathion on cell number of autotrophically growing Chlorella protothecoides. Methyl parathion was added to an eight day old culture. The time indicated in the figure is counted from the day of addition of the insecticide to the culture. The inhibition described in the text was calculated using the formula: Chlorophyll content (per ml of culture medium) decreased during the first 6 days after addition of 100 μμ methyl parathion and then showed an increase as compared to the initial value. As compared to the control culture the inhibition was about 80% on the 12th day (figure 2). With 200 μμ methyl parathion, the cells were completely bleached within 5 days after the addition of the insecticide. Figure 3 shows the rates of photosynthesis as a function of days of growth during treatment with various concentrations of methyl parathion. There was a prompt (in 40 min) inhibition of 25% to 55% depending on the concentration used (figure 3B). The inhibition increased further in a non-linear fashion during the rest of the incubation (figure 3A). With 50 and 100 μμ methyl parathion a partial reversal of
74 Saroja and Bose Figure 2. Effect of methyl parathion on total chlorophyll content (per ml) during growth of Chlorella protothecoides. Growth conditions were the same as in figure 1. % inhibition was calculated as in figure 1. Figure 3A. Effect of methyl parathion on photosynthetic O 2 evolution during the growth of Chlorella protothecoides. Growth conditions were the same as in figure 1. Assay conditions for the measurement of photosynthesis have been described in Materials and methods. Figure 3B. Short-term effect of methyl parathion on the rate of O 2 evolution in Chlorella. Methyl parathion at a given concentration was added to a control culture (8 days old) and aliquots were taken at various intervals (as indicated in the abscissa) and the rate was measured. Control rate of O 2 evolution: 224 μmol/mg chlorophyll. The rate was measured on chlorophyll basis, not on culturre volume basis.
, Methyl parathion inhibits photosynthesis 75 the inhibition was observed, while with 200 μμ no O 2 evolution was detectable after 7 days. It should be noted that in control the rate of photosynthesis did not change during the growth period when the rate was measured on chlorophyll basis. The results described so far showed that all the three parameters (cell number, chlorophyll content, and photosynthesis) measured were inhibited by the insecticide treatment. To examine whether there is any correlation between inhibition of these parameters the data were replotted in terms of inhibition of photosynthesis as a function of inhibition of either cell number or chlorophyll content (figure 4). For a meaningful comparison all the parameters were expressed Figure 4. Inhibition of photosynthesis as a function of inhibition of cell number and inhibition of chlorophyll content. Cell number and chlorophyll content were determined from the same batches of cells used for photosynthesis measurements as shown in figure 3. In this figure all the three parameters were expressed on the basis of culture volume and the inhibition was calculated as in figure 1 at various days during the treatment. The circles, triangles and rectangles represent data from treatment with 50,100 and 200 μμ respectively of methyl parathion. per ml of culture. A strong correlation between inhibition of photosynthesis and that of cell number (or chlorophyll content) was obtained over a wide range of inhibition measured. Insecticide-induced inhibition of cell number occurred also in case of nonphotosynthetic (heterotrophically grown) cells (figure 5). However, for a concentration below 200 μμ the inhibition was greater for autotrophic cells than for heterotrophic cells. For instance, with 100 μμ insecticide and at the 5th day, the inhibitions were 80% for the autotrophic cells and 50% for the heterotrophic cells. Table 1 shows that in treated (100 μμ) cells the diameter of the cells increased by about 10 to 20% as compared to the control both in the cases of autotrophic and heterotrophic growth. This increase was outside the normal variability in cell diameter. Discussion The parallelism observed between inhibition of photosynthesis and that of cell number (or chlorophyll content) strongly suggests that inhibition of photosynthesis by methyl parathion is primarily responsibile for inhibition of
76 Saroja and Bose Figure 5. Effect of methyl parathion on cell number of autotrophically and heterotrophically grown Chlorella protothecoides. Methyl parathion was added on the day of inoculation. The inhibition was calculated as in figure 1. The cell number at zero time was 1 10 6 for control and for treated samples under autotrophic and heterotrophic conditions. The cell numbers (in millions) at 5th day were 20.4 (control), 12.6 (100 μμ) and 1.2 (200 μμ) for heterotrophic growth, and 8.5 (control), 1.5 (100 μμ) and 0.4 (200 μμ) for autotrophic growth. Table 1. Relative cell diameter (as % of control) in the insecticide treated cultures. Each value was the mean of measurements of diameter of 15 cells. growth Chlorella. This observation, however, does not exclude other metabolic processes as possible sites of the insecticide action. In fact, an increase in cell diameter during methyl parathion treatment (table 1) suggests that the insecticide affects cell division or related processes.
Methyl parathion inhibits photosynthesis 77 The prompt inhibition of photosynthesis observed upon addition of methyl parathion in the assay mixture is attributed to inhibition of photosynthetic electron transport, because it has been reported recently that Hill reactions (H 2 O dichlorophenol-indophenol or ferricyanide) in thylakoids isolated from Sorghum was inhibited by methy parathion (Anbudurai et al., 1981). The inhibition observed during subsequent days is attributed to superimposed secondary effects, viz., inhibition of chlorophyll content, and changes in the thylakoid membrane organization as indicated by decrease in chlorophyll a to b ratio (Saroja et al., 1982). To examine if there are other sites of action, effects of the insecticide has been tested on the growth of non-photosynthetic cells. Inhibition of cell number has been observed in this case also, indicating clearly the existence of other sites of the insecticide action. As expected, however, the magnitude of inhibition is less as compared to the autotrophic counterpart. The partial reversal of inhibition of growth and photosynthesis observed during the last 4 to 5 days of the experimental period is of special interest. Although methyl parathion degrades rapidly especially within plants, we believe that this reversal was not attributable to the insecticide degradation. Our preliminary studies indicate that an insecticide resistant variant of Chlorella develops during the treatment because by transferring the treated cells every few days to fresh media containing insecticide and by continuing this operation for several weeks we obtain cells which are resistant to the insecticide with respect to growth and photosynthetic activity (to be published elsewhere). Reversal of cell growth inhibition occurred also at 200 μμ and at higher concentrations of methyl parathion when the treatment was carried for longer periods than reported in this communication. Acknowledgement This research was partially supported by the University Grants Commission, New Delhi [Research Scheme No: F.23 (1304)/81 (SR-II)]. References Anbudurai, P. R., Mannar-Mannan, R. and Bose, S. (1981) Biosci., 3, 23. Holden, Μ. Α. (1967) in Chemistry and Bio-chemistry of Plant Pigments, ed. Τ. W. Goodwin (London: Academic Press) p. 488. MacKinney, G. (1941) Biol. Chem., 140, 315. Saroja, G. and Bose, S. (1982) Environmental Pollution (Series A), 27, 297. Senger, H. and Oh-Hama, T. (1976) Plant Physiol., 17, 551.