Bull. Environ. Contam. Toxicol. (1992) 49:576-581 9 1992 Springer-Verlag New York Inc.
ConZamirmt;ion and Toxicology
Growth Pattern Changes of Chlorella vulgaris and Anabaena doliolum Due to Toxicity of Dimethoate and Endosulfan P. K. Mohapatra and R. C. Mohanty* Environmental Biology Laboratory, Department of Botany, Utkal University, Bhubaneswar-751004, India Effects of p e s t i c i d e s in the e c o s y s t e m do not remain r e s t r i c t e d to target o r g a n i s m s but rather extend to nontarget organisms like microorganisms which play an important role in the food chain v i s - a - v i s biological processes such as b i o g e o c h e m i c a l cycling, production, decomposition, interaction with other organisms, etc. A g r o c h e m i c a l s c o n t a m i n a t e surface waters of a g r i c u l t u r a l regions and e f f e c t i v e l y inhibit growth, pigment biomass and survival rate of freshwater p h y t o p l a n k t o n (Lal 1984; A d h i k a r y 1989; Mandal and M o h a n t y 1990: N e t r a w a l i and Gandhi 1990). However, other t o x i c i t y m e t h o d such as effect on growth pattern is useful to achieve standardization. The present paper d e s c r i b e s the effects of two pesticides, viz., d i m e t h o a t e and e n d o s u l f a n on the growth patterns of C h l o r e l l a v u l g a r i s and A n a b a e n a doliolum. MATERIALS
AND METHODS
The axenic cultures of C h l o r e l l a vulgaris Beij. and Anabaena doliolum Bhar. of this laboratory were maintained in a liquid and sterilized modified Chu + No.10 medium ( Safferman and Morris 1964) with A6 micronutrients of Allen and Arnon (1955) for A n a b a e n a and of G e r l o f f et al. (1950) for Chlorella. The stock cultures were grown in 250 -mL b o r o s i l i c a t e conical flasks containing i00 mL of the culture m a i n t a i n e d in a culture room at 27 • 2~ with l i g h t / d a r k 12/12 hr, photosynthetic photon flux density (PPFD) 70 ~ m o l / m 2 sec, p h o t o s y n t h e t i c a l l y active radiation (PAR: 400-700 nm), and relative h u m i d i t y (RH) 75%. The e x p e r i m e n t a l cultures were also grown under the same c o n d i t i o n s in 100-mL b o r o s i l i c a t e conical flasks containing 25 mL of medium and using e x p o n e n t i a l l y grown cultures (7 d old) as initial inoculum. An
organophosphorus
*Author
to whom
pesticide
correspondence
576
dimethoate
should
be made
[ 0,
0-
dimethyl-S(-N-methyl-carbamoylethyl)-dithiophosphate]and an o r g a n o c h l o r i n e p e s t i c i d e e n d o s u l f a n (6, 7, 8, 9, i0, 1 0 - h e x a c h l o r o - l , 5, 5a, 6, 9, 9 a - h e x a h y d r o - 6 , 9 - m e t h a n o 2, 4, 3 - b e n z o d i o x a t h i e p i n - 3 - o x i d e ) , made a v a i l a b l e from Rallis India Ltd., Bombay, were used as the test chemicals. The stock s o l u t i o n s (i g/L) of these (acetone dissolved) w e r e p r e p a r e d in the s t e r i l i z e d m e d i u m under aseptic conditions for r e p e a t e d use at p r e d e t e r m i n e d concentrations. The survival test of both the o r g a n i s m s was c o n d u c t e d in nutrient agar plates with certain varying c o n c e n t r a t i o n s of the pesticides. Sample of 0.I mL of the e x p o n e n t i a l l y grown culture (12 filaments /cm 2 and 15 cells /cm 2 in case of A n a b a e n a and Chlorella, respectively) was spread evenly and a s e p t i c a l l y on each plate and incubated in the culture room for i0 d. The survival rate was c a l c u l a t e d as d e s c r i b e d in an earlier report (Mohapatra et al. 1990). The test c h e m i c a l s were applied just at the beginning of log phase (8 hr and 30 hr after inoculation in case of Anabaena and Chlorella, respectively). The growth patterns were studied incubating the cultures separately with initial cell numbers 1.40 x 106 cells/mL (Chlorella) and 0.59 x 106 cells/mL (Anabaena), for 16 d and m e a s u r i n g the light t r a n s m i s s i o n of the h o m o g e n i z e d cultures at 660 nm every 4 d by a Carl Zeiss SPEKOL spectrophotometer. The t r a n s m i s s i o n values were then converted to ~ log 2 OD (Sor0kin 1975). RESULTS
AND D I S C U S S I O N
None of the p e s t i c i d e s had any inhibitory effect on C h l o r e l l a at low c o n c e n t r a t i o n s . However, reduction in survival rates of the alga was o b s e r v e d at nominal c o n c e n t r a t i o n of i0 mg/L of both pesticides, endosulfan being more toxic than dimethoate. The sublethal doses (LC50) for the alga were 51.0 • 0.79 mg/L and 41.5 • 1.39 mg/L of d i m e t h o a t e and endosulfan, respectively (Table i). Growth of C h l o r e l l a c o m p l e t e l y ceased on the agar plates at 125 mg/L of d i m e t h o a t e and at i00 mg/L of endosulfan. On the other hand, reduction of survivability of A n a b a e n a was found at all the tested c o n c e n t r a t i o n s of both the pesticides (Table 2). The LC50s for this c y a n o b a c t e r i u m were 28.50 • 0.61 mg/L and 2.15 • 0.07 mg/L of d i m e t h o a t e and endosulfan, respectively. The o r g a n i s m t o l e r a t e d a m a x i m u m c o n c e n t r a t i o n of 40 mg/L of d i m e t h o a t e and 3 mg/L of endosulfan, while all other higher doses were lethal. In case of Chlorella, d i m e t h o a t e at a c o n c e n t r a t i o n 1 mg/L did not cause any significant change in 577
of the
T a b l e i. S u r v i v a l rate of C h l o r e l l a v u l g a r i s at d i f f e r e n t nominal concentrations of the ( C u l t u r e s in t r i p l i c a t e ) . Conc. mg/L 0 1 i0 25 50 75 i00 125
in
Dimethoate Survival %
LC50 = 51.0•
Endosulfan Conc. in Survival % SE mg/L 0 I00.0 2.98 0.i i00.0 2.63 1 i00.0 2.51 i0 84.4 3.08 30 74.7 1.02 40 54.6 1.26 50 31.6 1.20 75 13.4 0.33 i00 0.0 0.0 LC50 = 41.5+1.39 mg/L (p=0.05)
SE
i00.0 i00.0 93.1 84.8 55.4 19.6 11.4 0.0
1.80 1.41 1.48 3.42 2.56 2.44 1.70 0.0
mg/L (p=0.05)
T a b l e 2. S u r v i v a l rate of A n a b a e n a d o l i o l u m at d i f f e r e n t n o m i n a l concentrations of the ( C u l t u r e s in t r i p l i c a t e ) . Conc. mg/L 0 1 i0 20 30 40 50
in
Dimethoate Survival i00.0 94.6 71.8 62.9 48.5 4.8 0.0
LC50 = 28.5• SE = S t a n d a r d
%
a f t e r i0 d pesticides
a f t e r i0 d pesticides
Endosulfan Conc. in Survival % SE mg/L 0 i00.0 1.12 0.i 96.4 1.18 0.5 93.9 0.45 1.0 92.8 1..41 1.5 60.9 0.70 2.0 52.1 0.70 2.5 5.1 1.42 3.0 1.0 0.0 3.5 0.0 0.0 LC50 = 2.15+0.07 mg/L(p=0.05)
SE 1.12 2.63 1.71 1.92 1.50 0.73 0.0
mg/L (p=0.05) error
growth pattern whereas endosulfan, at all the t e s t e d c o n c e n t r a t i o n s , had some k i n d of e f f e c t s on it (Fig. 1 and 2). D i m e t h o a t e at i0 and 25 m g / L also did not s h o w any c h a n g e in the s i g m o i d a l p a t t e r n of g r o w t h , w h i l e at 0.i and 1.0 m g / L of e n d o s u l f a n s h o r t e n i n g of log p h a s e and c h a n g e in s i g m o i d a l g r o w t h c u r v e w e r e o b s e r v e d a f t e r 8 d. D i m e t h o a t e at 40 and 50 m g / L c a u s e d s l i g h t d e l a y i n g of lag p h a s e and also s h o r t e n i n g of log phase, w h i l e more irregularities in l o g i s t i c g r o w t h p a t t e r n of the alga w e r e seen at o t h e r h i g h e r c o n c e n t r a t i o n s of both the p e s t i c i d e s . Unlike Chlorella, the g r o w t h pattern of A n a b a e n a was affected from initial stage of the culture by the p e s t i c i d e s . D i m e t h o a t e , h o w e v e r , did not s h o w any c h a n g e in the g r o w t h p a t t e r n at c o n c e n t r a t i o n s ~ i0 mg/L (Fig.3). At 20 and 30 m g / L of the p e s t i c i d e , partial
578
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Figure i. Effect of d i m e t h o a t e growth pattern of C. v u l g a r i s
concentrations
(mg/L)
on
Figure 2. Effect of e n d o s u l f a n growth pattern of C. vulgaris
concentrations
(mg/L)
on
growth inhibition during the initial stage (up to 4 d ) resulted in a p s e u d o - l e n g t h e n i n g of lag phase. Delayed growth coupled with l e n g t h e n i n g of lag phase of the c y a n o b a c t e r i u m was found at all other higher doses of the p e s t i c i d e ( ~ 40 mg/L). E n d o s u l f a n also at 0.i and 0.5 mg/L did not show any r e m a r k a b l e change in the g r o w t h pattern of A n a b a e n a though r e d u c t i o n in the growth rates was o b s e r v e d at such c o n c e n t r a t i o n s (Fig.4). At 1 mg/L of the pesticide, partial l e n g t h e n i n g of lag phase resulted due to growth inhibition at initial level of the culture but this c o n c e n t r a t i o n did not affect the rest of the logistic phase. However, while the p e s t i c i d e at 2 mg/L resulted in a totally different growth pattern by affecting both lag and log phases, with a p p l i c a t i o n of other higher concentrations ( ~ 2.5 mg/L), the c y a n o b a c t e r i u m was found to be at lag phase for the entire 16 d. Endosulfan was found to be more e f f e c t i v e in reducing the survivability of both the test organisms than d i m e t h o a t e and this finding supports the view that organochlorines are more effective than o r g a n o p h o s p h a t e s . Since both the groups of the pesticides have cell d e s t r u c t i v e action ( A n t u n e s - M a d e i r a et al.
579
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Figure 3. Effect of d i m e t h o a t e growth pattern of A. d o l i o l u m
concentrations
(mg/L)
on
Figure 4. Effect of endosulfan growth pattern of A. doliolum
concentrations
(mg/L)
on
1980; N e t r a w a l i and Gandhi 1990), the reduction rate, the present study, at higher doses might be due killing of some cells from the beginning.
in to
Four different types of growth patterns, in r e s p o n s e to the pesticides, of the test organisms were o b s e r v e d viz. i) partial inhibition and slight change in log phase, ii) delayed inhibition and s h o r t e n i n g of log phase, iii) initial growth inhibition with subsequent growth resumption and lengthening of lag phase, and iv) complete inhibition of growth. The delayed inhibition of growth was r e s p o n s i b l e for the shortening of log phase as noted in case of C h l o r e l l a in response to e n d o s u l f a n ( Fig.2). Probably the alga was very efficient in a c c u m u l a t i n g the o r g a n o c h l o r i n e pesticide as a result of which the concentration inside the cell increased steadily causing delayed effect (Hansen 1980). On the other hand, in both the test o r g a n i s m s the lengthening of lag phase, due to initial growth inhibition and resumption afterwards, might be because of (i) biodegradation of the pesticide, (ii) a u t o d e g r a d a t i o n of the chemicals, (iii) m o d i f i c a t i o n by the organisms, and/or (iv) decrease influx of the
580
chemicals into the cells, caused m o d i f i c a t i o n (Hansen 1980; Thomas and 1986; N e t r a w a l i and Gandhi 1990).
by cell wall Shanmugasundaram
A c k n o w l e d g m e n t s . We thank Prof. P.C.Tripathy, Head of the D e p a r t m e n t of Botany, Utkal U n i v e r s i t y for providing us the l a b o r a t o r y facilities and the UGC, New Delhi for the financial assistance. REFERENCES Adhikary SP (1989) Effect of p e s t i c i d e s on the growth, p h o t o s y n t h e t i c oxygen e v o l u t i o n and n i t r o g e n fixation of W e s t i e l l o p s i s prolifica. J Gen Appl M i c r o b i o l 35: 319-325 Allen MB, Arnon DI (1955) Studies on n i t r o g e n fixing b l u e - g r e e n algae I. growth and nitrogen fixation by Anabaena c y l i n d r i c a Lemm. Plant Physiol 30: 366-372 Antunes-Madeira MC, Carvalho AP, Madeira VMC (1980) Effect of insecticides on t h e r m o t r o p i c lipid phase transition. P e s t i c i d e B i o c h e m Physiol 1 4 : 1 6 1 - 1 6 9 Gerloff GC, F i t z g e r a l d GP, Skoog F (1950) The isolation, p u r i f i c a t i o n and culture of b l u e - g r e e n algae, Amer J Bot 3 7 : 2 1 6 - 2 1 8 Hansen PD (1980) Uptake and t r a n s f e r of c h l o r i n a t e d h y d r o c a r b o n Lindane ( T -BHC) in a l a b o r a t o r y fresh water chain. Environ Pollut 2 1 : 9 7 - 1 0 8 Lal S (1984) M i c r o b i a l a c c u m u l a t i o n of insecticides. Insecticide Microbiol, S p r i n g e r - V e r l a g , Berlin, pp 6185 Mandal J, Mohanty RC (1990) Effect of m a n c o z e b on growth, carbohydrate, protein and chlorophyll contents of the green alga C h l o r e l l a v u l g a r i s Beijerinck. Pollut Res 8: 159-162 Mohapatra, PK, Sethi PK, M o h a n t y RC (1990) Toxicity of d i m e t h o a t e to c y a n o b a c t e r i u m Anabaena d o l i o l u m Bhar. In: Dalela RC (ed) Trends ecotoxicol, AEB, M u z a f f e r n a g a r , pp 189-196 Netrawali MS, Gandhi SR (1990) Mechanism of cell destructive action of organophosphorus insecticide phosalone in C h l a m y d o m o n a s reinhardtii algal cells. Bull Environ C o n t a m Toxicol 4 4 : 8 1 9 - 8 2 5 S a f f e r m a n RS, Morris ME (1964) Growth c h a r a c t e r i s t i c s of b l u e - g r e e n algal virus LPP-I. J Bact 88: 771-775 Sorokin C (1975) Dry weight, packed cell v o l u m e and optical density. In: Stein JR (ed) Handbook of phycological methods, Cambridge University Press, Cambridge, pp 321-324 Thomas PS, Shanmugasundaram S (1986) Interaction of a g r o c h e m i c a l s with the c y a n o b a c t e r i u m A n a c y s t i s n i d u l a n s and patterns of growth. M i c r o b i o s Lett 33: 115-120 Received January 2, 1992; accepted March 6, 1992.
581
ConZamirmt;ion and Toxicology
Growth Pattern Changes of Chlorella vulgaris and Anabaena doliolum Due to Toxicity of Dimethoate and Endosulfan P. K. Mohapatra and R. C. Mohanty* Environmental Biology Laboratory, Department of Botany, Utkal University, Bhubaneswar-751004, India Effects of p e s t i c i d e s in the e c o s y s t e m do not remain r e s t r i c t e d to target o r g a n i s m s but rather extend to nontarget organisms like microorganisms which play an important role in the food chain v i s - a - v i s biological processes such as b i o g e o c h e m i c a l cycling, production, decomposition, interaction with other organisms, etc. A g r o c h e m i c a l s c o n t a m i n a t e surface waters of a g r i c u l t u r a l regions and e f f e c t i v e l y inhibit growth, pigment biomass and survival rate of freshwater p h y t o p l a n k t o n (Lal 1984; A d h i k a r y 1989; Mandal and M o h a n t y 1990: N e t r a w a l i and Gandhi 1990). However, other t o x i c i t y m e t h o d such as effect on growth pattern is useful to achieve standardization. The present paper d e s c r i b e s the effects of two pesticides, viz., d i m e t h o a t e and e n d o s u l f a n on the growth patterns of C h l o r e l l a v u l g a r i s and A n a b a e n a doliolum. MATERIALS
AND METHODS
The axenic cultures of C h l o r e l l a vulgaris Beij. and Anabaena doliolum Bhar. of this laboratory were maintained in a liquid and sterilized modified Chu + No.10 medium ( Safferman and Morris 1964) with A6 micronutrients of Allen and Arnon (1955) for A n a b a e n a and of G e r l o f f et al. (1950) for Chlorella. The stock cultures were grown in 250 -mL b o r o s i l i c a t e conical flasks containing i00 mL of the culture m a i n t a i n e d in a culture room at 27 • 2~ with l i g h t / d a r k 12/12 hr, photosynthetic photon flux density (PPFD) 70 ~ m o l / m 2 sec, p h o t o s y n t h e t i c a l l y active radiation (PAR: 400-700 nm), and relative h u m i d i t y (RH) 75%. The e x p e r i m e n t a l cultures were also grown under the same c o n d i t i o n s in 100-mL b o r o s i l i c a t e conical flasks containing 25 mL of medium and using e x p o n e n t i a l l y grown cultures (7 d old) as initial inoculum. An
organophosphorus
*Author
to whom
pesticide
correspondence
576
dimethoate
should
be made
[ 0,
0-
dimethyl-S(-N-methyl-carbamoylethyl)-dithiophosphate]and an o r g a n o c h l o r i n e p e s t i c i d e e n d o s u l f a n (6, 7, 8, 9, i0, 1 0 - h e x a c h l o r o - l , 5, 5a, 6, 9, 9 a - h e x a h y d r o - 6 , 9 - m e t h a n o 2, 4, 3 - b e n z o d i o x a t h i e p i n - 3 - o x i d e ) , made a v a i l a b l e from Rallis India Ltd., Bombay, were used as the test chemicals. The stock s o l u t i o n s (i g/L) of these (acetone dissolved) w e r e p r e p a r e d in the s t e r i l i z e d m e d i u m under aseptic conditions for r e p e a t e d use at p r e d e t e r m i n e d concentrations. The survival test of both the o r g a n i s m s was c o n d u c t e d in nutrient agar plates with certain varying c o n c e n t r a t i o n s of the pesticides. Sample of 0.I mL of the e x p o n e n t i a l l y grown culture (12 filaments /cm 2 and 15 cells /cm 2 in case of A n a b a e n a and Chlorella, respectively) was spread evenly and a s e p t i c a l l y on each plate and incubated in the culture room for i0 d. The survival rate was c a l c u l a t e d as d e s c r i b e d in an earlier report (Mohapatra et al. 1990). The test c h e m i c a l s were applied just at the beginning of log phase (8 hr and 30 hr after inoculation in case of Anabaena and Chlorella, respectively). The growth patterns were studied incubating the cultures separately with initial cell numbers 1.40 x 106 cells/mL (Chlorella) and 0.59 x 106 cells/mL (Anabaena), for 16 d and m e a s u r i n g the light t r a n s m i s s i o n of the h o m o g e n i z e d cultures at 660 nm every 4 d by a Carl Zeiss SPEKOL spectrophotometer. The t r a n s m i s s i o n values were then converted to ~ log 2 OD (Sor0kin 1975). RESULTS
AND D I S C U S S I O N
None of the p e s t i c i d e s had any inhibitory effect on C h l o r e l l a at low c o n c e n t r a t i o n s . However, reduction in survival rates of the alga was o b s e r v e d at nominal c o n c e n t r a t i o n of i0 mg/L of both pesticides, endosulfan being more toxic than dimethoate. The sublethal doses (LC50) for the alga were 51.0 • 0.79 mg/L and 41.5 • 1.39 mg/L of d i m e t h o a t e and endosulfan, respectively (Table i). Growth of C h l o r e l l a c o m p l e t e l y ceased on the agar plates at 125 mg/L of d i m e t h o a t e and at i00 mg/L of endosulfan. On the other hand, reduction of survivability of A n a b a e n a was found at all the tested c o n c e n t r a t i o n s of both the pesticides (Table 2). The LC50s for this c y a n o b a c t e r i u m were 28.50 • 0.61 mg/L and 2.15 • 0.07 mg/L of d i m e t h o a t e and endosulfan, respectively. The o r g a n i s m t o l e r a t e d a m a x i m u m c o n c e n t r a t i o n of 40 mg/L of d i m e t h o a t e and 3 mg/L of endosulfan, while all other higher doses were lethal. In case of Chlorella, d i m e t h o a t e at a c o n c e n t r a t i o n 1 mg/L did not cause any significant change in 577
of the
T a b l e i. S u r v i v a l rate of C h l o r e l l a v u l g a r i s at d i f f e r e n t nominal concentrations of the ( C u l t u r e s in t r i p l i c a t e ) . Conc. mg/L 0 1 i0 25 50 75 i00 125
in
Dimethoate Survival %
LC50 = 51.0•
Endosulfan Conc. in Survival % SE mg/L 0 I00.0 2.98 0.i i00.0 2.63 1 i00.0 2.51 i0 84.4 3.08 30 74.7 1.02 40 54.6 1.26 50 31.6 1.20 75 13.4 0.33 i00 0.0 0.0 LC50 = 41.5+1.39 mg/L (p=0.05)
SE
i00.0 i00.0 93.1 84.8 55.4 19.6 11.4 0.0
1.80 1.41 1.48 3.42 2.56 2.44 1.70 0.0
mg/L (p=0.05)
T a b l e 2. S u r v i v a l rate of A n a b a e n a d o l i o l u m at d i f f e r e n t n o m i n a l concentrations of the ( C u l t u r e s in t r i p l i c a t e ) . Conc. mg/L 0 1 i0 20 30 40 50
in
Dimethoate Survival i00.0 94.6 71.8 62.9 48.5 4.8 0.0
LC50 = 28.5• SE = S t a n d a r d
%
a f t e r i0 d pesticides
a f t e r i0 d pesticides
Endosulfan Conc. in Survival % SE mg/L 0 i00.0 1.12 0.i 96.4 1.18 0.5 93.9 0.45 1.0 92.8 1..41 1.5 60.9 0.70 2.0 52.1 0.70 2.5 5.1 1.42 3.0 1.0 0.0 3.5 0.0 0.0 LC50 = 2.15+0.07 mg/L(p=0.05)
SE 1.12 2.63 1.71 1.92 1.50 0.73 0.0
mg/L (p=0.05) error
growth pattern whereas endosulfan, at all the t e s t e d c o n c e n t r a t i o n s , had some k i n d of e f f e c t s on it (Fig. 1 and 2). D i m e t h o a t e at i0 and 25 m g / L also did not s h o w any c h a n g e in the s i g m o i d a l p a t t e r n of g r o w t h , w h i l e at 0.i and 1.0 m g / L of e n d o s u l f a n s h o r t e n i n g of log p h a s e and c h a n g e in s i g m o i d a l g r o w t h c u r v e w e r e o b s e r v e d a f t e r 8 d. D i m e t h o a t e at 40 and 50 m g / L c a u s e d s l i g h t d e l a y i n g of lag p h a s e and also s h o r t e n i n g of log phase, w h i l e more irregularities in l o g i s t i c g r o w t h p a t t e r n of the alga w e r e seen at o t h e r h i g h e r c o n c e n t r a t i o n s of both the p e s t i c i d e s . Unlike Chlorella, the g r o w t h pattern of A n a b a e n a was affected from initial stage of the culture by the p e s t i c i d e s . D i m e t h o a t e , h o w e v e r , did not s h o w any c h a n g e in the g r o w t h p a t t e r n at c o n c e n t r a t i o n s ~ i0 mg/L (Fig.3). At 20 and 30 m g / L of the p e s t i c i d e , partial
578
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Figure i. Effect of d i m e t h o a t e growth pattern of C. v u l g a r i s
concentrations
(mg/L)
on
Figure 2. Effect of e n d o s u l f a n growth pattern of C. vulgaris
concentrations
(mg/L)
on
growth inhibition during the initial stage (up to 4 d ) resulted in a p s e u d o - l e n g t h e n i n g of lag phase. Delayed growth coupled with l e n g t h e n i n g of lag phase of the c y a n o b a c t e r i u m was found at all other higher doses of the p e s t i c i d e ( ~ 40 mg/L). E n d o s u l f a n also at 0.i and 0.5 mg/L did not show any r e m a r k a b l e change in the g r o w t h pattern of A n a b a e n a though r e d u c t i o n in the growth rates was o b s e r v e d at such c o n c e n t r a t i o n s (Fig.4). At 1 mg/L of the pesticide, partial l e n g t h e n i n g of lag phase resulted due to growth inhibition at initial level of the culture but this c o n c e n t r a t i o n did not affect the rest of the logistic phase. However, while the p e s t i c i d e at 2 mg/L resulted in a totally different growth pattern by affecting both lag and log phases, with a p p l i c a t i o n of other higher concentrations ( ~ 2.5 mg/L), the c y a n o b a c t e r i u m was found to be at lag phase for the entire 16 d. Endosulfan was found to be more e f f e c t i v e in reducing the survivability of both the test organisms than d i m e t h o a t e and this finding supports the view that organochlorines are more effective than o r g a n o p h o s p h a t e s . Since both the groups of the pesticides have cell d e s t r u c t i v e action ( A n t u n e s - M a d e i r a et al.
579
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Figure 3. Effect of d i m e t h o a t e growth pattern of A. d o l i o l u m
concentrations
(mg/L)
on
Figure 4. Effect of endosulfan growth pattern of A. doliolum
concentrations
(mg/L)
on
1980; N e t r a w a l i and Gandhi 1990), the reduction rate, the present study, at higher doses might be due killing of some cells from the beginning.
in to
Four different types of growth patterns, in r e s p o n s e to the pesticides, of the test organisms were o b s e r v e d viz. i) partial inhibition and slight change in log phase, ii) delayed inhibition and s h o r t e n i n g of log phase, iii) initial growth inhibition with subsequent growth resumption and lengthening of lag phase, and iv) complete inhibition of growth. The delayed inhibition of growth was r e s p o n s i b l e for the shortening of log phase as noted in case of C h l o r e l l a in response to e n d o s u l f a n ( Fig.2). Probably the alga was very efficient in a c c u m u l a t i n g the o r g a n o c h l o r i n e pesticide as a result of which the concentration inside the cell increased steadily causing delayed effect (Hansen 1980). On the other hand, in both the test o r g a n i s m s the lengthening of lag phase, due to initial growth inhibition and resumption afterwards, might be because of (i) biodegradation of the pesticide, (ii) a u t o d e g r a d a t i o n of the chemicals, (iii) m o d i f i c a t i o n by the organisms, and/or (iv) decrease influx of the
580
chemicals into the cells, caused m o d i f i c a t i o n (Hansen 1980; Thomas and 1986; N e t r a w a l i and Gandhi 1990).
by cell wall Shanmugasundaram
A c k n o w l e d g m e n t s . We thank Prof. P.C.Tripathy, Head of the D e p a r t m e n t of Botany, Utkal U n i v e r s i t y for providing us the l a b o r a t o r y facilities and the UGC, New Delhi for the financial assistance. REFERENCES Adhikary SP (1989) Effect of p e s t i c i d e s on the growth, p h o t o s y n t h e t i c oxygen e v o l u t i o n and n i t r o g e n fixation of W e s t i e l l o p s i s prolifica. J Gen Appl M i c r o b i o l 35: 319-325 Allen MB, Arnon DI (1955) Studies on n i t r o g e n fixing b l u e - g r e e n algae I. growth and nitrogen fixation by Anabaena c y l i n d r i c a Lemm. Plant Physiol 30: 366-372 Antunes-Madeira MC, Carvalho AP, Madeira VMC (1980) Effect of insecticides on t h e r m o t r o p i c lipid phase transition. P e s t i c i d e B i o c h e m Physiol 1 4 : 1 6 1 - 1 6 9 Gerloff GC, F i t z g e r a l d GP, Skoog F (1950) The isolation, p u r i f i c a t i o n and culture of b l u e - g r e e n algae, Amer J Bot 3 7 : 2 1 6 - 2 1 8 Hansen PD (1980) Uptake and t r a n s f e r of c h l o r i n a t e d h y d r o c a r b o n Lindane ( T -BHC) in a l a b o r a t o r y fresh water chain. Environ Pollut 2 1 : 9 7 - 1 0 8 Lal S (1984) M i c r o b i a l a c c u m u l a t i o n of insecticides. Insecticide Microbiol, S p r i n g e r - V e r l a g , Berlin, pp 6185 Mandal J, Mohanty RC (1990) Effect of m a n c o z e b on growth, carbohydrate, protein and chlorophyll contents of the green alga C h l o r e l l a v u l g a r i s Beijerinck. Pollut Res 8: 159-162 Mohapatra, PK, Sethi PK, M o h a n t y RC (1990) Toxicity of d i m e t h o a t e to c y a n o b a c t e r i u m Anabaena d o l i o l u m Bhar. In: Dalela RC (ed) Trends ecotoxicol, AEB, M u z a f f e r n a g a r , pp 189-196 Netrawali MS, Gandhi SR (1990) Mechanism of cell destructive action of organophosphorus insecticide phosalone in C h l a m y d o m o n a s reinhardtii algal cells. Bull Environ C o n t a m Toxicol 4 4 : 8 1 9 - 8 2 5 S a f f e r m a n RS, Morris ME (1964) Growth c h a r a c t e r i s t i c s of b l u e - g r e e n algal virus LPP-I. J Bact 88: 771-775 Sorokin C (1975) Dry weight, packed cell v o l u m e and optical density. In: Stein JR (ed) Handbook of phycological methods, Cambridge University Press, Cambridge, pp 321-324 Thomas PS, Shanmugasundaram S (1986) Interaction of a g r o c h e m i c a l s with the c y a n o b a c t e r i u m A n a c y s t i s n i d u l a n s and patterns of growth. M i c r o b i o s Lett 33: 115-120 Received January 2, 1992; accepted March 6, 1992.
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