U P T A K E A N D B I O C O N C E N T R A T I O N O F D I E L D R I N ,

D I M E T H O A T E , A N D P E R M E T H R I N BY Tetrahymena pyr i formis

PRADEEP B H A T N A G A R

Department of Zoology, University of Rajasthan, Jaipur 302 015, India

SURENDAR KUMAR* and RUP LAL

Department of Zoology, Sri Venkateswara College, Dhaula Kuan, New Delhi 110 021, India

(Received February 12, 1988; revised August 31, 1988)

Abstract. A ciliate protozoan, Tetrahymenapyriformis was exposed to three insecticides, dieldrin, dimethoate, and permethrin for 12 hr to study the uptake and bioconcentration potential. Ciliates concentrated 922, 3547, and 1056 ~tg g- i dry wt. over an initial concentration of 1 lag mL- 1 of dieldrin, dimethoate, and permethrin, respectively. The highest bioconcentration factor for three insecticides was 2095, 3547, and 1110, respectively. It is suggested that if levels in the environment reach 1 lag mL- 1 the chief effects would be reduction of cell population, and accumulation of the toxicants by ciliates. Accumulation of insecticides by ciliates would permit the toxicants to enter aquatic food chains. Thus the compounds could exert toxic effects at higher trophic levels.

1. Introduction

Very few results have been published on the uptake and bioconcentration of insecticides

in pro tozoa (Gregory et al., 1969; Lal, 1984); despite the fact that protozoans occupy

an important trophic level where accumulation and/or bioconcentration are potential

problems (Cooley et al., 1972). Tetrahymena, a representative microfauna of aquatic

ecosystem, plays an important role in the turnover of organic detritus. It is one o f the

most extensively studied eukaryote and a vast literature is available on virtually every

aspect o f its biology (Elliott, 1973). In the laboratory, it can be cultured axenically both

easily and economically and therefore forms a suitable experimental tool. In this study an attempt is made to determine the uptake and bioconcentration potential of this ciliate

towards three insecticides, each representative of three major classes, organochlorine

(dieldrin), organophosphate (dimethoate) and a pyrethroid (permethrin). This is for the

first time that uptake and bioconcentration of a pyrethroid insecticide in a ciliate

protozoan is reported.

* Author for all correspondence.

Water, Air, and Soil Pollution 40 (1988) 345-349. © 1988 by Kluwer Academic Publishers.

346 PRADEEP BHATNAGAR El" AL.

2. Materials and Methods

2.1. CHEMICALS

All the three insecticides were a gift by Dr Robert E. Thompson, NSI, US Environ- mental Protection Agency, Triangle Research Park, U.S.A. Stock solutions of these insecticides were prepared in analytical grade acetone. The purity of each insecticide was more than 97 ~o.

2.2. O R G A N I S M A N D C U L T U R E S

Stock cultures of Tetrahymenapyriformis (Syngen-I) was obtained from Dr J. G. Jones, Department of biochemistry, University of Hull, U.K. Subsequently the ciliates were maintained axenicaUy in sterilized medium consisting of 1 ~ proteose peptone (Difco) supplemented with 0.5 ~/o NaC1 and 0.3 ~o yeast extract (Difco). The pH of the medium was 7.0. Accumulation studies were carried out with culture grown in 15 mL centrifuge tubes containing 2 mL medium. Cultures were incubated for 72 hr in the dark in 27 + 1 ° C. Three mL of autoclaved medium were transferred to log phase cultures of Tetrahymena and treated simultaneously with appropriate amounts of each insecticide to give a final concentration of 0.1, 0.5, and 1 gg m L - 1 for dieldrin and permethrin and 1, 5, and 10 gg m L - 1 for dimethoate.

2.3. EXPERIMENTAL

Tetrahyrnena cultures were regularly sampled at an interval of 2 hr till 12 hr. At each sampling time Tetrahymena was inactivated by chilling and then centrifuged at 5000 rpm for 10 min. The supernatant was decanted and the pellet was washed thrice with distilled water. Cells were extracted for insecticides with 2 mL acetone. The acetone extract was completely evaporated and the residue was dissolved in hexane before analysis by gas liquid chromatography. Since the uptake was expressed in terms of dry weight, the washed pellet was transferred to boat shaped aluminium cups, dried at 80 °C for 12 hr and weighed. The quantitative and qualitative analysis of dieldrin and permethrin was performed with electron capture detector (Packard-438 GLC). The detector, injector and column temperatures were 250, 220, and 200 ° C, respectively. Nitrogen at a flow rate of 40 mL min- 1 was used as the carrier gas. Dimethoate was analyzed with flame photometer detector (FPD) and the instrument was operated at temperatures 220, 220, and 200 °C for detector, injector, and column, respectively. The flow rate of carrier gas (N2) was 15mLmin -1 and for external gases (H 2 and air) it was 145 and 105 mL min- 1, respectively.

The bioconcentration factor was calculated as follows:

Bioconcentration factor = Insecticide concentration (pg g - 1 dry wt.) in the organism

Initial insecticide concentration in the medium (gg mL-1)

BIOCONCENTRATION OF INSECTICIDE IN TETRAHYMENA 347

3 . R e s u l t s

The uptake of dieldrin in Tetrahymena ranged between 30 and 922 ~tg g - 1 dry wt. (Table I). The kinetics of bioconcentration showed a steady increase in the bio-

TABLE I

Accumulation ~ (pg g - 1 dry weight) of dieldrin byTetrahymena pyriformis for 12 hr

Exposure Initial concentration in medium (pg m L - l ) (hr)

0.1 0.5 1

Accumulation BF Accumulation BF Accumulation BF

2 43.6 + 6.4 436 40.0 + 5.2 80 53.8 + 6.0 54 4 36.5 + 0.7 365 84.7 + 7.4 169 264.2 + 44.5 264 6 70.1 + 4.7 701 213.9 + 40.6 428 460.7 + 22.7 461 8 209.5 + 70.4 2095 503.7 + 70.6 1007 770.3 + 77.6 770

10 30.4 + 6.8 304 168.1 + 14.3 336 922.4 _+ 56.0 922 12 34.0 + 3.3 340 181.1 + 38.5 362 619.0 _+ 88.1 619

Mean of triplicate (+ standard deviation). BF = bioconcentration factor.

concentration factor between 8 and 10 hr and was inversely proportional to the dosage. The bioconcentration factor in Tetrahymena ranged from 54 to 2095.

Table II shows the uptake and bioconcentration factor of dimethoate in Tetrahymena. Its uptake was very rapid in the beginning and reached threshold level of 3547 gg g- 1

after 8 hr of treatment at 1 gg m L - 1 concentration. Like the rapid uptake, the fall in its concentration was also very sharp and in next 2 hr, insecticide contents were one-fourth (804 Ixg g - 1) at 1 ~tg m L - 1 concentration. Tetrahymena accumulated very little dimethoate from the medium containing 5 and 10 ~tg mL-~ insecticide. The

TABLE II

Accumulation a (~tg g - 1 dry weight) of dimethoate by Tetrahymena pyriformis for 12 hr

Exposure Initial concentration in medium (~tg m L - 1) (hr)

1 5 10

Accumulation B F Accumulation B F Accumulation BF

2 471.3 + 83.5 471 180.2 + 42.1 36 267.7 + 23.1 27 4 865.8 +_ 67.7 866 886.0 _+ 131.8 175 461.4 + 21.3 46 6 1090.4 + 153.2 1090 469.1 + 73.9 94 207.4 + 25.1 21 8 3547.0 + 282.2 3547 250.8 + 13.9 50 387.3 + 34.5 39

10 804.4 + 173.0 804 279.2 + 56.4 56 305.4 + 45.9 37 12 459.3 + 45.5 459 256.4 + 15.2 51 539.9 + 54 54

a Mean of triplicate ( + s tandard deviation). BF = bioconcentration factor.

348 PRADEEP BHATNAGAR ET AL.

maximum accumulation was 886 and 540 ~tg g - 1 at 4 and 12 hr of treatment, respec-

tively. The bioconcentration factor for dimethoate in Tetrahymena varied from 21 to 3547 and showed an inverse relation with the concentration of dimethoate added to the

medium.

In contrast, uptake of permethrin showed no definite pattern (Table III). The bi t- concentration factor was highest (1110) at 0.1 ~tg m L - 1 after 4 hr and was lowest (70)

at 1 txg m L - ~ after 2 hr of treatment.

TABLE III

Accumulation s (~tg g-1 dry weight) of permethrin by Tetrahymena pyriformis for 12 hr

Exposure Initial concentration in medium (~tg mL- 1) (hr)

0.1 0.5 1

Accumulation BF Accumulation BF Accumulation BF

2 54.2 + 6.0 542 174.4 + 2.6 349 69.6 + 14.1 70 4 111.0 + 16.3 1110 131.3 + 19.1 263 169.8 + 10.2 170 6 58.6 + 9.2~ 586 80.2 + 6.4 160 1055.9 + 25.0 1056 8 97.4 + 17.9 974 153.6 + 11.0 307 593.0 + 27.8 593

10 82.3 + 5.3 823 94.9 + 11.9 190 425.1 + 38.8 425 12 73.9 + 13.1 739 76.8 + 18.8 154 269.8 + 11.4 270

a Mean of triplicate (+ standard deviation). BF = bioconcentration factor.

4. Discussion

Tetrahymena has a very high capacity to pick-up and concentrate insecticides within its

cellular organization. This uptake was dependent upon the physical and chemical

properties of the insecticides. The kinetics of bioconcentration show that three insecti- cides, dieldrin, dimethoate and permethrin reached threshold level 922, 3547, and 1056 g g g - 1 , respectively, all at 1 gg m L - 1 treatment level. In general, equilibrium (maximum absorption) was reached between 6 and 10 hr, thereafter, insecticide concen-

tration in the organism declined. The maximum amounts of DDT, fenitrothion, and chlorpyrifos accumulated in Tetrahymena was 335, 2260, and 15400 btg g - 1, respectively

(Lal et al., 1987). In this case maximum uptake occurred between 2 and 6 hr. The maximum absorption of dieldrin in Ankistrodesmus amalloides was rapid and was achieved between 30 to 60 rain (Neudorf and Khan, 1975) whilst in Cy¢lotella nana in less than 1 hr (Rice and Sikka, 1973). However, fresh water alga, Chlorellapyrenoidosa showed maximum absorption of dieldrin after 48 hr (Wheeler, 1970).

Among the three insecticides, dimethoate has the highest water solubility (25 gg m L - 1 ) followed by permethrin (0.25 Bg m L - 1 ) and dieldrin (0.186 Ixg g -1 ) at 30 °C. The uptake of three insecticides followed the sequence: d imethoate> permethrin > dieldrin. However, the bioconcentration factor was highest for dimethoate (3547) followed by dieldrin (2095) and permethrin (1110), indicating an inverse relation

BIOCONCENTRATION OF INSECTICIDE IN TETRAHYMENA 349

to water solubility (exception dimethoate). An inverse relation between water solubility and bioconcentration in microorganisms has also been reported (Grimes and Morrison, 1975; Geyer et al., 1981).

The uptake of insecticides in Tetrahymena compared to algae (Gregory et aL, 1969; Lal, 1984) is very high. Pierce and Lichfield (1980) also showed that maximum percentage of DDT accumulated was greater in the bacteria than in the fungi by 51 ~o which was achieved through a six to seven fold increase in cell dry weight. Tetrahymena also has a very high mobility and rapid growth rate suggesting that increase in its cellular mass and the ease with which it disperses in the aqueous system influences the uptake of an insecticide. A dose of 1 Ixg mL- 1 appears to be the most realistic where maximum absorption of three insecticides occur. An interesting aspect emerging from the present finding is that non-toxic insecticides and toxic ones at sub-lethal doses are absorbed with rapidity and concentrated to many folds in the cellular organization. The ability of these ciliates to concentrate insecticides would enable them to enter aquatic food chains, thereby permitting the toxicants to pass to, and possibly exert their effect at higher trophic levels.

Acknowledgment

This research was supported by grants from the Ministry of Environment and Forests, Government of India. S. Kumar expresses his appreciation to Dr V. Krishnamoorthy, Principal, Sri Venkateswara College, New Delhi, for his enthusiastic support.

References

Cooley, N. R., Keltner, J. M., Jr., and Forester, J.: 1972, J. Protozool 19, 636. Elliott, A. M.: 1973, in Biology of Tetrahymena, Downden, Hutchinson and Ross. Geyer, H., Vishwanathan, R., Freitag, D., and Korte, F.: 1981, Chemosphere 10, 1307. Gregory, W. W., Jr., Reed, J. K., and Priester, L. E., Jr.: 1969, J. Protozool. 16, 69. Grimes, D. J. and Morrison, S. M.: 1975, Microbiol. Ecol. 2, 43. Lal, S.: 1984, in R. Lal (ed.), Insecticide Microbiology, Springer-Verlag, Berlin, pp. 61-85. Lal, S., Saxena, D. M., and Lal, R.: 1987, Pestic. Sci. 21, 181. Neudorf, S. and Khan, M. A. Q.: 1975, Bull. Environ. Contain. Toxicol. 13, 443. Pierce, G. E. and Lichfield, J. H.: 1980, Developments in Industrial Microbiology 20, 419. Rice, C. P. and Sikka, H. C.: 1973, J. Agric. Food. Chem. 21, 148. Wheeler, W. B.: 1970, J. Agric. Food. Chem. 18, 416.