Indian Journal of Experimental Biology Vol. 39, January 200 I, pp. 90-94

Influence of hexaconazole, carbofuran and ethion on soil microflora and dehydrogenase activities in soil and intact cell

A Kalam & A K Mukherjee*

Department of Botany, Burdwan Univers ity, Burdwan 71 3 104, India

Received 22 November 1999; revised 7 September 2000

Total mi crobial count was hi ghly affected (up to 61 % at I 000 11g level) in presence of hcxaconazole and persisted up to 2 1 days. Bacteria were more suscept ible than actinomycetcs. Carbofu ran and cthion were moderatel y toxic to soi l micro­nora. Inhi bit ory effects of all the three pes ti cides gradu all y decreased after 21 days as was evident by increase in total mi­crobial count except in carbofuran. GDH activit y in so il was also affected initially (up tol4 days) by all the three pesti cides (60.3 % in hexaconazo le at I 000 11g level) and inhibition gradu all y decreased to zero except in carbofuran ( 15-20 % tox icity persisted up to 35 days). GDH and LDH act ivi ty in presence of hexaconazole was strongly affected in intact cel ls of some standard culture of bacteria like Rhizobium sp. (host Doliclws sp., 32 .1 and 72.5%), Bacillus subtilis Cohn (86.75 and 76.5 %), Azotobacter sp. (36.9 and 55.4%) and /3. ~phaericus (67.6% GDH ) respect ively. Carbofuran inhib ited the en zyme ac tivit y in /3 .. whtilis (55.55 and 35.3 %) and to some exten t in 13. sphaeriws. Ethion moderately inhibited LD H acti vit y in Rhodococcus sp. AK I ( 17.1 and 33 .3'i'i>), Rhizobium (27.6% LD H), E. coli H B I 0 I (34.2% LDH) as evidenced by formazan formation. From the result , it mi gh t be concluded that among the above three pesticides tested hcxaconazole strongly inhib­ited the dehydrogenase system in bacteri:1 including nitrogen fixing bacteria of soil and thus may affect soil fcni lit y. It was concluded that hexac:onazole was more tox ic than cth ion to dehydrogenase enzymes.

Pestic ides are be ing commonl y used in agriculture for protecting crops from vari ous insec ts, diseases and weeds. Use of pes ticides be ne fits the farme rs but causing environmental polluti on. Since so il fe rtility is dependent to a large ex tent on the activ ities o f so il microorganisms, it is essent ia l to investigate the in­fluence of pesti c ides on the microbiology and bio­chemistry of soils. Therefore, al l approved pesti c ide products are subject to rout ine rev iew, but one may rev iew at any time if any ev idence e merges concern­ing the ir safety. If poss ible, an approval can be re­stricted or revoked e ntire ly 1

•

An inves ti gation was undertaken to study the e f­fects of three wide ly used pesticides name ly, hexaco­nazole, carbofuran and e thi on on total microbial count (TMC), de hydrogenase sys tem of soil and of some individual bac terium. Hexaconazole is a pro­tectant and eradicant sys temic fun g icide be longing to the c lass of triazo les [2-(2,4-dichloropheny l)-1-( I H­I ,2,4-triazo le-1-yl) hexan-2-o l] . It is a potent inhibitor of ergoste rol bi osynthesis having broad spec trum ac­tivity and spec ially active aga inst powdery mildews, basidiomycetes and ascomycetes 2

. Carbofuran [2,3-dihydro 2,2-dimethyl-7-benzofurany l methyl carba-

* Correspondent author

mate] is widely used rice insecti c ide and is active aga inst c hew ing and suck ing in sects-'. Ethi on [0,0 ,0,0-tetraethy I S-S meth y lene-bi s-phospho rocl i th ioate ] is an organophosphorous insec ti c ide and acaricide with knock clown and long last ing residua l activity again st pests of tea, cotton, vegetables etc.4

. According to WHO, carbofuran is highly hazardous, ethi on is mod­erate ly haza rdou s and hexaconazole is less haza rdous pesticides5

. An assay of de hyd rogenase activity has been recommended as an index of genera l activity o f so ilmicroorgani sms6

·7

, and can be used as a sens itive marker of soi I degradation and soil microbial acti v­it /. In the present in vest igati on, the e ffects of three pesticides on TMC, g lucose dehydrogenase (GDH) activ ity of so il and GDH and LDH (lac tate dehydro­genase) in intact ce lls of some standard bacteri a l strains have been reported .

Soil samp le was co ll ected in po lyethy lene bags from the loca l rice-fie ld . It was air dried in the shade, ground and sieved through a mesh ne t (No. I 00) . S tandard bacte ria were collected from the stock cul­ture of thi s Departme nt. Hexaconazo le, e thi on and carbofuran of tec hnica l grade was a g ift from Ralli s (fnclia) Ltd ., Banga lore.

To enumerate the co lony form ing unit s (CFU) o f the microorganisms in treated soil s, I 00 g of soil (pH

NOTES 9 1

6.9) was mixed with different concentrations (50,

I 00, 250, 500 and I 000 flg fin al concentrations of active ingredient) of each pesti cide separate ly and

incubated up to 35 days at 28° ± 2°C. Aliquots ( I 0 g) were removed at weekly intervals, suspended in 90 mL sterile d isti lled water, serially diluted and plated on soi l extract agar [ 1000 g fresh rice-field so il was suspended in I 000 mL tap water, autoc laved ( II 0 Kpa for 30 min), coo led to room temp. A pinch of CaS04 was added and filtered (Whatman . No . I). Yeast ex tract ( 1.0 g) and K2HP04 (0.2 g) were added to the filtrate , the vo lume was made up to I L, ad­justed to pH 6.8]. Agar (20 g) was added and steri l­ized ( II 0 Kpa for 15 min). Plates were incubated at

30° ± 2°C and co loni es were counted after 72 hr.

Dehydrogenase acti vity was measured co lorimetri­cally using triphenyl te trazolium chl oride (TTC). A li­quots of soil sample (2 g each) were weighed into 15 mL culture tu bes and mixed with 0 .2 mL of 3.0 % aqueous TTC so lution , 0.5 mL of I % glucose solu­ti on and 2.0 mL of di stilled water. The mixtures were thoroughly mi xed and incubated in dark for 96 hr at

28° ± 2°C. Afte r incubation, the developed co lour was ex tracted in methanol and kept for 6 hr in clark and measured at 485 nm in Shimadzu UV/VIS-

1 9 10 E .. spectrop 1otometer · . nzyme acti VIty was expressed in terms of formazan produced and the quantity of fo rmazan was calcul ated from a standard curve by using triphenyl formazan (TPF, Sigma) in methanol 11

.

The results are mean of six replicates.

Dehydrogenase ac ti vity in intact ce ll was measured [2,3 ,5-triphenyl tetrazo lium chl oride (TTC) reduc ti on to triphenyl formazan (TPF)] and used fo r comparing dehydrogenase acti vity of whole cells in culture tubes

12• The tubes conta ined ce ll suspension equi va­

lent to I 00-140 jlg protein/mL, ph osphate buffer (33 mM, pH 7.0, 2.0 mL ), substrate (50 mM, 0.1 mL) and TIC (0.3 mM, 0.5 mL). Pesticides were di sso lved in methanol or acetone (appropri ate so lvent) and des ired amou nt was poured in cu lture tubes. The tubes were

then dried at 50°C and the solvent was evaporated . These tubes were used in enzyme assay. The protein contents of cell suspens ions were determined by dye binding methodl.1. The con tents of the tubes were

thoroughly mi xed and incubated at 37°C for 2 hr. After incubati on, g lac ial acetic ac id (7 .5 mL) was added followed by to luene (3.0 mL) . The tubes were shaken vigorously and centrifuged at 4000g for 5 min. The layer of toluene was separated and measured at

485 nm in spectrophotometer. Quantity of formazan was calculated from the standard curve of formazan (Sigma) .

To determine minimum inhibitory concentrations (MICs) of the bac teria in solid medium 15 mL of molten agar medium containing 0-200 mg/L of pesti­c ide were poured on to sterile 9 e m petri pl ates. The

plates were dried at 37°C for I hr. Suspensions of the bacteria were streaked on agar surface and the pl ates were examined visually for growth of bacteri a fo l­

lowing incubati on at 37°C for 48 hr. Total microbial populati on (TMC) of microorgan­

isms in the so il samples was inhibited by hexaco­nazo le (Fig. I A). When inhibiti on was measured after 7 days, maximum effect was not iced after 2 1 days (up to 61 %) and subsequentl y, the inhibitory effect de­clined. Bacteria were more susceptible than actino­mycetes and were concentration dependent. Increase in TMC value might be due to increase in acti nomy­cetes popul ation only. TMC value declined under the influence of increas ing concentration of carbofuran

( I 00 to I 000 jlg) and durati on of treatment. Inhibition

was up to 66% after 2 1 days at I 000 jlg cone. and thereafter, there was diminishing toxic effec t (Fig. I B). Ethion had moderate effec t on TMC. It persisted only up to 14 days (max imum upto 26.5 % level) and

late r, the tox icity graduall y decreased even at 1000 jlg level as evidenced by increased microbi al population over control after 2 1 days (Fig. I C).

Among the three pest ic ides, hexaconazo le had maximum effect (60.3 %) on GDH enzyme after 7 days (Fig. 2A), persisted up to 14 days and decreased thereafter (measured in terms of formazan formation).

In presence of fungicide ( I 000 flg), formazan forma­tion was 50% more than in control. Carbofuran had moderate effect on GDH. Maximum inhibiti on was in

the tune of 36 % at I 000 fl g level of insect icide after 7 days and thereafter, the extent of inhibition de­clined . A little amou nt of tox ic ity ( 15 %) persisted ti ll

35 days of incubati on at I 000 jlg pesticide (Fig. 2B) . In case of ethion , dehydrogenase ac tivity was inhib­ited (66%) at the initial stage (7 days), hut the in­hibitory effec t dec! i ned after 14 days onward (Fig. 2C).

Activities of GDH and LDH enzymes were sub­stantially inhibited by hexaconazo le in a ll the s ix bacterial strains under investi gation . Hexaconazole strong ly inhibited the activiti es of above enzymes in Rhizobium (32. 1 and 72.5 % respective ly), R. suhtilis

92 I DIAN J EXP BIOL, JANUARY 200 1

A .Hexo cono zo le 80

60

40

20

0 0 !::: -20 t::

8 -40 ..... ~ -60 0 l

G -8o -·-~--~-----~---------;

::;s B. Corbofu ron ,.... 20 ,------------·--·-·-···-------- ----------------···--------·------~-----,

"--'

§ 10 0 (.)

(;j 0

:E -10 0 ..... (.)

'§ -20

(;j -30 § <... -40 0

2 -50 <1.l

"' C<:l -60 <1.l ..... g -70 .J.--·-~·---·---·---·-·-·------··--- ------·-·-------·- ·--

"0 ..... 0

,.-.-. 80 C. Eth in

+ "--'

<1.l • 50 pg • I 00 pg ~ 250 pg llil:I SOO ).lg IIID I 000 j.lg "' 60 C<:l <1.l ..... (.)

·= 40 ~ <1.l (.)

20 ..... I!)

Q...

0

-20

' -40 -·-------------------------------------------------------- ; 2 3 4 5

Incubation period (IAIMics)

Fig. 1- lnlluence of hcxaconazolc (A) , carbofuran (B) and et hion (C) on total microbial count (TMC).

(86.75 and 76.5 %), Azotobacter (36.9 and 55.5%) and B. sphaericus (67 .6 % GDH), while moderate or littl e inhibition was noti ced in E. coli HB I 0 I (23.2 and 2 1.1 %) and Rhudococcus (4.9 and 12.5 %). While carbofuran inhibited GDH and LDH enzyme ac ti vity in B. subtilis (55.5 and 35 .3%), E. coli (23.7 % LDH) and B.sphaericus ( 17 . I% GDH), some promoting ef­fect on Rhizobium and Azotobacter was prominent. Ethi on moderate ly a ffec ted the acti ities of those e n­zymes in Rhodococcus sp. ( 17. 1 and 33.3 %), Rhi;:o­hillln (27.6 % LDH), B. subtilis ( 16.86 % LDH ) and

£.coli (34.2% LDH), but promoting effec t was re­corded in Azotobacter (T able I).

Pesti c ides have some inhibitory effect on so il mi ­cro fl ora and thu s may have an effec t on so il fertili t/~. There a re some adverse repo rts concern ing the above pesti c ides on non-target organi sms. Et hi on and carbo­furan pers ist for longer peri od in ac idic and late rite so il fo ll owed by red and black so ils and degrade fas ter with increas ing pH of the mecl ium

15. Res idues

o f e thi on in tomato, okra and tea pers is ted beyond 15 days and became non-detectab le after 22 days . T here

NOTES 93

A. Hexoconozole 80

60

40 . IOOpg ~ 250pg §1500pg 20

0

-20 0 .... -40 t: 0 -60 (.)

.... ., -80 >

0

Oil 0 _::,

c -5 .2 ;;;

-10 E ..s -15 c

"' -20 ~ E -25 ..s .... -30 0 ~

' -35 '-" ., "' "' -40 ~ u .,

"'0 30 ....

0

£ 20 ., 10 "' "' ~ 0 (.)

c ·~ -10 ., ~ -20

P-o -30 -40 -50 +-----60 -l-----70 +---------- ------ ------------i -80 J _ ______________________________________ _

2 3 4 5

Incubation period (weeks)

Fig. 2-lnllucnce of hexaconazole (A), carbofuran (8 ) and ethi on (C) on glucose dehydrogenase activity of soi l.

are reports that ethion has severe effect on gennina­tion of Passif!ora edufa pollen 10

• Acetylcholine es­te rase (AchE) activity is inhibited in Bombyx mori by ethion

17• Carbofuran is highly tox ic after acute oral

administration . LD50 va lues ranges from 3 to 19 mg/kg body weight. WHO has classified carbofuran as highly hazardous as it inhibits AchE activity at the lowest dose

18. Ri sk assessment of carbofuran has

been reported to be 5' according to Inte rpretative Structural Mode lling (ISM) in so il 19

. Carbofuran is known to be c ho line es te rase inhibitor in contact and stomach action and highl y tox ic to mammals20

. Hexa-

conazole is a stero l biosynthe ti c inhibitor2 1 and its antibacterial effect has not been studied in detail s. One of the present autho r has reported the inhibitory effect of tridemorph on soil dehydrogenase activity22

.

LDH and GDH enzyme studi es in whole cells in pres­ence of tridemorph have shown that it inhibited both of the enzymes and ultimate ly microbial respirati on 11

.

The present investi gation revealed that above three pesticides had moderate e ffec t on TMC that pe rsisted upto 2-3 weeks and the reafte r degraded as envi saged from the da ta of increased numbe r o f TMC. In case of hexaconazo le, the inc re ment of TMC was due to in-

94 I DIAN J EXP BIOL, JANUARY 2001

Table 1-EITcct of hexaconazolc, carbofuran and cthion on glucose and lactate dehydrogenase activities in who le cel ls [Values arc arithmetic mean of three replicati ons]

Organi sms Minimum inhibitory conc.(MIC)" Pest icides and substrates for dehydrogena~e activit/' Hcxa co- Carbofuran Ethion Hexaconazolc Carbofuran Ethion nazole Glucose Pyruvate Glucose Pyruvate Glucose Pyruvate

Rhodococcus sp. AK I so >200 >200 4.9 12.5 4.9 0 17 .1 33.3 Rhi:obittllt sp. 100 200 200 32. 1 72.5 +1 8 +13 .1 +9.4 27.6 Bacillus subtilis 60 >200 >200 86.75 76.5 55 .5 35.3 16.86 +5.8 C. coli HB 101 200 >200 >200 23.2 21. 1 +3.65 23.7 +4.87 34.2 Azotobacter sp. lOO >100 >200 36.9 55.4 +24.4 +1.2 +37.2 +4.46 B. Sf!lwcricus >50 >200 >200 67.6 +10.2 17.1 17.1 14.5 10.48

a MIC for growth in so lidi lied medium for respective pesticides. 11 Figures indicate per cent inhibition of dehyd rogenase activity in presence of pesticides (50 )..lg le vel each) in compari son to control. Dehydrogenase activity was measured by the amount of formazan formation from triphenyl tetazolium chloride (T fC). '+' indicates % increase over control.

crease in actinomycetes population only. The so il respiration data corroborate well with the said find­ings. Hexaconazole exhibited marked inhibitory ef­fect on TMC, soil respiration , GDH and LDH activ ity in intact cell s of some standard bacteria like Rhizo­biwn sp., B. suhtilis, Azotobacter sp. and B. splweri­cus. Inhibiti on of dehydrogenase enzyme system in these bacteria, including nitrogen fixers by hexaco­nazole might be a plausible cause of' inhibition. Car­bofuran and ethion had moderate inhibitory effect on TMC and dehyd rogenase enzyme system of so il mi­croflora.

The authors are grateful to CSIR, New Delhi for financial assistance, to UGC for facilities created un­der the Special Assistance Programme and to the Rallis (India) Ltd for the gift of sat pies of hexaco­nazole, carbofuran and ethion .

References I Pesticides sn fety directorate literature, Vol. 3 (The Pesticides

Safety Directorate, Peasholme Green , York , U.K) 1998. 2 Hexaconazo le-technical literature, (Ralli s (India) Ltd ., Bom­

bay) 1998, 2. 3 Furadan-tcchnical literature, (Rallis (Indi a) Ltd ., Bombay)

1998, I. 4 Ethi on-tcchnical literature, (Rallis (Indi a) Ltd. , Bombay)

1998, I. 5 International programme on chemical safety, (W HO Report )

1993. 17. 6 Casida L E. Jr. , Methods for the Isolati on and Estimation of

activity of soi l bacteria, in The ecology of .wit bacteria, ed­ited by T R G Grey and D. Parkinson. (Liverpool Univ. Press, London) 1968. 97.

7 Frankenberger W T & Dick W A, Relationship between en­zyme ac ti vities and microbial growt h and activity indices in soil. Soil Sci Soc Am 1, 47 ( 1983) 945.

8 Garcia C, Hernandez T & Costa F, Potent ial usc of dehydro­genase activity as an index of microbi al act ivity in degraded soil s, Soil Sci Plant A nair, 28 ( 1997) 123 .

9 Casida L E, Klein D A & Santoro T, Soil dehydrogenase acti vity, Soil Sci, 98 ( I 964) 371.

I 0 Kl ein D A, Loh T C & Goulding, RL, A rapid procedure to evaluate the dehydrogenase activity in so il low in organic matter, Soil Bioi Biocltet/1 , 3 ( 197 1) 385.

II Stevenson I L, Dehydrogenase activity in soi l. Cmt 1 Micro­bioi, 5 ( 1959) 229.

12 Kalam A & Banerjee A K, Action of th ·~ fungicide tride­morph on the glucose, lac tate and succin· te dehydrogenase activiti es of some tridemorph sensitive and resi stant bacteria, Pest Sci, 43 ( 1995) 41.

13 Bracll'ord M M, A rapid and sensitive method for the quanti­llcation of microgram quantities of protein utili zing the prin­ciple of protein dye binding, A11al Bioclte11t , 72 ( 1976) 248.

14 Wainwright M & Pugh G J F. Effects of fun gicides on the number of microorgan isms and frequency of cellullolyt ic fun gi in soil , Plant Soil, 43 ( 1975) 561.

I 5 Melinkov N N, Chemi stry of pesticides , Residue Rev, 36 ( 1971 ). I.

16 Dasilva M M, Bruckner C H, Picanco M & Cruz C D, Fac­tors affecting the grain pollen germinal ion C1 f ye ll ow passion fruit cu lture medium and agrotoxices, r'esqui Agropecu Bras, 34 ( 1999) 347.

17 Nath B S & Kumar R P S, Toxic impact C1f organophospho­rus insecticides on acetylcholine esterase activity in silk worm, Bombyx muri L. Ecotux En viron Safe, 42 ( 1999) I 57.

18 FAO and WHO Report on Pesticide Residue ( 1996). 19 Kumar A S M & Mahadcvan A V. Ri sk assessment of pesti­

cides in the Madurai Distri ct of Tamil Nad u, Pollw Res, 12 (1993) 91.

20 WHO dat a sheet on pesticides- carbo luran. 56 ( 1985). 21 Shephard M C, Noon R A, Worthington P A, McClellan W

D & Lever B G, Hexaconazole : a novel triazolc fungicide. Proc Br CrotJ Prot Cot({- Pests Dis. I ( 1996) 19.

22 Kalam A & Banerjee A K, lnlluence of calix in on urease and dehydrogenase act ivities of soi l, 1 Ecoto.ricol E111·iron Monit 6( 1996)247.