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Effects of methyl parathion on the growth, cell size, pigment and protein content of Chlorella protothecoides
Enrironmemal Pollution(Series A) 27 (1982) 297-308
EFFECTS OF METHYL PARATHION ON THE GROWTH, CELL SIZE, PIGMENT AND PROTEIN CONTENT OF C H L O R E L L A P R O TO T H E C O I D E S
G. SAROJA • SALIL BOSE
School o f Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India
ABSTRACT
The unicellular green alga Chlorella protothecoides, grown autotrophically in the presence of methyl parathion, showed inhibition in cell number, packed cell volume, pigment (chlorophylls and carotenoids) contents and protein content. Inhibition was slight with 0"001% (lOppm), moderate with 0.002°//0 and severe with 0"003% or higher. For any concentration of the insecticide, chlorophyll content was inhibited more than any other parameter measured. Chlorophyll a appeared to be inhibited more than chlorophyll b, yielding a decrease in the chlorophyll a/b ratio. Cell number was inhibited more than paeked cell volume or protein content~ resulting in an increase in packed cell volume and protein content per cell. The results suggest multiple sites o/ action o/the insecticide on cell metabolism, including inhibition o[ cell division.
INTRODUCTION
In modern agriculture insecticides are widely used to increase plant productivity by controlling pests, but there is also growing concern that the insecticides may adversely affect plant growth and metabolism. Many organochlorine insecticides affect photosynthesis and other cell metabolic events and leave persistent residues in the plant. Organophosphorus insecticides are biodegraded (Fest, 1977) comparatively more readily and hence are replacing the organochlorine pesticides in agricultural use. It is therefore important to examine the effects of organophosphorus insecticides on plant metabolism. Recently it has been shown that methyl parathion, one of the most widely used organophosphorus insecticides, inhibits photosynthetic electron transport in isolated chloroplasts (Anbudurai et al., 1981). We have described the effects of methyl parathion on the growth and metabolism of Chlorella pro297 Enliron. Pollut. Set. A. 0143-1471,82/0027-0297/$02.75 Printed in Great Britain
(" Applied Science Publishers Ltd. England, 1982
298
G. SAROJA, SALIL BOSE
tothecoides, a common unicellular alga which readily contacts pesticides carried by water from fields into streams or ponds near application sites (Brown, 1978).
MATERIALS AND METHODS
Organism and growth conditions A 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 1 g K H / P O 4, 1 g K2HPO4, 0.3 g MgSO 4, 3 mg FeSO 4 . 7H10, 1 ml of Arnon's A s solution and 10 #g thiamine hydrochloride per litre, plus 0.1 NH4C1 as the nitrogen source (Senger & Oh-hama, 1976). The medium (140 ml) in a 250-ml culture flask was inoculated with 10 ml cells from 8-day-old stock culture and grown autotrophically (control). The culture was shaken reciprocally at 120 strokes per minute at 25 °C under illumination of 3000 lux intensity. Duplicate flasks containing methyl parathion at 0.001% (10ppm), 0.002 %, 0-003 ~o and 0.004 were maintained. All the flasks were closed by cotton plugs. All experiments were performed aseptically. Measurement of growth parameters Cell number was counted with a Neubauer double haemocytometer. Packed cell volume (PCV) was determined in haematocrit tubes by centrifugation at 2000 x g for 5 min. Pigment analysis For pigment analyses, 10 ml aliquots were centrifuged at 2000 x g for 3 min to obtain a pellet. Chlorophylls were extracted from the cells with hot methanol and estimated according to the method of Holden (1965), using the extinction coefficient of MacKinney (1941). Total carotenoids were estimated accordingto the method of Robbelen (1957) with additional data by Metzner et al. (1965). Protein estimation Protein was estimated according to the method of Lowry et al. (1951) using Bovine Serum Albumin fraction V as the standard. Source of materials The chemicals were purchased from BDH (KH2PO4, K2HPO 4 and CuSO4), Sarabhai M. Chemicals, Baroda, India (NH4C1, MgSO 4 . 7H20 , FeSO 4 . 7H20 and KNaC4H40 6.4H20), SD'S (NaOH) and Bayer India Ltd, Bombay, India (Metacid-50, the commercial product, containing 50 ~o parathion in a base (50 dimethyl p-nitrophenyl thiophosphate)). Concentrations are of methyl parathion in
% (w/v).
EFFECTS OF METHYL PARATHION ON
Chlorella
299
RESULTS
Effect on growth Cells in the exponential growth phase were transferred to media containing various concentrations of methyl parathion and grown under autotrophic conditions. In the control medium, the cells showed exponential growth after a lag of one day (Fig. 1). On replotting the data on a semilog graph the growth appeared to
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Growth of autotrophically growing Chlorellaprotothecoidesexposed to various concentrations (9/0) of methyl parathion.
be biphasic, the first phase being twice as fast as the second (inset, Fig. 1). In the presence of the insecticide, growth was inhibited. At 0.002 ~ (20 ppm), the cells started growing only after 2 days and growth appeared to be monophasic, the faster phase being absent. Growth was severely inhibited at 0.003 ~ and 0.004 ~. No recovery of growth was observed until day 19 after inoculation.
300
G. SAROJA, SAUL BOSE
Effect on packed cell volume Packed cell volume (PCV) (per culture volume) increased at a slower rate than the control in cultures treated with 0.001 ~o and 0.002 ~o of insecticide whilst, with 0"003 % and 0.004 % of insecticide, PCV was inhibited (Fig. 2) compared with the
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Effect of methyl parathion on packed cell volume (per 10ml) during growth of Chlorella protothecoides. Inset: relative PCV on day 19.
PCV at zero time. PCV calculated on a cell number basis increased, compared with the control, at all concentrations of the insecticide used. For instance, on day 19 the PCV per cell at 0.003 ~ and 0.004 ~o was 43 ~o higher than the control value (inset, Fig. 2).
EFFECTS OF METHYL P A R A T H I O N
ON
Chlorella
301
Effect on chlorophyll content Figure 3 illustrates the change in chlorophyll content (volume of culture basis) during growth after insecticide treatment. At 0.002 %, chlorophyll content started increasing after a lag of 2 days. At 0.003 % and 0.004 % chlorophyll content was severely reduced. Cells were completely bleached by day 8 and remained so until day 19. In the control medium and at 0.001% the chlorophyll content showed a biphasic exponential increase (inset, Fig. 3), the first phase being faster than the second. At 0.002 % there was no faster phase but a lag of 2 days, followed by a slow phase with a rate constant similar to that of the slower phase in the control. 48
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Fig. 3. Effoct of methyl parathion on total chlorophyl| content (l~r miililitr¢) during growth of CMorella protothecoides. Inset: Chlorophyll content plotted on a logarithm scale.
302
G. SAROJA, SALIL BOSE
On insecticide treatment chlorophyll content per treated cell was inhibited compared with the control (Fig. 4). At 0.002 ~ the chlorophyll content per cell remained approximately unchanged during the growth observed up to 19 days. Chlorophyll content decreased drastically at 0.003 ~ and 0-004
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Effect of methyl p a r a t h i o n on c h l o r o p h y l l c o n t e n t (per 10 6 cells) d u r i n g g r o w t h of Chlorella
protothecoides.
Both chlorophyll a and chlorophyll b contents in the treated cells decreased compared with the control (Figs 5 and 6). Chlorophyll a appeared to be more inhibited than chlorophyll b, resulting in a decrease in the chlorophyll a/b ratio (Fig. 7). The preferential inhibition of chlorophyll a, compared with chlorophyll b, predominated with increasing concentration of the insecticide treatment, clearly demonstrated by a gradual and drastic decrease in the chlorophyll a/b ratio with increasing concentrations of insecticide.
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Fig. 5. Effect of methyl parathion on chlorophyll Fig. 6. Effect of methyl parathion on chloroa (per millilitre) during growth of Chlorella phyllb(permillilitre) during growth of Ch/orella
protothecoides,
protothecoides.
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Effect of methyl parathion on chlorophyll a/b ratio during growth of Chlorellaprotothecoides.
304
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Fig. 8. Effect of methyl parathion on carotenoid content during growth of Chlorella protothecoides. Inset: carotenoid content plotted on a logarithm scale. EJJect on carotenoid content
Carotenoids were also inhibited by insecticide treatment compared with the control (Fig. 8). As with chlorophyll, carotenoids also increased biphasically (inset, Fig. 8) in the control and 0-001% but with different rate constants. At 0.002 ~o the increase was monophasic with a rate constant approximately the same as that of the control in the second phase. At 0-003 % and 0-004 % no carotenoid was detected from day 4 onwards. Effect on protein content
Figure 9 shows that the protein content was inhibited in the treated cultures as compared with the control. When expressed on a cell basis, protein content shows a rapid increase followed by a slow decrease in the control. A similar trend was
EFFECTS OF M E T H Y L P A R A T H I O N O N
Chlorella
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Effect of methyl parathion on protein content (per millilitre) during growth of Chiorella protothecoides. Inset: protein content per 10 6 cells plotted against age.
observed with treated cells but with the following differences: (a) the magnitude of the initial increase was less; (b) the peak was postponed toward later days; and (c) the value on day 19 was higher than the control in the case of 0"001 ~o and 0.002 ~o. At 0.003 ~o and 0.004 ~o the protein content per cell was inhibited compared with the control (inset Fig. 9).
DISCUSSION
Methyl parathion at 0.001/°/o concentration does not greatly affect cell metabolism. Cell number, cell volume, protein and pigment contents and biphasic characteristic of growth are not greatly affected compared with controls.
306
G. SARO,JA, SALIL BOSE
X-PCV 0 - PROTEIN O - C E L L NUMBER &-TOTAL CHL O-CHL- 0 A - CHL- b
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Fig. 10. Relative amount (as compared with the control) of protein, pigment contents, PCV and cell number in 0.002 ~ methyl parathion-treated Chlorellaprotothecoides as a function of age of the culture.
At a concentration of 0.002 ~ (20 ppm) cell metabolism is substantially affected. The rapid initial phase of growth is abolished. At 0-003 ~o or higher concentrations cell growth is completely arrested, cell number, pigment and protein contents decreasing during treatment. Chlorinated insecticides, including DDT, DDE, dieldrin and endrin, also inhibit phytoplankton growth (Wurster, 1968; Menzel et al., 1970; Bowes, 1972; Mosser et al., 1972; Powers et al., 1977b). The parameters measured are differentially inhibited by methyl parathion treatment. At 0.002 ~o, for example, pigment content is more affected than are other parameters, with chlorophylls being more affected than carotenoids. Chlorophyll a was inhibited more than chlorophyll b. Similar effects were reported with PCB-
EFFECTS OF METHYL PARATHION ON
Chlorella
307
treated Thalassiosira pseudonana, a marine diatom (Powers et al., 1977a). Since the concentration dependences of inhibition for different parameters are not identical, it is suggested that the insecticide has multiple sites of action on cell metabolism. It may be noted that the time course of inhibition for one parameter is different from that of the other: for example, the rate of inhibition of the protein content is highest whilst that of PCV is least during the early days of growth (Fig. 10). Cell number is inhibited more than PCV or protein content: Figure 10 suggests that the pigment content per cell decreased while protein content and PCV increased. PCV per cell increased, indicating that cell division is inhibited. Increase in protein content per cell has been reported with DCMU-treated Euglena (Calvayrac et al., 1979). The observation that the pigment contents, particularly of chlorophyll, are inhibited most indicates that the photosynthetic activities are affected by insecticide treatment. The significant decrease in the chlorophyll a/b ratio, together with the decrease in total pigment contents, suggests a change in membrane organisation (Salil Bose et al., 1977). Change in the chlorophyll a/b ratio may lead to changes in photosynthetic unit size and/or changes in the concentration of photochemical reaction centres of the two photosystems. It has been reported recently that methyl parathion inhibits the Hill reaction in isolated chloroplasts in higher plants (Anbudurai et al., 1981). It would be worth investigating the cause-effect relationship between the direct inhibition of photosynthesis by the insecticide and its effect on various aspects of cell metabolism as presented in this paper. We have consistently noticed a biphasic characteristic of growth (cell number and pigment contents) of this species under our culture conditions. The biological significance of this biphasic growth is not clear at present but it suggests that the factor(s) controlling the overall growth rate is altered from one phase to the other.
REFERENCES ANBUDURAI, P. R., MANNARMANNAN, R. & SALIL BOSE(1981). The inhibition of photosynthetic electron transport by methyl parathion. J. Biosci., 3, 23-7. BowEs, G. W. (1972). Uptake and metabolism of 2,2-bis-(p-chlorophenyl)-l,l ,l-trichloroethane (DDT) by marine phytoplankton and its effect on growth and chloroplast electron transport. Pl. Physiol., Lancaster, 49, 172-6. BROWN, A. W. A. (Ed.) (1978). Ecology of pesticides. New York, John Wiley. CALVAYRAC,R., BOMSEL,J. L. & L^V^L-M^RTIN, D. (1979). Analysis and characterization of 3-(3,4Dichlorophenyl)-l,l-Dimethyl urea (DCMU)-resistant Euglena. Pl. Physiol., Lancaster, 63, 857-65. FEST, C. (1977). Recent developments of organophosphorus pesticides. Proc. int. Congr. on Phosphorus Compounds, 1st, 17-21 October 1977, 633-48. HOLDEN, M. A. (1965). Chlorophylls. In Chemistry and biochemistry of plant pigments, ed. by T. W. Goodwin, 488-561. London, Academic Press.
308
G. SAROJA, SALIL BOSE
LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L. & RANDALL,R. J. (1951). Protein measurement with folin-phenol reagent. J. biol. Chem., 193, 265-75. MAcKINNEY, G. (1941). Absorption of light by chlorophyll solutions. Y. biol. Chem., 140, 315-22. MI~NZEL,D. W., ANDERSON,J. & RANDTKE,A. (1970). Marine phytoplankton vary in their response to chlorinated hydrocarbons. Science, N.Y., 167, 1724-6. METZNER, H., RAU, H. & SENGER, H. (1965). Untersuchungen zur Synchronisierbarkeit einzelner Pigment Mangelmutanten yon Chlorella. Planta Berl., 65, 186-94. MossEs, J. L., FIsrmR, N. S. & WURSTER,C. F. (1972). Polychlorinated biphenyls and DDT alter species composition in mixed cultures of algae. Science, N.Y., 176, 533-5. PowERS, C. D., ROWLAND,R. G., O'CoNNoRS, H. B. Jr. & WURST~R, C. F. (1977a). Response to polychlorinated biphenyls of marine phytoplankton isolates cultured under natural conditions. Appl. & environ. Microbiol., 34, 760-4. PowERS, C. D., ROWLAND,R. G. & WURSTER,C. F. (1977b). Dieldrin-induced destruction of marine algal cells with concomitant decrease in size of survivors and their progeny. Environ. Pollut., 12, 17-25.
ROgBWLEN,G. (1957). Untersuchungen an Strahlen in dozierten Blatffarbmutanten" yon Arabidopsis thaliana L. Heynh. Z. VererbLehre, 88, 113-20. SAHLBOSE,BURr~, J. J. & ARNTZEN,C. J. (1977). Cation-induced microstructural changes in chloroplast membranes: Effects on photosystem II activity. In Membrane bioenergetics, ed. by L. Packer, A. Trebst and G. Papageorgiou, 245-56. Amsterdam, Elsevier/North Holland. SENGER,H. & On-nXMA,T. (1976). Quantum yield and conformational changes during greening and bleaching of Chlorella protothecoides. PI. Cell Physiol., 17, 551-6. WURSr~R, C. F. (1968). DDT reduces photosynthesis by marine phytoplankton. Science, N. Y., 159, 1474-5.
EFFECTS OF METHYL PARATHION ON THE GROWTH, CELL SIZE, PIGMENT AND PROTEIN CONTENT OF C H L O R E L L A P R O TO T H E C O I D E S
G. SAROJA • SALIL BOSE
School o f Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India
ABSTRACT
The unicellular green alga Chlorella protothecoides, grown autotrophically in the presence of methyl parathion, showed inhibition in cell number, packed cell volume, pigment (chlorophylls and carotenoids) contents and protein content. Inhibition was slight with 0"001% (lOppm), moderate with 0.002°//0 and severe with 0"003% or higher. For any concentration of the insecticide, chlorophyll content was inhibited more than any other parameter measured. Chlorophyll a appeared to be inhibited more than chlorophyll b, yielding a decrease in the chlorophyll a/b ratio. Cell number was inhibited more than paeked cell volume or protein content~ resulting in an increase in packed cell volume and protein content per cell. The results suggest multiple sites o/ action o/the insecticide on cell metabolism, including inhibition o[ cell division.
INTRODUCTION
In modern agriculture insecticides are widely used to increase plant productivity by controlling pests, but there is also growing concern that the insecticides may adversely affect plant growth and metabolism. Many organochlorine insecticides affect photosynthesis and other cell metabolic events and leave persistent residues in the plant. Organophosphorus insecticides are biodegraded (Fest, 1977) comparatively more readily and hence are replacing the organochlorine pesticides in agricultural use. It is therefore important to examine the effects of organophosphorus insecticides on plant metabolism. Recently it has been shown that methyl parathion, one of the most widely used organophosphorus insecticides, inhibits photosynthetic electron transport in isolated chloroplasts (Anbudurai et al., 1981). We have described the effects of methyl parathion on the growth and metabolism of Chlorella pro297 Enliron. Pollut. Set. A. 0143-1471,82/0027-0297/$02.75 Printed in Great Britain
(" Applied Science Publishers Ltd. England, 1982
298
G. SAROJA, SALIL BOSE
tothecoides, a common unicellular alga which readily contacts pesticides carried by water from fields into streams or ponds near application sites (Brown, 1978).
MATERIALS AND METHODS
Organism and growth conditions A 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 1 g K H / P O 4, 1 g K2HPO4, 0.3 g MgSO 4, 3 mg FeSO 4 . 7H10, 1 ml of Arnon's A s solution and 10 #g thiamine hydrochloride per litre, plus 0.1 NH4C1 as the nitrogen source (Senger & Oh-hama, 1976). The medium (140 ml) in a 250-ml culture flask was inoculated with 10 ml cells from 8-day-old stock culture and grown autotrophically (control). The culture was shaken reciprocally at 120 strokes per minute at 25 °C under illumination of 3000 lux intensity. Duplicate flasks containing methyl parathion at 0.001% (10ppm), 0.002 %, 0-003 ~o and 0.004 were maintained. All the flasks were closed by cotton plugs. All experiments were performed aseptically. Measurement of growth parameters Cell number was counted with a Neubauer double haemocytometer. Packed cell volume (PCV) was determined in haematocrit tubes by centrifugation at 2000 x g for 5 min. Pigment analysis For pigment analyses, 10 ml aliquots were centrifuged at 2000 x g for 3 min to obtain a pellet. Chlorophylls were extracted from the cells with hot methanol and estimated according to the method of Holden (1965), using the extinction coefficient of MacKinney (1941). Total carotenoids were estimated accordingto the method of Robbelen (1957) with additional data by Metzner et al. (1965). Protein estimation Protein was estimated according to the method of Lowry et al. (1951) using Bovine Serum Albumin fraction V as the standard. Source of materials The chemicals were purchased from BDH (KH2PO4, K2HPO 4 and CuSO4), Sarabhai M. Chemicals, Baroda, India (NH4C1, MgSO 4 . 7H20 , FeSO 4 . 7H20 and KNaC4H40 6.4H20), SD'S (NaOH) and Bayer India Ltd, Bombay, India (Metacid-50, the commercial product, containing 50 ~o parathion in a base (50 dimethyl p-nitrophenyl thiophosphate)). Concentrations are of methyl parathion in
% (w/v).
EFFECTS OF METHYL PARATHION ON
Chlorella
299
RESULTS
Effect on growth Cells in the exponential growth phase were transferred to media containing various concentrations of methyl parathion and grown under autotrophic conditions. In the control medium, the cells showed exponential growth after a lag of one day (Fig. 1). On replotting the data on a semilog graph the growth appeared to
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O.OOZ
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o0 x t2
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4 AGE
Fig. 1.
6 OF
8 THE
I0
12
X
14
CULTURE
16
18
20
(DAYS)
Growth of autotrophically growing Chlorellaprotothecoidesexposed to various concentrations (9/0) of methyl parathion.
be biphasic, the first phase being twice as fast as the second (inset, Fig. 1). In the presence of the insecticide, growth was inhibited. At 0.002 ~ (20 ppm), the cells started growing only after 2 days and growth appeared to be monophasic, the faster phase being absent. Growth was severely inhibited at 0.003 ~ and 0.004 ~. No recovery of growth was observed until day 19 after inoculation.
300
G. SAROJA, SAUL BOSE
Effect on packed cell volume Packed cell volume (PCV) (per culture volume) increased at a slower rate than the control in cultures treated with 0.001 ~o and 0.002 ~o of insecticide whilst, with 0"003 % and 0.004 % of insecticide, PCV was inhibited (Fig. 2) compared with the
30-
2~
Treatmerit
I
1 PCV
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I00
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132
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2
; 4 OF
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; I0
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CULTURE
I I 18 2 0
(DAYS)
Effect of methyl parathion on packed cell volume (per 10ml) during growth of Chlorella protothecoides. Inset: relative PCV on day 19.
PCV at zero time. PCV calculated on a cell number basis increased, compared with the control, at all concentrations of the insecticide used. For instance, on day 19 the PCV per cell at 0.003 ~ and 0.004 ~o was 43 ~o higher than the control value (inset, Fig. 2).
EFFECTS OF METHYL P A R A T H I O N
ON
Chlorella
301
Effect on chlorophyll content Figure 3 illustrates the change in chlorophyll content (volume of culture basis) during growth after insecticide treatment. At 0.002 %, chlorophyll content started increasing after a lag of 2 days. At 0.003 % and 0.004 % chlorophyll content was severely reduced. Cells were completely bleached by day 8 and remained so until day 19. In the control medium and at 0.001% the chlorophyll content showed a biphasic exponential increase (inset, Fig. 3), the first phase being faster than the second. At 0.002 % there was no faster phase but a lag of 2 days, followed by a slow phase with a rate constant similar to that of the slower phase in the control. 48
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20
(DAYS)
Fig. 3. Effoct of methyl parathion on total chlorophyl| content (l~r miililitr¢) during growth of CMorella protothecoides. Inset: Chlorophyll content plotted on a logarithm scale.
302
G. SAROJA, SALIL BOSE
On insecticide treatment chlorophyll content per treated cell was inhibited compared with the control (Fig. 4). At 0.002 ~ the chlorophyll content per cell remained approximately unchanged during the growth observed up to 19 days. Chlorophyll content decreased drastically at 0.003 ~ and 0-004
0-4
O-CONTROL -0'001 A- 0"002
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4 AGE
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Effect of methyl p a r a t h i o n on c h l o r o p h y l l c o n t e n t (per 10 6 cells) d u r i n g g r o w t h of Chlorella
protothecoides.
Both chlorophyll a and chlorophyll b contents in the treated cells decreased compared with the control (Figs 5 and 6). Chlorophyll a appeared to be more inhibited than chlorophyll b, resulting in a decrease in the chlorophyll a/b ratio (Fig. 7). The preferential inhibition of chlorophyll a, compared with chlorophyll b, predominated with increasing concentration of the insecticide treatment, clearly demonstrated by a gradual and drastic decrease in the chlorophyll a/b ratio with increasing concentrations of insecticide.
151 0 -CONTROL • - 0"001
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Fig. 5. Effect of methyl parathion on chlorophyll Fig. 6. Effect of methyl parathion on chloroa (per millilitre) during growth of Chlorella phyllb(permillilitre) during growth of Ch/orella
protothecoides,
protothecoides.
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Effect of methyl parathion on chlorophyll a/b ratio during growth of Chlorellaprotothecoides.
304
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Fig. 8. Effect of methyl parathion on carotenoid content during growth of Chlorella protothecoides. Inset: carotenoid content plotted on a logarithm scale. EJJect on carotenoid content
Carotenoids were also inhibited by insecticide treatment compared with the control (Fig. 8). As with chlorophyll, carotenoids also increased biphasically (inset, Fig. 8) in the control and 0-001% but with different rate constants. At 0.002 ~o the increase was monophasic with a rate constant approximately the same as that of the control in the second phase. At 0-003 % and 0-004 % no carotenoid was detected from day 4 onwards. Effect on protein content
Figure 9 shows that the protein content was inhibited in the treated cultures as compared with the control. When expressed on a cell basis, protein content shows a rapid increase followed by a slow decrease in the control. A similar trend was
EFFECTS OF M E T H Y L P A R A T H I O N O N
Chlorella
305
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Effect of methyl parathion on protein content (per millilitre) during growth of Chiorella protothecoides. Inset: protein content per 10 6 cells plotted against age.
observed with treated cells but with the following differences: (a) the magnitude of the initial increase was less; (b) the peak was postponed toward later days; and (c) the value on day 19 was higher than the control in the case of 0"001 ~o and 0.002 ~o. At 0.003 ~o and 0.004 ~o the protein content per cell was inhibited compared with the control (inset Fig. 9).
DISCUSSION
Methyl parathion at 0.001/°/o concentration does not greatly affect cell metabolism. Cell number, cell volume, protein and pigment contents and biphasic characteristic of growth are not greatly affected compared with controls.
306
G. SARO,JA, SALIL BOSE
X-PCV 0 - PROTEIN O - C E L L NUMBER &-TOTAL CHL O-CHL- 0 A - CHL- b
90-
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40-
50.
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I 4
AGE
I 8
OF
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THE
I 16
CULTURE
I 20
(DAYS)
Fig. 10. Relative amount (as compared with the control) of protein, pigment contents, PCV and cell number in 0.002 ~ methyl parathion-treated Chlorellaprotothecoides as a function of age of the culture.
At a concentration of 0.002 ~ (20 ppm) cell metabolism is substantially affected. The rapid initial phase of growth is abolished. At 0-003 ~o or higher concentrations cell growth is completely arrested, cell number, pigment and protein contents decreasing during treatment. Chlorinated insecticides, including DDT, DDE, dieldrin and endrin, also inhibit phytoplankton growth (Wurster, 1968; Menzel et al., 1970; Bowes, 1972; Mosser et al., 1972; Powers et al., 1977b). The parameters measured are differentially inhibited by methyl parathion treatment. At 0.002 ~o, for example, pigment content is more affected than are other parameters, with chlorophylls being more affected than carotenoids. Chlorophyll a was inhibited more than chlorophyll b. Similar effects were reported with PCB-
EFFECTS OF METHYL PARATHION ON
Chlorella
307
treated Thalassiosira pseudonana, a marine diatom (Powers et al., 1977a). Since the concentration dependences of inhibition for different parameters are not identical, it is suggested that the insecticide has multiple sites of action on cell metabolism. It may be noted that the time course of inhibition for one parameter is different from that of the other: for example, the rate of inhibition of the protein content is highest whilst that of PCV is least during the early days of growth (Fig. 10). Cell number is inhibited more than PCV or protein content: Figure 10 suggests that the pigment content per cell decreased while protein content and PCV increased. PCV per cell increased, indicating that cell division is inhibited. Increase in protein content per cell has been reported with DCMU-treated Euglena (Calvayrac et al., 1979). The observation that the pigment contents, particularly of chlorophyll, are inhibited most indicates that the photosynthetic activities are affected by insecticide treatment. The significant decrease in the chlorophyll a/b ratio, together with the decrease in total pigment contents, suggests a change in membrane organisation (Salil Bose et al., 1977). Change in the chlorophyll a/b ratio may lead to changes in photosynthetic unit size and/or changes in the concentration of photochemical reaction centres of the two photosystems. It has been reported recently that methyl parathion inhibits the Hill reaction in isolated chloroplasts in higher plants (Anbudurai et al., 1981). It would be worth investigating the cause-effect relationship between the direct inhibition of photosynthesis by the insecticide and its effect on various aspects of cell metabolism as presented in this paper. We have consistently noticed a biphasic characteristic of growth (cell number and pigment contents) of this species under our culture conditions. The biological significance of this biphasic growth is not clear at present but it suggests that the factor(s) controlling the overall growth rate is altered from one phase to the other.
REFERENCES ANBUDURAI, P. R., MANNARMANNAN, R. & SALIL BOSE(1981). The inhibition of photosynthetic electron transport by methyl parathion. J. Biosci., 3, 23-7. BowEs, G. W. (1972). Uptake and metabolism of 2,2-bis-(p-chlorophenyl)-l,l ,l-trichloroethane (DDT) by marine phytoplankton and its effect on growth and chloroplast electron transport. Pl. Physiol., Lancaster, 49, 172-6. BROWN, A. W. A. (Ed.) (1978). Ecology of pesticides. New York, John Wiley. CALVAYRAC,R., BOMSEL,J. L. & L^V^L-M^RTIN, D. (1979). Analysis and characterization of 3-(3,4Dichlorophenyl)-l,l-Dimethyl urea (DCMU)-resistant Euglena. Pl. Physiol., Lancaster, 63, 857-65. FEST, C. (1977). Recent developments of organophosphorus pesticides. Proc. int. Congr. on Phosphorus Compounds, 1st, 17-21 October 1977, 633-48. HOLDEN, M. A. (1965). Chlorophylls. In Chemistry and biochemistry of plant pigments, ed. by T. W. Goodwin, 488-561. London, Academic Press.
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G. SAROJA, SALIL BOSE
LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L. & RANDALL,R. J. (1951). Protein measurement with folin-phenol reagent. J. biol. Chem., 193, 265-75. MAcKINNEY, G. (1941). Absorption of light by chlorophyll solutions. Y. biol. Chem., 140, 315-22. MI~NZEL,D. W., ANDERSON,J. & RANDTKE,A. (1970). Marine phytoplankton vary in their response to chlorinated hydrocarbons. Science, N.Y., 167, 1724-6. METZNER, H., RAU, H. & SENGER, H. (1965). Untersuchungen zur Synchronisierbarkeit einzelner Pigment Mangelmutanten yon Chlorella. Planta Berl., 65, 186-94. MossEs, J. L., FIsrmR, N. S. & WURSTER,C. F. (1972). Polychlorinated biphenyls and DDT alter species composition in mixed cultures of algae. Science, N.Y., 176, 533-5. PowERS, C. D., ROWLAND,R. G., O'CoNNoRS, H. B. Jr. & WURST~R, C. F. (1977a). Response to polychlorinated biphenyls of marine phytoplankton isolates cultured under natural conditions. Appl. & environ. Microbiol., 34, 760-4. PowERS, C. D., ROWLAND,R. G. & WURSTER,C. F. (1977b). Dieldrin-induced destruction of marine algal cells with concomitant decrease in size of survivors and their progeny. Environ. Pollut., 12, 17-25.
ROgBWLEN,G. (1957). Untersuchungen an Strahlen in dozierten Blatffarbmutanten" yon Arabidopsis thaliana L. Heynh. Z. VererbLehre, 88, 113-20. SAHLBOSE,BURr~, J. J. & ARNTZEN,C. J. (1977). Cation-induced microstructural changes in chloroplast membranes: Effects on photosystem II activity. In Membrane bioenergetics, ed. by L. Packer, A. Trebst and G. Papageorgiou, 245-56. Amsterdam, Elsevier/North Holland. SENGER,H. & On-nXMA,T. (1976). Quantum yield and conformational changes during greening and bleaching of Chlorella protothecoides. PI. Cell Physiol., 17, 551-6. WURSr~R, C. F. (1968). DDT reduces photosynthesis by marine phytoplankton. Science, N. Y., 159, 1474-5.