Excretion of unchanged organophosphorus compounds after their consumption by larvae of the Egyptian cotton leafworm, Spodoptera littoralis Boisd

PBSTICIDT:

BIOCHEMISTRY

Excretion Their

AND

PHYSIOLOGY

6, 58-64 (1976)

of Unchanged Organophosphorus Consumption by Larvae of the Leafworm, Spodoptera littoralis

A. BEN-AZIZ,~ J. MEISNER, Agricultural

N. AHARONSON,

Research Organization,

The Volcani

Compounds after Egyptian Cotton B0isd.l AND K. R. S. ASCHRR

Center,

Bet Dagan,

Israel

Received April 22, 1975; accepted July 9, 1975 The ingestion and excretion of sublethal doses of phosfolan, monocrotophos, parathion, and leptophos were studied in larvae of the Egyptian cotton leafworm, Spodo-ptera littoralis Boisd. Styropor (foamed polystyrene) lamellae treated with insecticide-sucrose mixtures were fed to the larvae and recovery of the undecomposed insecticides from the feces could be estimated by gas-liquid chromatography, without any cleanup of the sample. The insecticidal residues on Styropor were found to be stable for 5 days. As regards the percentage of insecticide recovered from the feces, two groups could be distinguished: (a) leptophos, practically complete; parathion, 59-67%; b) phosfolan, 10-23%; monocrotophos, 4-7%. A tentative hypothesis was advanced that both oral toxicitv of the four compounds for S. Zittoralis larvae and their subsequent recovery in the feces were related to-water solubility.

the feces voided after consumption of treated leaves requires an extensive extracMuch work was done on the excretion tion and cleanup procedure [see, for rate of insecticides by insects (5, 7, 11, 12). example, Hanna and Atallah (6)]. In certain cases excretion may play an The aims of the present study were important role in the insect’s survival to twofold: (i) to find a simple and rapid insecticide exposure. The beetle Coleomethod for determination of the amount of megilla maculata, for example, disposes of insecticides excreted by some lepidopterous DDT, apart from metabolizing it to DDE, larvae which would constitute a significant by excreting DDT and DDE in the feces. improvement over the conventional methFor 34 days after topical application of ods ; and (ii) to establish (with the help of DDT, the feces contain greater amounts of such a method) the excretion rate of two DDT than DDE (3). Bowman and Casida water-soluble and two practically water(4) studied the metabolism of phorate by insoluble insecticides in the feces of SpoProdenia eridania larvae ; they showed that doptera littoralis larvae. In this study we the composition of the metabolites in the confined ourselves to the excretion of the feces was similar to that in the whole pure substances only. insect, even after 12 days. The determination of the insecticides in MATERIALS AND METHODS INTRODUCTION

1 Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. 1975 Series, No. 142-E. z Died on October 11, 1973.

Cherkcals The following insecticides which are currently used as pesticides against the 58

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

RECOVERY

Egyptian

OF INSECTICIDES

cott,on leafworm,

Spodoptwa

FROM

Zit-

toralis, in this count’ry were assayed : 1. phosfolan, [2- (diethoxyphosphinylimino)-1,3-dithiolane], as ‘Cyolane’ 25% E.C., American Cyanamid Company, Princeton, N.J. 2. parathion, [O,O-diethyl O-p-nitrophenyl phosphorothioate], as ‘Folidol’ 46.7oj, E.C., Bayer A.G., Leverkusen, West Germany. 3. leptophos, [0-(2,5-dichloro-4-bromophenyl) O-methyl phenylphosphonothioat’?], as ‘Phosvel 367, E.C., Makhteshim, Be’er Sheva’, Israel. 4. monocrotophos, [c&3-(dimethoxyphonphingloxy)-N-methylcrotonamide], as ‘Nuvacron’ 40y0 S.C., Milchan Bros., Tel Aviv, Israel. Sucrose, which was found in previous studies to be highly phagostimulatory for larvae of 8. ZittolaZis (9), served as phagostimulant. l3iolo~qical Material Larvae of a susceptible strain of S. littoralis were grown on alfalfa at’ 27°C. Experiments were carried out with individually weighed larvae according t’o the weight range desired, unless stated other&t>. IkWn~itmtion of the Toxicity pounds hy Different Methods

of the Com-

1. (‘or~suv~ption of treated Sty~opor Zamellae. Lamellae of St#yropor (foamed polystyrene) of the PI6 type (2) were used as carrier for the insecticides. Thin, r&angular (6 X 4 cm) Iamellae were cut wit,h the help of a manually driven meat slicer. Dilutions in water-ethanol (70 : 30) of the compounds at the required concentrat,ions were prepared from the formulations and 5’j; sucrose was then dissolved in them. The lamellae were dipped in t’he dilut,ions and left to dry for 1 day. They Lvere then placed singly in petri dishes (diamet.er 15 cm) and one larva weighing bet,wren 260 and 310 mg was int,roduced

FECES

OF

SIwl~ol~TER.~l

I,ITTOR.ll,l.~

.iCl

ptbr dish. For each c*ompound i- 13 (~OIICPIItrations were test,ed, and for each concentrat’ion an experiment was conducted with 20-25-larva replications. Mortality counts were taken after a 4%h-feeding on the lamellae, and the LCao as well as the sublethal (20 T] kill or less) conc~entrations of each compound on Styropor lamellae were determined by constructing :t probitlog. concentration curve. 2. Sippitrg wethod. Using the sipping method first suggested for A’. littwalis larvae by Abe-Elghar et a/. (l), the larva,? were allowed to sip up Y-p1 droplets of a diffrrcnt8 ./c sucrose solution containing concentrations of insecticides ; 5’:) sucrose served as cont#rol in all experimelbt,s. The droplets were offered to the larvae with a 0.0002 syringe mirroburet, (Jlicrcj Atrtric Instrument Co.,, Cleveland, Ohio. l?. S. A.) with a 25-gauge hypodermic nrt~tlle. S. littwalis larvae, when held c:osr to the droplets formed on the needle ill c.)rder to allow their mouth part,s to touch thca solution, imbibe it willingly. Larvae weighing approximattely ZOO mg \vere chosen by visual &inlation. After t,reatment they were t,ransfrrre(l in groups of ten to glass jars (S (m hasta clixmeter, 14 cm ht). The jars c~ontained ctlfalfa placed over :I. layer of sa\vdust (10 absorb frass) nntl were covered with ca!otli. Ii:arh concentration leas assayed \vith :Iboiit8 50 larvae per experiment. JIortalit T counts were made after 24 and 48 h and thta IJ)Y,o’s were calculated for the 1attc.r. ,?. Topical application. T\VO microlit ers of acetone solutions of the insect icidras at, different, concentrations were applied topirally to the dorsum of the larva hetwrhen t,he two black spots on the first at~dominal segment using the same type of microburet as in the sipping method. For eacsh r,t’ the S-13 concentrations tested of each insecticide, an experiment was conduc@trtl with 40 larvae, each one of which weighed ahout 200 mg. Aft,er treatment. the larvse were transferred as well to glass jars containing alfalfa. \lortality counts \vert’ lll:~& 5'1,

i3ftPr

60

BEN-AZIZ

24 and 48 h and LDSO’S were calculated for the latter. 4. Contact e$ect of insecticide residues on Styropor and glass. Big disks of Styropor (diameter 15 cm, thickness 0.7-0.9 mm) were cut and then dipped in 0.05, 0.1, 0.25, and 0.5y0 dilutions of parathion and leptophos (without sucrose) and left to dry for 24 h. The dried lamellae were introduced singly into petri dishes (diameter 15 cm) so that they covered the bottom of the dishes completely. Three larvae, each weighing 26@-310 mg, were introduced per petri dish and for each concentration of the compounds seven replications were done. Parallel experiments were carried out on glass surfaces: petri dish (diameter 15 cm) lower halves were treated with 1.6 ml of an acetone solution of the concentrations of parathion and leptophos mentioned above and the acetone was evaporated by swirling. After 1 h, larvae were introduced into the dishes which were then covered. In both cases larvae were exposed to contact with the poison for 3 h and then transferred to glass jars containing alfalfa. Mortality was recorded both at the end of the exposure to the insecticide in the petri dishes and after 48 h. 5. The recovery rate in fecal pellets of the insecticides ingested by larval feeding on treated lamellae. Styropor lamellae were weighed individually and dipped in aqueousethanolic dilutions (70:30) of the insecticides containing 5% sucrose at the following sublethal concentrations : phosfolan, 0.00025% ; parathion, 0.007y0 ; leptophos, 0.00085% ; and monocrotophos, 0.00055ojo. The lamellae for the residual chemical determinations and the bio!ogical assay of a given insecticide were treated in one batch. After treatment, the lamellae were left to dry in air, allowed to age for 1, 3, 5, or 7 days, and reweighed. For larval consumption experiments the lamellae were then placed singly in petri dishes (diameter 15 cm). Larvae ranging in weight between 260 and 310 mg were introduced into the dishes, one larva per dish, and for each

ET AL.

compound an experiment was conducted with 30-60 larvae. After 24 h, the lamellae were taken out, weighed, and replaced in the dishes ; the lamellae were finally removed from the dishes after 48 h, but the larvae were left in the dishes till 72 h. The fecal pellets were collected and weighed after 24, 48, and 72 h and used for chemical analysis; the samples were introduced into small brown bottles with 10 ml acetone. For parallel quantitative determination of the insecticide deposits 15 treated lamellae were introduced separately into a petri dish (without larvae). Each day, five of the lamellae were removed from the dishes, cut into thin strips and placed in bottles with acetone. The bottles with the fecal pellets or the strips were shaken in a mechanical shaker for 15 min. Ten microliters of each of these solutions was injected into a gas chromatograph without any further cleanup. Care was exercised when taking the samples not to stir up any of the powdery precipitate and the plastic residue found on the bottom of the bottles with the fecal pellets and the strips, respectively. To allow detection of very small quantities, the acetone solution had to be concentrated in certain cases to 0.5 ml prior to glc analysis, by carefully blowing in a mild stream of nitrogen. A Tracer gas chromatograph equipped with an electron capture detector and a flame phot’ometric detector for the analysis of organophosphorus insecticides was used. For parathion and leptophos the chromatographic column used was 4 ft X 0.25 in. glass packed with 3% OV-17 on 80-100 mesh gas chrom Q. Phosfolan was determined on 4 ft X 0.25 in. glass column packed with 20% OV-1 on 80-100 mesh gas chrom Q. Monocrotophos was injected into a gas chromatographic column 6 ft X 0.25 in. glass packed with 5% Reoplex on 80-100 mesh gas chrom Q. RESULTS

The toxicity to S. littoralis larvae of the four OP-compounds assayed by three

RECOVERY

OF

INSECTICIDES

FROM

TABLE 7’hc ‘I’oricily

Tnsert,kides

of your

FECES

OF Sf’OlN~f’TRKd

for H.

Hipping,

--

Parathion Leptophos _-. ---------~__ (1A(ncording to Martin

lit~loritlih krt~r

ppm”

f&larva

f’hosfolan Monocrotophos

Ii1

IA\‘

I

()rgu?wphosphmus Insecticidrs Assayed by Di$rrcnt M&orls

Water solubility,

ZJTT0K.I

soluble in water miscible in any concentration with water 24.0 2.4

Topirnl applkaticm, pg/larva

Fredi11g

on

trraletl slyr~‘p”r, ,,

0.40

4.0

o.oof):~

I ..5 2.0 2.6

5.G 7.0 4.2

0.000(i 0.01 0.00 IIi

(8).

different, application methods is given in Table 1. On topical application the difference in t,he toxicity of the compounds was not very large, the order of toxicity being : phosfolan = leptophos 2 monocrotjophos 2. parathion. With the sipping method, the following order of toxicity emerged: phosfolan > monocrotophos > leptophos > parathion. The LCjo’s determined following feeding on treated Styropor showed that phosfolan was by far the most toxic by this mode of application, followed closely by monocrotophos; leptophos was less toxic, and parathion much less so. By the latter method, differences between the toxicity of the four compounds were the most conspicuous. The probit-log. concentration curves for this method were very steep, and sublethal concentrations were rather close to the I,C50’s, especially with phosfolan and monocrotophos. Parathion was less toxic by topical application than leptophos. The same tendency, but much more pronounced, was noted in feeding experiments on treated Styropor. By the sipping method, however, parathion was slightly more toxic than leptophos. To explain this discrepancy, forced contact experiments were done with parathion and leptophos, to find whether on treated Styropor lamellae,

contact only with either of the two substances plays a role in toxicity (Table 2). Although both compounds were highly toxic on forced 3-h contact on glass (down to 0.00570 and 0.017, in parathion and leptophos, respectively), such contact with insecticide-only treated Styropor surfaces, in which DO feeding at all took place, caustad no kill even with residues from 0..5(‘;, solutions ; in other words, at a concentration of about 50-fold and 300-fold of the LC ,&O’sof parathion and leptophos, rclspectively, when fed to larvae on insecticidesucrose-treated Styropor (cf. Table I). Therefore, the enhanced toxicity of lel)tophos as compared with parathion in feeding experiments on Styropor lamellae is probably not attributable to contact toxicity. Gaseous phase experiments (unpublished data) showed that leptophos does indeed have a moderate gaseous effect (41’)‘; kill of S. littoralis la.rvae weighing 200 mg exposed to the vapors only of 0.45 g a.i. per 15-cm petri dish for 4 h), where:~s parathion wa,s nontoxic undrr t,llcAse conditions. The consumption and eXCr&Jn rat(l of the larvae fed on lamellae treated \vit,h aqueous-ethanolic dilutions of the insvctitides containing 574 sucrose, are give11 in Table 3. Table 3 shows that feeding rut’e was not markedly influenced eithf>r h?; t/It*

62

BEN-AZ111

ET

TABLE Comparison

between Treated

AL.

2

Kill of S. littoralis Larvae Which Were Kept in Forced Contact with Lame&e with Varying Concentrations of Parathion and Leptophos with That of Larvae Which Were in Contact with Glass Treated with the Same Insecticides

Insecticides

On glass y. kill after 3.3 and 48 h

of Styropor

3.3 h

48 h

On lamellae of styropor ‘% kill after 48 h

Parathion-

0.1 0.25 0.5

71 81 95

100 100 100

0 0 0

Leptopho@

0.1 0.25 0.5

81 95 95

100 100 100

0 0 0

Control

0

0 Full kill after 48 hr following 3-h contact on glass down to 0.005yo. b Full kill after 48 hr following 3-h contact on glass down to 0.01%.

type of the insecticide used or by the age of its residue. Moreover, there were no differences between the amount consumed from lamellae treated with sublethal con-

centrations of insecticide plus 5% sucrose, or with sucrose alone. Table 4 shows the amount of the insectitides deposited on the lamellae and the

TABLE Consumption

and Excretion containing

Treatment

3

Rate of S. littoralis Larvae Fed on Lame&e of Styropor Treated Aqueous-ethanolic Dilutions (70 : SO) of Four Organophosphorus Compounds at Sublethal Concentrations

with 5%-Sucrose-

Concentration of insecticide, y.

Age of the residue, days

Number of larvae employed

Weight of lamella consumed during 48 h, mg/larva

Weight of fecal pellets excreted after 72 hr, mg/larva

Leptophos

0.00085

60 30 60

26.0 25.7 22.3

17.0 23.0 21.2

Monocrotophos

0.00055

1 3 5 1 3 5

45 30 30

16.5 13.9 13.0

Phosfolan

0.00025

Parathion

0.007

1 3 5 1 3 7 1

45 45 20 55 47 24

19.5 20.0 18.4 22.3 23.6 23.7

13.8 11.9 12.0 15.2 17.3 17.6 18.0 18.0 21.0

100

24.3

18.0

Control (5% sucrose only)

-

TABLE Percentage

Treatment

Recovery of the Insecticides

from

4 the Feral

Pr/l&

o.f P.

Concentration of the insecticide, %

Residual age of insectitide on lamellae of styropor, days

Amount of insecticide found on lamellae, b%/g Btyropor

Leptophos

0.0008.i

1 3 6

44.6 42.8 46.2

0.77 O..Xi o.:i7

0.77 0,.X O.r,l

Monocrotophos

0.000.55

1 3 6

43.1) 38.0 30.0

0.83 0.w I)..57

0.032 0.040 0.038

Phosfolan

0.0002.i

1 3

0.51 O.-i2 0.59

0.o.i 0.o.i 0.13

Parathion

0.007

1 3 7

27.0 27.0 27.0 350.0 370.0 220.0

--

rate of their recovery from the fecal pellets. The amount of insecticide deposited on the lamellae remained fairly constant notwithstanding whether the residue was 1, 3, or 5 days old. With parathion these determinations were done after 7 instead of after 5 days and some loss of the compound from the lamellae seems to have occurred then. At the sublethal concentrations tested, the recovery of leptophos from the feces ranged from S&100% of the ingested quantity and that of parathion from 59-67y0. On the other hand, recovery of phosfolan and monocrotophos in the feces ranged from low (9.S-22.7%) to very low (3.9~6.7y0) rates, respectively. DISCUSSION

The recovery from the feces of insecticides ingested by S. littoralis larvae can be studied with facility by feeding the insects with insecticide plus phagostimulanttreated Styropor lamellae. This method has the following advantages over feeding

Insecticide ingested, fig/larw

lit tomlis I.nrcwr:

8.9 10.6 li .o

Insecticide excreted in fecal pellets, pg/l:tr\.:L

Rem\-er,x, rate in frw, I

1110.0 !)4 ..i so 4

5.2 6.7 4.0

treated leaves : (a) Styropor is an inert carrier and is not altered chemically in the digest,ive tract of S. littoralis (10). (b) Styropor treated with high concentrations of insecticide had no contact toxicity during 4 h of forced contact. (c) The insecticidal residues on Styropor are stable insofar as there was no loss of substance between 1 and 5 days after application. (d) At sublethal concentrations, the quantity of sucrose-insecticide-treat4 lamells consumed is relatively constant, irrespective of the insecticide applied. (e) The fecal pellets obtained as a result of feeding on the treated lamellae are dry and can be handled easily. When trsnsferred into acetone, there is no need for any clea.nup procedure, and direct, injection into the gas chromatograph for dekrminntion of the insecticides is possible. The results show that, among the insecticides tested two (leptophos and pa.rathion) were excreted either totally or at very high

64

BEN-AL12

rate, whereas two others had a very low excretion rate (phosfolan and monocrotophos). Comparing the water-solubility of the compounds (Table 1) with their rate of recovery in the feces (Table 4), it can be seen that in the two compounds with low water solubility, excretion of the pure compound was very high, whereas the contrary was true for the water-soluble compounds. A similar relationship seems to exist between solubility and oral toxicity of the four compounds: the compounds with low water solubility were less toxic, with both the Styropor-lamellae feeding and sipping feeding methods. REFERENCES

1. M.

R. Abo-Elghar, M. M. Zaki, S. M. Hassan, and M. A. Hanna, Establishment of a susceptibility level of the cotton leaf-worm Prodenia litura (F.) to toxaphene, Bull. Entomol. Sot. Egypt, Econ. Ser. 2, 1 (1968). 2. K. R. S. Ascher and J. Meisner, Evaluation of a method for assay of phagostimulants with Spodoptera littoralis larvae under various conditions, Entomol. Exp. Appl. 16, 101 (1973). 3. Y. H. Atallah and W. C. Nettles, Jr., DDTmetabolism and excretion in Coleomegilla rnaculata De Geer, J. Econ. Entomol. 59, 560 (1966). 4. J. S. Bowman and J. E. Casida, Further studies on the met,abolism of Thimet by plants, insects, and mammals, J. Ewn. Entomol. 51, 838 (1958).

ET

AL.

5. H. E. Fernando, C. C. R,oan, and C. W. Kearns, The penetration and metabolism of organic phosphates in the American roach, Periplaneta americana (Linn.), Ann. Entomol. Sot. Amer. 44, 551 (1951). 6. M. A. Hanna and J. H. Atallah, Penetration and biodegradation of carbaryl in susceptible and resistant strains of the Egyptian cotton leafworm, J. Econ. Entomol. 64, 1391 (1971). 7. Te-yeh Ku and J. L. Bishop, Penetrat’ion, excretion and metabolism of carbaryl in susceptible and resistant German cockroaches, J. Ewn. Entomol. 60, 1328 (1967). 8. H.

Martin, 303, 349, Council, 1972.

“Pest,icide 378, 395, Droitwich,

Manual,” 3rd ed., pp. British Crop Protection Worcester, England,

9. J. Meisner, K. R. S. Ascher, and H. M. Flowers, The feeding response of the larva of the Egypt,ian cotton leafworm Spodoptera littoralis Boisd. to sugars and related compounds. I. Phagostimulatory and deterrent effects, Cowq. Biochem. Physiol. 42A, 899 (1972). 10. J. Meisner, H. M. Flowers, K. R. S. Ascher, and I. Ishaaya, The feeding response of the larva of the Egypt)ian cotton leafworm, Spodoptera &torah Boisd., to sugars and related compounds. III. Biochemical and enzymological aspects of sucrose consumption, Comp. Biochew&. Physiol. 44A, 793 (1973). 11. C. C. Roan, H. E. Fernando, and C. W. Kearns, A radiobiological study of four organic phosphates, J. Econ. Entomol. 43, 319 (1950). 12. L. G. Sellers and F. E. Guthrie, Distribution and metabolism of I%-dieldrin in the resistant and susceptible house Ay. J. Econ. Entomol. 65, 378 (1972).