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Effects of carbofuran and carbaryl on the growth of a green alga and two cyanobacteria isolated from a rice soil
Agriculture, Ecosystems and Environment, 25 (1989) 329-336 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
329
E f f e c t s of C a r b o f u r a n a n d C a r b a r y l on the G r o w t h of a G r e e n A l g a a n d T w o C y a n o b a c t e r i a I s o l a t e d f r o m a R i c e Soil M. MEGHARAJ 1, K. VENKATESWARLU2. and A.S. RAO 1
1Department of Botany, Nagarjuna University, Nagarjunanagar 522 510 (India) 2Department of Microbiology, S.K. University, Anantapur 515 003 (India) (Accepted for publication 7 November 1988) ABSTRACT Megharaj, M., Venkateswarlu, K. and Rao, A.S., 1989. Effects of carbofuran and carbaryl on the growth of a green alga and two cyanobacteria isolated from a rice soil. Agric. Ecosystems Environ., 25: 329-336. Two methylcarbamate insecticides, carbofuran and carbaryl, were assessed for their effects on a green unicellular alga, Scenedesmus bijugatus, and two cyanobacteria, Synechococcus elongatus and Nostoc linckia, all isolated from a rice soil. In laboratory studies, both insecticides up to 20 zg ml-1 concentration significantly increased the cell number of S. bijugatus. Synechococcus elongatus was highly sensitive, 5/~g ml- 1of each chemical being lethal. Concentrations, up to 20 or 50 #g ml-', of the two insecticides initially increased chlorophyll a in N. linckia significantly, which again led to a subsequent inhibition. In general, carbaryl was more toxic than carbofuran to all the test organisms.
INTRODUCTION In r e c e n t years, c a r b o f u r a n ( 2 , 3 - d i h y d r o - 2 , 3 - d i m e t h y l - 7 - b e n z o f u r a n y l N m e t h y l c a r b a m a t e ) is i n c r e a s i n g l y b e i n g u s e d in rice c u l t u r e as, p r o b a b l y , t h e m o s t effective i n s e c t i c i d e for c o n t r o l l i n g b r o w n p l a n t h o p p e r (Nilaparvata lugens Stal. ), a m a j o r p e s t o f rice ( R a j a g o p a l et al., 1984). C a r b o f u r a n is a p p l i e d a t 2 kg a.i. h a -1 as g r a n u l e s ( F u r a d a n 3 G ) or in gelatin c a p s u l e s to t h e soils or as s p r a y s to t h e lower p a r t o f t h e rice l e a f s h e a t h t h a t h a r b o r s t h e i n s e c t p e s t ( I R R I , 1975 ). C a r b a r y l ( 1 - n a p h t h y l N - m e t h y l c a r b a m a t e ) , a b r o a d - s p e c t r u m insecticide, b o t h c o n t a c t a n d s y s t e m i c , a g a i n s t o v e r 150 m a j o r p e s t s (Beck, 1965) is also e x t e n s i v e l y u s e d in t h e t r o p i c s a n d s u b - t r o p i c s a t r a t e s r a n g i n g f r o m 0.57 to 4.5 kg a.i. h a - 1 for c o n t r o l l i n g rice b r o w n p l a n t h o p p e r ( R a j a g o p a l et al., 1984). M i c r o a l g a e , a n ecologically b e n e f i c i a l g r o u p b y v i r t u e of t h e i r h a b i t a t i o n of *Author to whom correspondence should be addressed.
0167-8809/89/$03.50
© 1989 Elsevier Science Publishers B.V.
330 moist soils (Round, 1965; McCann and Cullimore, 1979), may encounter the methylcarbamate insecticides aimed at other biotic targets. A perusal of the literature clearly indicates that reports related to the effects of carbofuran and carbaryl on pure cultures of terrestrial algae are virtually unavailable (Wright, 1978; Lal, 1984; Rajagopal et al., 1984) although these insecticides are presently used extensively. Very recently, however, the toxic effect of carbofuran on native algal population in soil was reported (Megharaj et al., 1988). Because of great concern over the environmental hazard of carbamate (Rajagopal et al., 1984), the present study was undertaken to determine the influence of carbofuran and carbaryl on a green alga and two cyanobacteria, isolated from rice soil. MATERIALSAND METHODS
Test cultures and insecticides Cultures of Scenedesmus bijugatus (Turpin) Kuetzing (unicellular green alga ), Synechococcus elongatus Nageli (unicellular cyanobacterium) and Nostoc linckia (Roth) B and F (filamentous, heterocystous nitrogen-fixing cyanobacterium) were isolated from a black soil used to cultivate rice, and were raised to axenic population by employing standard microbial techniques (Deason and Bold, 1960). The dinitrogen fixer was propagated in modified nitrogen-free Chu-10 medium supplemented with trace elements, with the unicellular forms grown in Bold's basal medium (BBM) as outlined earlier (Megharaj et al., 1986). Stock solutions from technical grade carbofuran (75% pure) and carbaryl (99.9% pure) were prepared in acetone.
Bioassay procedure Aliquots from stock solutions of carbofuran and carbaryl were added to sterilized culture tubes (25 × 150 mm) to provide final concentrations ranging between 0 and 100 pg ml-1 culture medium. The carrier solvent was completely evaporated to dryness and 20-ml portions of steam-sterilized BBM or Chu-10 medium were dispensed, equilibrated and the tubes were left for 1 day to obtain aqueous solutions (Megharaj et al., 1986). Late log phase cultures of the test organisms were used as inocula as described previously (Megharaj et al., 1986). The initial cell culture of unicellular forms was adjusted to contain more than 1.5 × 10~cells m l - 1 medium (Goulding and Ellis, 1981 ). Untreated but inoculated culture tubes served as controls. The cultures were incubated at room temperature (28 _+ 4°C) in a growth chamber in a slanted position under continuous fluorescent illumination (200 /~E m -2 s - ' PPFD ). The culture solution was aerated 4 times daily by thorough shaking. Triplicate samples from treatments and controls were with-
331
drawn each time for growth measurements at intervals until 32-35 days. The phototrophic growth response of unicellular forms was determined by periodically counting the cells using a haemocytometer and the growth of N. linckia was measured in terms of acetone-soluble chlorophyll a (Megharaj et al., 1987). The data were subjected to analysis of variance, and the means were compared by Duncan's new multiple range test at the 5% level. RESULTS
The effect of different concentrations of carbofuran and carbaryl on the phototropic growth of the selected test organisms was determined periodically almost until the stationary phase in their growth cycle. The growth, measured as cell number, of S. bijugatus was either unaffected or slightly enhanced with carbofuran treatment at 1-5 pg ml -~ at the end of 6 days (Table 1 ). Carbofuran, at 10/lg m l - ~ level, caused about 28% inhibition. By 32 days after treatment, all these concentrations significantly increased the cell number of the alga. Thus, even 10 pg ml -~ led to an almost 1% increase over control. The higher concentrations used (20 and 50 pg ml -~) effected a significant inhibition throughout whereas 100 pg m l - ~ was totally aligicidal. However, the iniTABLE
1
Effect of carbofuran and carbaryl on cell number Insecticide
( X 105 m l - ~ medium)
ofS. bijugatus
Sampling time after treatment (days)
(Bgml 1) 6
20
26
32
Untreated Carbofuran
14.4 c
150.5 d
260.3 d
301.6 f
1
15.5 a
153.7 b
268.2 ~
401.6 ~
2
15.4 a
151.3 c
255.3 ~
4 0 1 . 8 L'
5 10
14.5 " 10.4 d
150.7 d 80.5 f
264.8' 173.0 i
394.V 304.5 ~
20
5.7 f
51.2 h
138.0 j
211.2 ~
50 100
4.7 ~ 0j
21.6 j Ok
83.01 0m
209.8 h
15.5 a 14.8 ~'
155.3 ~ 150.0 d
273.2 ~ 250.4 f
403.9 ~ 309.0 d
0i
Carbaryl 1 2 5
7.5 ~
112.2 e
223.3 ~
309.0 d
10
2.2 h
65.V
207.8 h
309.3 d
20 50
0.5 e (P
37.1 i Ok
117.5 k 0m
309.3 d 0i
Initial cell number, immediately after inoculation was 1.6 × 105 m l - ~. Means ( n = 3 ) in each column followed by the same superscript are not significantly different 4 0 . 0 5 ) according to Duncan's new multiple range ( D M R ) test.
(P
332
tial 67% inhibition from 50 Bg m1-1 was later alleviated, accounting for only 30% depression in growth at the end of the experiment. On the other hand, carbaryl exerted a different effect on the green alga. Excepting the concentrations of 1 and 2/~g m l - 1, all levels of carbaryl were either growth inhibitory or algicidal at the end of 6 days after treatment. Thus, 5, 10 and 20 /lg ml-1 concentrations led to 48, 85 and 95% growth inhibition, respectively. However, these algistatic concentrations again exhibited a significant increase in subsequent growth by Day 32. Microscopic observations revealed a clump formation of the algal cells at higher concentrations of both insecticides. The growth response of S. elongatus to the insecticide treatment was noteworthy. The lower concentrations (0.5 and 1 /~g m1-1) of carbofuran were found to enhance the cell number of the cyanobacterium throughout the period of study (Table 2). A significant toxic effect (with 67% inhibition) was observed even at 2 pg m1-1 concentration initially but only 17% depression in growth was evident by Day 32. The highest concentration employed (5 /~g m1-1) was associated with an initial 89% inhibition which became lethal at the end of 20 days. As with carbofuran, carbaryl also exhibited a similar effect on the test organism. Cells treated with only 0.1 and 0.5/~g ml-1 concentrations of carbaryl significantly increased in their number. A progressive decrease in growth was observed with increasing concentrations of the insecticide. Thus, the initial 89-91% inhibition with the 1 and 2 /~g m1-1 levels increased to 98-99% towards the end of the period. However, 5/~g m1-1 had killed the cells by Day 6. TABLE 2 E f f e c t o f c a r b o f u r a n a n d c a r b a r y l o n cell n u m b e r ( × l 0 b m l - 1 m e d i u m ) of Insecticide (/tg m l 1)
S. elongatus
Sampling time after treatment (days) 6
20
26
32
Untreated
19.7 e
497.7 e
607.1 e
611.4 e
Carbofuran 0.5 1 2 5
22.7 a 21.8 b 6.5 f 2.1 g
551.3 a 503.0 ° 306.3 f Oh
666.9 c 613.5 d 413.4 f 0i
703.8 a 621.7 a 508.9 f 0i
Carbaryl 0.1 0.5
20.8 c 20.5 d
526.0 b 519.8 c
673.2 a 670.3 b
681.V 686.4 b
1 2
2.1 g 1.7 h
1A g 0.5 g
4.2 g 2.4 h
14.1 ~ 8.7 h
5
0i
Oh
0i
0i
I n i t i a l cell n u m b e r w a s 3.5 × l 0 b m1-1 m e d i u m . M e a n s ( n = 3 ) in e a c h c o l u m n f o l l o w e d b y t h e s a m e s u p e r s c r i p t a re n o t s i g n i f i c a n t l y d i f f e r e n t (P~<0.0 5) a c c o r d i n g to D M R t e s t .
333 TABLE 3 Chlorophyll a (#g g-1 fresh weight) of N. linckia as influenced by treatment of carbofuran and carbaryl Insecticide (,ug m1-1 ) Untreated Carbofuran 1 2 5 10 20 50 100 Carbaryl 1 2 5 10 20 50
Sampling time after treatment (days) 10
15
20
25
35
1042 e
1338 h
1615 f
1831 g
143T
1117 b 1103 e 1095 ¢ 1062 d 1024 f 972 g 0j
1484 a 1461 b 1432 c 1417 d 1394 ef 10925 01
1880 a 1687 d 1665 e 1613 ~ 1592 g 13925 01
1965 b 1964 b 1946 ca 1937 de 1938 cd 1906 ~ Oh
880 j 927 h 879 j 894 i 941 g 1027 f Ok
1290 ~ 1049 e 1023 f 945 h 410 i 05
1405 d~ 1382 fg 1374 g 1117 i 809 k 01
1856 b 1797 ¢ 1562 h 1416 i 1233 k 01
1994 ~ 1946 cd 1956 bc 1919 de
1303 ~ 1494 h 1508 a 1129 e 1117 e Ok
1885 f
Oh
Means (n = 3) in each column followed by the same superscript are not significantly different (P ~<0.05 ) according to DMR test. Table 3 summarizes the data on quantitative estimation of acetone-soluble c h l o r o p h y l l a i n N. linckia. T h e v a l u e s o f c h l o r o p h y l l a s h o w e d a s i g n i f i c a n t increase when carbofuran was treated at 1-20 pg ml-1 concentrations. Even 50 p g m 1 - 1 , w h i c h w a s t o x i c u p t o 20 d a y s a f t e r t r e a t m e n t , s i g n i f i c a n t l y e n h a n c e d c h l o r o p h y l l a b y t h e e n d o f 25 d a y s . T h e r e w a s , h o w e v e r , a m a r k e d d e c r e a s e i n c h l o r o p h y l l a w i t h a l l t h e c o n c e n t r a t i o n s t o w a r d s t h e e n d o f 35 days. The cyanobacterium could not survive at the highest concentration used ( 100 p g m l - 1 ). C a r b a r y l , a t 10 a n d 20 p g m l - 1 l e v e l s , w a s t o x i c i n i t i a l l y t o t h e d i n i t r o g e n f i x e r . H o w e v e r , a f t e r 25 d a y s o f t r e a t m e n t , a l l c o n c e n t r a t i o n s r a n g i n g b e t w e e n 1 a n d 20 p g m l - 1 e x h i b i t e d a s i g n i f i c a n t i n c r e a s e i n c h l o r o p h y l l a. F o r i n s t a n c e , t h e o b s e r v e d 6 1 % i n h i b i t i o n w i t h 20 p g m 1 - 1 b y 10 d a y s w a s f u r t h e r r e d u c e d e f f e c t i n g a b o u t 3 % i n c r e a s e i n g r o w t h t o w a r d s t h e e n d o f 25 d a y s . B y D a y 35, t h i s l e v e l h o w e v e r l e d t o a 2 2 % i n h i b i t i o n i n s y n t h e s i s o f t h e p i g m e n t . C o m p l e t e i n h i b i t i o n w a s a c h i e v e d w i t h 50 ttg m l - 1 c o n c e n t r a t i o n . DISCUSSION C a r b o f u r a n a n d c a r b a r y l , a t c o n c e n t r a t i o n s r a n g i n g f r o m 1 t o 20 p g m l - 1 , s i g n i f i c a n t l y i n c r e a s e d t h e c e l l n u m b e r o f S. bijugatus. I n c o n t r a s t , t h e a v a i l -
334
able evidence indicates that most of the cultures of fresh-water green algae were found to be sensitive to carbaryl at concentrations as low as 0.5 or 1 ppm (Lal, 1984). The significant enhancement in the growth of S. bijugatus observed here in the presence of carbaryl, up to 20/~g m l - 1 level, may involve the alga in metabolism of the insecticide especially in view of the reported implication of a closely related fresh-water green alga, Scenedesmus quadricauda, in the assimilation of carbaryl (Stadnyk et al., 1971). A comparison of the data on response of the two unicellular forms (Tables 1 and 2 ) clearly indicates that the green alga was relatively insensitive to carbofuran and carbaryl with the growth of the cyanobacterium being inhibited even at concentrations of 1 or 2/~g ml- 1. On the contrary, it has recently been reported that organophosphorus insecticides, monocrotophos and quinalphos (Megharaj et al., 1986), and synthetic pyrethroids, cypermethrin and fenvalerate (Megharaj et al., 1987 ) are less toxic to S. elongatus than to S. bijugatus. Interestingly, carbofuran and carbaryl, up to concentrations of 50 and 20/~g m l - 1, respectively, significantly enhanced the concentrations of chlorophyll a in N. linckia at the end of 25 days. The growth stimulation in N. linckia by carbofuran, in particular, accords with the reported enhancement in survival, growth and nitrogen fixation of a similar cyanobacterium, N. muscorum, when carbofuran was added at 25 ppm in culture medium (Kar and Singh, 1978). Likewise, very recent investigation revealed that carbofuran application to the soil, even at 5 kg ha-1 level, increased the population of filamentous cyanobacteria (Megharaj et al., 1988). However, even the lowest concentration (1 /lg ml- 1) of the two insecticides exhibited a pronounced toxic effect by the end of 35 days. It would seem from the present observation that until about 25 days after treatment the cyanobacterium is able to utilize carbofuran and carbaryl, presumably yielding their hydrolytic products, as with a similar pathway of primary hydrolysis at the carbamate linkage for both insecticides. Consequently, the products of hydrolysis might have exerted a toxic effect on the organism. For instance, carbofuran was hydrolyzed to carbofuran phenol during bacterial metabolism (Venkateswarlu and Sethunathan, 1984, 1985) and 1-naphthol was the major product of hydrolysis in the degradation of carbaryl by bacteria (Bollag and Liu, 1971; Sud et al., 1972). Also, the available evidence suggests that 1-naphthol is more toxic to bacteria than its parent compound, carbaryl (Ramakrishna and Sethunathan, 1983). Again, of particular interest is the reaction of carbofuran and carbaryl towards the cyanobacteria. Whereas the filamentous dinitrogen fixer, N. linckia, was less sensitive to carbofuran and carbaryl, the cells of S. elongatus were killed even at 5 ttg ml-1 concentration of the two insecticides. The overall response of the test organisms to the insecticides conforms to the fact that carbaryl is more toxic to algae than carbofuran (Lal, 1984). Thus, the results presented here clearly identify a potential problem in arriving at a generalization on the environmental impact of carbofuran and carbaryl on soil algae.
335
More research is therefore needed, involving a wide array of soil isolates, belonging to Chlorophyta and Cyanophyta, in order to predict the effect of an insecticide in the soil environment more accurately. However, extrapolation of data on pure cultures of soil isolates to the field requires caution because the actual and effective concentration of an insecticide in the laboratory may be lowered in the soil because of absorption, chemical degradation, degradation by soil microorganisms and co-metabolism, leading to different results. ACKNOWLEDGEMENT
The senior author is grateful to the Council of Scientific and Industrial Research, New Delhi for financial assistance.
REFERENCES Beck, R.C., 1965. Significant developments in eight years with sevin insecticide. J. Agric. Food Chem., 13: 198-202. Bollag, J.M. and Liu, S.Y., 1971. Degradation of sevin by soil microorganisms. Soil Biol. Biochem., 3: 337-345. Deason, T. and Bold, H.C., 1960. Phycological studies. I. Explanatory studies of Texas soil algae. Univ. Texas Publ. No. 6022, Austin, Texas. Goulding, K.H. and Ellis, S., 1981. The interaction of DDT with two species of water algae. Environ. Pollut., A25: 271-290. IRRI, 1975. International Rice Research Institute, Los Bafios, Philippines. Annual Report for 1974. Kar, S. and Singh, P.K., 1978. Toxicity of carbofuran to blue-green alga Nostoc muscorum. Bull. Environ. Contam. Toxicol., 20: 707-714. Lal, S., 1984. Effects of insecticides on algae. In: R. Lal (Editor), Insecticide Microbiology. Springer-Verlag, Berlin, pp. 203-236. McCann, A.E. and Cullimore, D.R., 1979. Influence of pesticides on soil algal flora. Residue Rev., 72: 1-31. Megharaj, M., Venkateswarlu, K. and Rao, A.S., 1986. Growth response of four species of soil algae to monocrotophos and quinalphos. Environ. Pollut., A42: 15-22. Megharaj, M., Venkateswarlu, K. and Rao, A.S., 1987. Influence of cypermethrin and fenvalerate on a green alga and three cyanobacteria isolated from soil. Ecotoxicol. Environ. Saf., 14: 142146. Megharaj, M., Venkateswarlu, K. and Rao, A.S., 1988. Tolerance of algal population in rice soil to carbofuran application. Curr. Sci., 57: 100-102. Rajagopal, B.S., Brahmaprakash, G.P., Reddy, B.R., Singh, U.D. and Sethunathan, N., 1984. Effect and persistence of selected carbamate pesticides in soil. Residue Rev., 93: 1-19. Ramakrishna, C. and Sethunathan, N., 1983. Inhibition of heterotrophic and autotrophic nitrification in bacterial cultures by carbaryl and 1-naphthol. J. Appl. Bacteriol., 54: 191-196. Round, F.E., 1965. The Biology of Algae. Edward Arnold, London. Stadnyk, I., Campbell, R.S. and Johnson, B.T., 1971. Pesticide effect on growth and 14C assimilation in a fresh water alga. Bull. Environ. Contam. Toxicol., 6: 1-8. Sud, R.K., Sud, A.K. and Gupta, K.G., 1972. Degradation of sevin (1-naphthyl N-methylcarbamate) by Achromobacter sp. Arch. Mikrobiol., 87: 353-358. Venkateswarlu, K. and Sethunathan, N., 1984. Degradation of carbofuran by Azospirillum lipo-
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ferum and Streptomyces spp. isolated from flooded alluvial soil. Bull. Environ. Contam. Toxicol., 33: 556-560. Venkateswarlu, K. and Sethunathan, N., 1985. Enhanced degradation of carbofuran by Pseudomonas cepacia and Nocardia sp. in the presence of growth factors. Plant Soil, 84: 445-449. Wright, S.J.L., 1978. Interactions of pesticides with microalgae. In: I.R. Hill and S.J.L. Wright (Editors), Pesticide Microbiology. Academic Press, New York, NY, pp. 535-602.
329
E f f e c t s of C a r b o f u r a n a n d C a r b a r y l on the G r o w t h of a G r e e n A l g a a n d T w o C y a n o b a c t e r i a I s o l a t e d f r o m a R i c e Soil M. MEGHARAJ 1, K. VENKATESWARLU2. and A.S. RAO 1
1Department of Botany, Nagarjuna University, Nagarjunanagar 522 510 (India) 2Department of Microbiology, S.K. University, Anantapur 515 003 (India) (Accepted for publication 7 November 1988) ABSTRACT Megharaj, M., Venkateswarlu, K. and Rao, A.S., 1989. Effects of carbofuran and carbaryl on the growth of a green alga and two cyanobacteria isolated from a rice soil. Agric. Ecosystems Environ., 25: 329-336. Two methylcarbamate insecticides, carbofuran and carbaryl, were assessed for their effects on a green unicellular alga, Scenedesmus bijugatus, and two cyanobacteria, Synechococcus elongatus and Nostoc linckia, all isolated from a rice soil. In laboratory studies, both insecticides up to 20 zg ml-1 concentration significantly increased the cell number of S. bijugatus. Synechococcus elongatus was highly sensitive, 5/~g ml- 1of each chemical being lethal. Concentrations, up to 20 or 50 #g ml-', of the two insecticides initially increased chlorophyll a in N. linckia significantly, which again led to a subsequent inhibition. In general, carbaryl was more toxic than carbofuran to all the test organisms.
INTRODUCTION In r e c e n t years, c a r b o f u r a n ( 2 , 3 - d i h y d r o - 2 , 3 - d i m e t h y l - 7 - b e n z o f u r a n y l N m e t h y l c a r b a m a t e ) is i n c r e a s i n g l y b e i n g u s e d in rice c u l t u r e as, p r o b a b l y , t h e m o s t effective i n s e c t i c i d e for c o n t r o l l i n g b r o w n p l a n t h o p p e r (Nilaparvata lugens Stal. ), a m a j o r p e s t o f rice ( R a j a g o p a l et al., 1984). C a r b o f u r a n is a p p l i e d a t 2 kg a.i. h a -1 as g r a n u l e s ( F u r a d a n 3 G ) or in gelatin c a p s u l e s to t h e soils or as s p r a y s to t h e lower p a r t o f t h e rice l e a f s h e a t h t h a t h a r b o r s t h e i n s e c t p e s t ( I R R I , 1975 ). C a r b a r y l ( 1 - n a p h t h y l N - m e t h y l c a r b a m a t e ) , a b r o a d - s p e c t r u m insecticide, b o t h c o n t a c t a n d s y s t e m i c , a g a i n s t o v e r 150 m a j o r p e s t s (Beck, 1965) is also e x t e n s i v e l y u s e d in t h e t r o p i c s a n d s u b - t r o p i c s a t r a t e s r a n g i n g f r o m 0.57 to 4.5 kg a.i. h a - 1 for c o n t r o l l i n g rice b r o w n p l a n t h o p p e r ( R a j a g o p a l et al., 1984). M i c r o a l g a e , a n ecologically b e n e f i c i a l g r o u p b y v i r t u e of t h e i r h a b i t a t i o n of *Author to whom correspondence should be addressed.
0167-8809/89/$03.50
© 1989 Elsevier Science Publishers B.V.
330 moist soils (Round, 1965; McCann and Cullimore, 1979), may encounter the methylcarbamate insecticides aimed at other biotic targets. A perusal of the literature clearly indicates that reports related to the effects of carbofuran and carbaryl on pure cultures of terrestrial algae are virtually unavailable (Wright, 1978; Lal, 1984; Rajagopal et al., 1984) although these insecticides are presently used extensively. Very recently, however, the toxic effect of carbofuran on native algal population in soil was reported (Megharaj et al., 1988). Because of great concern over the environmental hazard of carbamate (Rajagopal et al., 1984), the present study was undertaken to determine the influence of carbofuran and carbaryl on a green alga and two cyanobacteria, isolated from rice soil. MATERIALSAND METHODS
Test cultures and insecticides Cultures of Scenedesmus bijugatus (Turpin) Kuetzing (unicellular green alga ), Synechococcus elongatus Nageli (unicellular cyanobacterium) and Nostoc linckia (Roth) B and F (filamentous, heterocystous nitrogen-fixing cyanobacterium) were isolated from a black soil used to cultivate rice, and were raised to axenic population by employing standard microbial techniques (Deason and Bold, 1960). The dinitrogen fixer was propagated in modified nitrogen-free Chu-10 medium supplemented with trace elements, with the unicellular forms grown in Bold's basal medium (BBM) as outlined earlier (Megharaj et al., 1986). Stock solutions from technical grade carbofuran (75% pure) and carbaryl (99.9% pure) were prepared in acetone.
Bioassay procedure Aliquots from stock solutions of carbofuran and carbaryl were added to sterilized culture tubes (25 × 150 mm) to provide final concentrations ranging between 0 and 100 pg ml-1 culture medium. The carrier solvent was completely evaporated to dryness and 20-ml portions of steam-sterilized BBM or Chu-10 medium were dispensed, equilibrated and the tubes were left for 1 day to obtain aqueous solutions (Megharaj et al., 1986). Late log phase cultures of the test organisms were used as inocula as described previously (Megharaj et al., 1986). The initial cell culture of unicellular forms was adjusted to contain more than 1.5 × 10~cells m l - 1 medium (Goulding and Ellis, 1981 ). Untreated but inoculated culture tubes served as controls. The cultures were incubated at room temperature (28 _+ 4°C) in a growth chamber in a slanted position under continuous fluorescent illumination (200 /~E m -2 s - ' PPFD ). The culture solution was aerated 4 times daily by thorough shaking. Triplicate samples from treatments and controls were with-
331
drawn each time for growth measurements at intervals until 32-35 days. The phototrophic growth response of unicellular forms was determined by periodically counting the cells using a haemocytometer and the growth of N. linckia was measured in terms of acetone-soluble chlorophyll a (Megharaj et al., 1987). The data were subjected to analysis of variance, and the means were compared by Duncan's new multiple range test at the 5% level. RESULTS
The effect of different concentrations of carbofuran and carbaryl on the phototropic growth of the selected test organisms was determined periodically almost until the stationary phase in their growth cycle. The growth, measured as cell number, of S. bijugatus was either unaffected or slightly enhanced with carbofuran treatment at 1-5 pg ml -~ at the end of 6 days (Table 1 ). Carbofuran, at 10/lg m l - ~ level, caused about 28% inhibition. By 32 days after treatment, all these concentrations significantly increased the cell number of the alga. Thus, even 10 pg ml -~ led to an almost 1% increase over control. The higher concentrations used (20 and 50 pg ml -~) effected a significant inhibition throughout whereas 100 pg m l - ~ was totally aligicidal. However, the iniTABLE
1
Effect of carbofuran and carbaryl on cell number Insecticide
( X 105 m l - ~ medium)
ofS. bijugatus
Sampling time after treatment (days)
(Bgml 1) 6
20
26
32
Untreated Carbofuran
14.4 c
150.5 d
260.3 d
301.6 f
1
15.5 a
153.7 b
268.2 ~
401.6 ~
2
15.4 a
151.3 c
255.3 ~
4 0 1 . 8 L'
5 10
14.5 " 10.4 d
150.7 d 80.5 f
264.8' 173.0 i
394.V 304.5 ~
20
5.7 f
51.2 h
138.0 j
211.2 ~
50 100
4.7 ~ 0j
21.6 j Ok
83.01 0m
209.8 h
15.5 a 14.8 ~'
155.3 ~ 150.0 d
273.2 ~ 250.4 f
403.9 ~ 309.0 d
0i
Carbaryl 1 2 5
7.5 ~
112.2 e
223.3 ~
309.0 d
10
2.2 h
65.V
207.8 h
309.3 d
20 50
0.5 e (P
37.1 i Ok
117.5 k 0m
309.3 d 0i
Initial cell number, immediately after inoculation was 1.6 × 105 m l - ~. Means ( n = 3 ) in each column followed by the same superscript are not significantly different 4 0 . 0 5 ) according to Duncan's new multiple range ( D M R ) test.
(P
332
tial 67% inhibition from 50 Bg m1-1 was later alleviated, accounting for only 30% depression in growth at the end of the experiment. On the other hand, carbaryl exerted a different effect on the green alga. Excepting the concentrations of 1 and 2/~g m l - 1, all levels of carbaryl were either growth inhibitory or algicidal at the end of 6 days after treatment. Thus, 5, 10 and 20 /lg ml-1 concentrations led to 48, 85 and 95% growth inhibition, respectively. However, these algistatic concentrations again exhibited a significant increase in subsequent growth by Day 32. Microscopic observations revealed a clump formation of the algal cells at higher concentrations of both insecticides. The growth response of S. elongatus to the insecticide treatment was noteworthy. The lower concentrations (0.5 and 1 /~g m1-1) of carbofuran were found to enhance the cell number of the cyanobacterium throughout the period of study (Table 2). A significant toxic effect (with 67% inhibition) was observed even at 2 pg m1-1 concentration initially but only 17% depression in growth was evident by Day 32. The highest concentration employed (5 /~g m1-1) was associated with an initial 89% inhibition which became lethal at the end of 20 days. As with carbofuran, carbaryl also exhibited a similar effect on the test organism. Cells treated with only 0.1 and 0.5/~g ml-1 concentrations of carbaryl significantly increased in their number. A progressive decrease in growth was observed with increasing concentrations of the insecticide. Thus, the initial 89-91% inhibition with the 1 and 2 /~g m1-1 levels increased to 98-99% towards the end of the period. However, 5/~g m1-1 had killed the cells by Day 6. TABLE 2 E f f e c t o f c a r b o f u r a n a n d c a r b a r y l o n cell n u m b e r ( × l 0 b m l - 1 m e d i u m ) of Insecticide (/tg m l 1)
S. elongatus
Sampling time after treatment (days) 6
20
26
32
Untreated
19.7 e
497.7 e
607.1 e
611.4 e
Carbofuran 0.5 1 2 5
22.7 a 21.8 b 6.5 f 2.1 g
551.3 a 503.0 ° 306.3 f Oh
666.9 c 613.5 d 413.4 f 0i
703.8 a 621.7 a 508.9 f 0i
Carbaryl 0.1 0.5
20.8 c 20.5 d
526.0 b 519.8 c
673.2 a 670.3 b
681.V 686.4 b
1 2
2.1 g 1.7 h
1A g 0.5 g
4.2 g 2.4 h
14.1 ~ 8.7 h
5
0i
Oh
0i
0i
I n i t i a l cell n u m b e r w a s 3.5 × l 0 b m1-1 m e d i u m . M e a n s ( n = 3 ) in e a c h c o l u m n f o l l o w e d b y t h e s a m e s u p e r s c r i p t a re n o t s i g n i f i c a n t l y d i f f e r e n t (P~<0.0 5) a c c o r d i n g to D M R t e s t .
333 TABLE 3 Chlorophyll a (#g g-1 fresh weight) of N. linckia as influenced by treatment of carbofuran and carbaryl Insecticide (,ug m1-1 ) Untreated Carbofuran 1 2 5 10 20 50 100 Carbaryl 1 2 5 10 20 50
Sampling time after treatment (days) 10
15
20
25
35
1042 e
1338 h
1615 f
1831 g
143T
1117 b 1103 e 1095 ¢ 1062 d 1024 f 972 g 0j
1484 a 1461 b 1432 c 1417 d 1394 ef 10925 01
1880 a 1687 d 1665 e 1613 ~ 1592 g 13925 01
1965 b 1964 b 1946 ca 1937 de 1938 cd 1906 ~ Oh
880 j 927 h 879 j 894 i 941 g 1027 f Ok
1290 ~ 1049 e 1023 f 945 h 410 i 05
1405 d~ 1382 fg 1374 g 1117 i 809 k 01
1856 b 1797 ¢ 1562 h 1416 i 1233 k 01
1994 ~ 1946 cd 1956 bc 1919 de
1303 ~ 1494 h 1508 a 1129 e 1117 e Ok
1885 f
Oh
Means (n = 3) in each column followed by the same superscript are not significantly different (P ~<0.05 ) according to DMR test. Table 3 summarizes the data on quantitative estimation of acetone-soluble c h l o r o p h y l l a i n N. linckia. T h e v a l u e s o f c h l o r o p h y l l a s h o w e d a s i g n i f i c a n t increase when carbofuran was treated at 1-20 pg ml-1 concentrations. Even 50 p g m 1 - 1 , w h i c h w a s t o x i c u p t o 20 d a y s a f t e r t r e a t m e n t , s i g n i f i c a n t l y e n h a n c e d c h l o r o p h y l l a b y t h e e n d o f 25 d a y s . T h e r e w a s , h o w e v e r , a m a r k e d d e c r e a s e i n c h l o r o p h y l l a w i t h a l l t h e c o n c e n t r a t i o n s t o w a r d s t h e e n d o f 35 days. The cyanobacterium could not survive at the highest concentration used ( 100 p g m l - 1 ). C a r b a r y l , a t 10 a n d 20 p g m l - 1 l e v e l s , w a s t o x i c i n i t i a l l y t o t h e d i n i t r o g e n f i x e r . H o w e v e r , a f t e r 25 d a y s o f t r e a t m e n t , a l l c o n c e n t r a t i o n s r a n g i n g b e t w e e n 1 a n d 20 p g m l - 1 e x h i b i t e d a s i g n i f i c a n t i n c r e a s e i n c h l o r o p h y l l a. F o r i n s t a n c e , t h e o b s e r v e d 6 1 % i n h i b i t i o n w i t h 20 p g m 1 - 1 b y 10 d a y s w a s f u r t h e r r e d u c e d e f f e c t i n g a b o u t 3 % i n c r e a s e i n g r o w t h t o w a r d s t h e e n d o f 25 d a y s . B y D a y 35, t h i s l e v e l h o w e v e r l e d t o a 2 2 % i n h i b i t i o n i n s y n t h e s i s o f t h e p i g m e n t . C o m p l e t e i n h i b i t i o n w a s a c h i e v e d w i t h 50 ttg m l - 1 c o n c e n t r a t i o n . DISCUSSION C a r b o f u r a n a n d c a r b a r y l , a t c o n c e n t r a t i o n s r a n g i n g f r o m 1 t o 20 p g m l - 1 , s i g n i f i c a n t l y i n c r e a s e d t h e c e l l n u m b e r o f S. bijugatus. I n c o n t r a s t , t h e a v a i l -
334
able evidence indicates that most of the cultures of fresh-water green algae were found to be sensitive to carbaryl at concentrations as low as 0.5 or 1 ppm (Lal, 1984). The significant enhancement in the growth of S. bijugatus observed here in the presence of carbaryl, up to 20/~g m l - 1 level, may involve the alga in metabolism of the insecticide especially in view of the reported implication of a closely related fresh-water green alga, Scenedesmus quadricauda, in the assimilation of carbaryl (Stadnyk et al., 1971). A comparison of the data on response of the two unicellular forms (Tables 1 and 2 ) clearly indicates that the green alga was relatively insensitive to carbofuran and carbaryl with the growth of the cyanobacterium being inhibited even at concentrations of 1 or 2/~g ml- 1. On the contrary, it has recently been reported that organophosphorus insecticides, monocrotophos and quinalphos (Megharaj et al., 1986), and synthetic pyrethroids, cypermethrin and fenvalerate (Megharaj et al., 1987 ) are less toxic to S. elongatus than to S. bijugatus. Interestingly, carbofuran and carbaryl, up to concentrations of 50 and 20/~g m l - 1, respectively, significantly enhanced the concentrations of chlorophyll a in N. linckia at the end of 25 days. The growth stimulation in N. linckia by carbofuran, in particular, accords with the reported enhancement in survival, growth and nitrogen fixation of a similar cyanobacterium, N. muscorum, when carbofuran was added at 25 ppm in culture medium (Kar and Singh, 1978). Likewise, very recent investigation revealed that carbofuran application to the soil, even at 5 kg ha-1 level, increased the population of filamentous cyanobacteria (Megharaj et al., 1988). However, even the lowest concentration (1 /lg ml- 1) of the two insecticides exhibited a pronounced toxic effect by the end of 35 days. It would seem from the present observation that until about 25 days after treatment the cyanobacterium is able to utilize carbofuran and carbaryl, presumably yielding their hydrolytic products, as with a similar pathway of primary hydrolysis at the carbamate linkage for both insecticides. Consequently, the products of hydrolysis might have exerted a toxic effect on the organism. For instance, carbofuran was hydrolyzed to carbofuran phenol during bacterial metabolism (Venkateswarlu and Sethunathan, 1984, 1985) and 1-naphthol was the major product of hydrolysis in the degradation of carbaryl by bacteria (Bollag and Liu, 1971; Sud et al., 1972). Also, the available evidence suggests that 1-naphthol is more toxic to bacteria than its parent compound, carbaryl (Ramakrishna and Sethunathan, 1983). Again, of particular interest is the reaction of carbofuran and carbaryl towards the cyanobacteria. Whereas the filamentous dinitrogen fixer, N. linckia, was less sensitive to carbofuran and carbaryl, the cells of S. elongatus were killed even at 5 ttg ml-1 concentration of the two insecticides. The overall response of the test organisms to the insecticides conforms to the fact that carbaryl is more toxic to algae than carbofuran (Lal, 1984). Thus, the results presented here clearly identify a potential problem in arriving at a generalization on the environmental impact of carbofuran and carbaryl on soil algae.
335
More research is therefore needed, involving a wide array of soil isolates, belonging to Chlorophyta and Cyanophyta, in order to predict the effect of an insecticide in the soil environment more accurately. However, extrapolation of data on pure cultures of soil isolates to the field requires caution because the actual and effective concentration of an insecticide in the laboratory may be lowered in the soil because of absorption, chemical degradation, degradation by soil microorganisms and co-metabolism, leading to different results. ACKNOWLEDGEMENT
The senior author is grateful to the Council of Scientific and Industrial Research, New Delhi for financial assistance.
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ferum and Streptomyces spp. isolated from flooded alluvial soil. Bull. Environ. Contam. Toxicol., 33: 556-560. Venkateswarlu, K. and Sethunathan, N., 1985. Enhanced degradation of carbofuran by Pseudomonas cepacia and Nocardia sp. in the presence of growth factors. Plant Soil, 84: 445-449. Wright, S.J.L., 1978. Interactions of pesticides with microalgae. In: I.R. Hill and S.J.L. Wright (Editors), Pesticide Microbiology. Academic Press, New York, NY, pp. 535-602.