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Penetration and metabolism of topically applied permethrin and cypermethrin in pyrethroid-tolerant Wiseana cervinata larvae
PESTICIDE
BIOCHEMISTRY
AND
17, 196-204
PHYSIOLOGY
(1982)
Penetration and Metabolism of Topically Applied Permethrin and Cypermethrin in Pyrethroid-Tolerant Wiseana cerwinata Larvae C.K. Biochemistry
CHANGAND
Department,
T.W.
JORDAN
Victoria University, Wellington, New Zealand
Received October 6, 1981; accepted January 4, 1982 The penetration, excretion, and metabolism of topically applied [Wlpermethrin and rV]cypermethrin have been examined in larvae of the porina moth Wiseana cervinata to determine the factors which affect body levels of unchanged pyrethroids. Metabolism was by hydrolysis and to a lesser extent oxidation and the primary metabolites were quickly conjugated to water-soluble products. Little excretion occurred and body levels of unchanged pyrethroids were dependent on the interaction of penetration and metabolism. cis-Cypermethrin was more resistant to metabolism than trans-cypermethrin and cis- and trans-permethrin. trans-Permethrin most readily penetrated into larvae. The body levels of unchanged pennethrin were enhanced by pretreatment of larvae with the metabolic inhibitors carbaryl or piperonyl butoxide. Tolerance of the pasture pest porina to the synthetic pyrethroids is discussed in relation to these findings. INTRODUCTION
Larvae of the Hepialid moth porina, Wiseana cervinata (Walker), are a major New Zealand insect pest competing effectively with livestock for pasture feed (1). The larvae live in subterranean burrows and emerge at night to feed on grasses, clovers, lucerne, tussocks, and pampas. Porina damage is characterized by open patches of sward and may result in permanent changes in the composition of pasture plants. Good control can be achieved with diazinon or fenitrothion but the synthetic pyrethroids are not effective in economical quantities. Porina caterpillars show tolerance to the synthetic pyrethroids, LD,, values being approximately 6 pg/insect for cypermethrin and greater than 20 pg/insect for permethrin (2). Resistance to the synthetic pyrethroids is now well documented in a number of insect strains. Several-hundredfold resistances to permethrin and other pyrethroids in Cufex quinqefusciutus are accompanied by cross-resistance to a range of insecticides and a Kdr-type mechanism has been suggested as an important component of resistance in this species (3). Reduced penetration or increased metabolism of topically 196 0048-3575/82/020196-09$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
applied permethrin did not appear to contribute significantly to the fourfold resistance in a permethrin-resistant strain of Egyptian cotton-leafworm (Spodopteru littoralis) larvae (4) but the greater tolerance of tobacco budworm (Heliothis virestens) compared with the bollworm (Heliothis zeu) to permethrin has been attributed to the greater rate of metabolism in budworm larvae (5). The widespread occurrence of oxidative and hydrolytic pyrethroid metabolism in intact insects (4-9) suggests that metabolic resistances to the synthetic pyrethroids may be expected. We have studied the fate of topically applied pyrethroids in late-instar porina caterpillars and here report the penetration, distribution, and metabolism of permethrin and cypermethrin in these insects. MATERIALS
AND
METHODS
Insects. Porina caterpillars weighing 0.4-0.8 g were collected from infested patures and kept individually at 4°C for up to 12 weeks before use. Experiments carried out with freshly collected larvae gave results identical to those obtained with insects previously stored at 4°C.
PYRETHROID
PENETRATION
AND METABOLISM
IRS-cis, trans-Permethrin (93.6%, w/w), IRS- cis, trans-cypermethrin (98.5%, w/w), and cis, trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (DCVA) (99.6%, w/w) were gifts from Shell, Sittingboume, Kent, United Kingdom. 2’-Hydroxypermethrin (3-[2’-hydroxyphenoxylbenzyl-cis, trans-3-[2,2-dichlorovinyl]-2,2-dimethylcyclopropanecarboxylate) and 4’-hydroxypermethrin (3-[4’-hydroxyphenoxylbenzyl-trans-3-[2,2dichlorovinyl] - 2,2 - dimethylcyclopropanecarboxylate) were provided by FMC Corporation, Middleport, New York. Cyclopropane-I-14C-labeled cis- and rrans-permethrin and cis- and trans-cypermethrin with specifc radioactives 50 and 54 mCi/ mmol, respectively, were gifts from ICI, Jealott’s Hill, Berkshire, United Kingdom, and were greater than 97% pure when chromatographed in three separate thin-layer chromatography (TLC) systems. cis and trans isomers of permethrin were separated from cis, trans-permethrin by TLC on activated silica gel GF,,, (MERCK) using n-hexane:diethyl ether (lO:l, by vol) as the developing solvent. cis and trans isomers were similarly separated from cis, trans-cypermethrin by TLC using benzene:carbon tetrachloride (l:l, by vol) as the developing solvent. Purities of sepaChemicals.
of Pyrethroids
and Metabolites
Insect dosing and analysis of penetration and excretion. [14C]Pyrethroids (approxi-
mately 105 dpm) were topically applied, dissolved in 2 ~1 of n-heptane, to the dorsal abdominal surface of insects suspended by cotton threads above lOO-ml beakers. At intervals up to 24 hr after dosing groups of five insects were individually rinsed with 2 x 1 ml of n-heptane. The n-heptane extracts were transferred to 20-ml scintillation vials and 10 ml of a scintillation cocktail containing 0.5% 2,5-diphenyl oxazoie and 0.03% 1,4-bis-(4-methyl-5-phenoxyloxazol-2-yl)benzene in toluene was added. After storage for 24 hr in the dark, radioactivity was measured in a Beckman LS3100 liquid scintillation counter at ambient temperature. Rates of penetration were thus measured as loss of n-heptane-soluble radioactivity from porina surfaces. TLC of the n-heptane extracts showed that greater than 97% of the surface radioactivity was identical with the applied pyrethroid. Heptane washings of the beakers over which larvae had been suspended were also ana-
1 after
TLC on Activated
Silica
Gel GFps
&
Compound cis-Pennethrin rrans-Permethrin cis-Cypermethrin trans-Cypermethrin cis-DCVA trans-DCVA 2’-Hydroxypermethrin 4’-Hydroxypermethrin
Solvent system:
197
rated isomers, checked by TLC and by NMR using a Varian FT 80A NMR spectrometer, were greater than 97%. The Rf values of reference compounds in several TLC systems are reported in Table 1.
TABLE RI Values
IN PORINA CATERPILLARS
1
2
3
4
5
0.37 0.28 0.07 0.05 -
0.68 0.61 -
0.82 0.82 0.80 0.80 0.65 0.59 0.71 0.58
0.87 0.87 0.80 0.80 0.57 0.47 0.80 0.77
0.80 0.80 0.75 0.75 0.60 0.57 0.73 0.61
:-
-
-h -0.77 0.77 0.73 0.73 0.40 0.30 0.67 0.57
Note. Solvent systems were: (I) n-hexane:diethyl ether (ilkl, by vol); (2) benzene:carbon tetrachloride (1: 1, by vol); (3) toluene saturated with 98% formic acid:diethyl ether (10:3, by vol); (4) benzene:ethyl acetate:methanol (15:5:1, by vol); (5) formic acid-saturated benzene:diethyl ether (10:3, by vol); (6) benzene:ethyl acetate (6:1, by vol).
198
CHANG
AND
lyzed by liquid scintillation counting to detect pyrethroids or metabolites excreted in feces. Analysis of body radioactivity for unchanged pyrethroids and metabolites. Groups of heptane-rinsed larvae were added to 10 ml methanol and immediately homogenized in a Polytron PT-10 homogenizer (Kinematica, GmbH, Lucerne, Switzerland). Homogenized extracts were centrifuged at 5OOOg for 10 min and the supernatant was concentrated to a small volume by rotary evaporation at 50°C. Aliquots were separated by TLC on activated silica gel GFZs4 plates using toluene saturated with 98% formic acid:diethyl ether (10:3, by vol) as the developing solvent. Up to five major bands of radioactivity (bands l-5) from permethrinor cypermethrindosed caterpillars were detected either by radioautography or by liquid scintillation counting of OS-cm zones successively scraped from the origin to the solvent front of chromatograms developed for 10 cm. For radioactivity determination of the silica gel scrapings the scintillation cocktail contained 0.5% 2,5-diphenyloxazole and 0.03% 1,4-bis-(4-methyl-5-phenoxyloxazol-2-yl)benzene dissolved in toluene:Triton X-100 (2:2, by vol). In some experiments radioactivity retained in the body after heptane washing was measured following bleaching and solubilization. Bodies from heptane-washed insects were homogenized in methanol. Aliquots of the homogenates were concentrated to dryness and heated at 50°C for 1 hr in stoppered vials with 1 ml of 30% (w/v) H,Oz. Hyamine 10-X hydroxide (lo%, w/v, 2 ml) was added and the stoppered vials were incubated at 50°C for 2 hr. Under these conditions the total recovery of radioactivity in the methanol extracts used for quantitative metabolite analysis was 90-100% of the radioactivity measured after bleaching and solubilization. Identification of metabolites in body extracts. The 24-hr methanol extracts of heptane-rinsed bodies from larvae dosed with known amounts of either cypermethrin
JORDAN
or permethrin were pooled and concentrated by rotary evaporation. The five major metabolite bands were separated by preparative TLC on l-mm-thick layers of silica gel GF,, using formic acid-saturated toluene:diethyl ether (10:3) as the developing solvent. The separated bands were eluted and analyzed by TLC and by acid, base, and enzymatic hydrolysis. Bands were acid or base hydrolyzed by heating in either 0.2 M NaOH, 1.O M NaOH, or 0.5 M HCl in sealed tubes in a boiling water bath for 10 min or by reflux in 6 M HCl for 1 hr. Under these conditions the hydrolysis of [14C]permethrin to DCVA as measured by TLC and radioactive zone counting was 69% in 0.2 M NaOH, 80% in 1 M NaOH, 5% in 0.5 M HCl, and 17% in 6 M HCl. Enzymatic hydrolyses were carried out by incubating separated metabolite bands with 2 mg each of P-glucuronidaselaryl sulfatase (BHD Chemicals, Poole, Dorset, U.K.) and P-glucosidase (Koch-Light, Colnbrook, Bucks., U.K.) in 1 ml of 0.1 M sodium acetate-acetic acid buffer, pH 4.6, at 37°C for 6-24 hr in a rocking water bath. After acid, base, or enzyme hydrolysis incubation mixtures were adjusted to pH 2 and extracted with 3 x 3 vol of diethyl ether. Ether extracts were analyzed by TLC and radioactive zone counting. RESULTS
Penetration and Excretion Rates of penetration of topically applied permethrin and cypermethrin are shown in Figs. 1 and 2. Doses used were less than the LDso but at the higher doses some insects became moribund and showed water loss. Porina larvae did not produce substantial quantities of solid feces and in all cases less than 5% of the applied pyrethroid was found in the beakers containing excreted feces. trans-Permethrin was absorbed faster than cis-permethrin and 0.5~pg doses were absorbed faster than 5 .O-pg doses. cis :trans (1: 1, by, weight) mixtures of permethrin were absorbed at the slower rate of the cis isomer for both 0.5- and 5.0-pg doses. For
PYRETHROID
PENETRATION
AND
METABOLISM
12 Time (hr)
IN
PORINA
199
CATERPILLARS
24
FIG. 1. Penetration of topically applied permethrin in porina larvae. Groups offive insects were analyzed at each time interval. Vertical bars represent standard deviations. cis-Permethrin, 0.5 pg. l ; trans-permethrin, 0.5 gg, 0; cis-permethrin, 5.0 pg, m; trans-permethrin, 5.0 pg. 0.
cypermethrin 0.5 pg of the tram isomer was absorbed faster than 0.5 pg of the cis isomer but 0.5 pg of a 1:l (by weight), cis:truns mixture was absorbed at an intermediate rate. No apparent differences between rates of penetration of 2.5 pg of cis-; truns-, or cis, trans-cypermethrin were observed. Separation and Partial Characterization of Pyrethroid Metabolites
The five major metabolite bands separated from the heptane-washed bodies of larvae dosed with 5 pg of cis, trans[14C]permethrin (Fig. 3A) were partially characterized by their TLC behavior, sol-
vent solubility, and susceptibility to hydrolysis. Band 1 contained 10% of the body radioactivity and was identified as unchanged permethrin by cochromatography with permethrin in solvent systems 1 and 3-6 (Table 1) and by acid and base hydrolysis to DCVA. Band 2 contained approximately 30% of the body radioactivity and was identified as more than 95% DCVA by cochromatography in solvent systems 1 and 3-6 and by resistance to acid, base, and enzymatic hydrolysis. Band 3 (15% of the body radioactivity) contained at least four compounds distinguishable by radioautography of extracts chromatographed in solvent systems 1-6. Band 3 was partially
CHANG
AND
JORDAN
24
12 Time
(hrsj
FIG. 2. Penetration of topically applied cypermethrin in porina larvae. Groups ofJve insects were analyzed at each time interval. Vertical bars represent srandard deviations. cis-Cypermethrin, 0.5 +g, 0; trans-cypermefhrin, 5.O’kg, 0; ck-cypermethrin, 2.5 pg, n ; trans-cypermethrin, 2.5 pg, 0.
cleaved to radioactive compounds of higher & only by reffuxing in 6 M HCl for 1 hour, Band 4 contained 5% of the radioactivity and was hydrolyzed, only in 6 h4 HCl, to a radioactive compound migrating with DCVA in TLC systems 1-6. Band 5 (40% of the body radioactivity) contained water-soluble metabolites which could not be extracted from acidic, basic, or neutrai solution into diethyl ether and which remained at the origin after TLC in formic acid-saturated toluene:diethyl ether (10:3). After overnight enzymatic hydrolysis of band 5, 7% of the radioactivity (band HI) migrated in the permethrin zone, 34% (band H2) with DCVA, 26% (band H3)
slightly behind DCVA, and 32% remained at the origin (band H4). Small amounts of radioactive material migrating with 4’hydroxypertnethrin in solvent systems 3 -6 were also present in band H2. Band H3 could not be further cleaved by acid or base hydrolysis and could only be extracted into ether from acidic solutions. Band H4 was refluxed in 6 M HCI for 1 hr, the extract cooled, and the radioactivity extracted into diethyl ether. About 75% of the extracted radioactivity migrated with DCVA after TLC. The rest of the ether extracted radioactivity from refluxed band H4 had an Rf similar to that of band H3. Five major metabolite bands were also
PYRETHROID
PENETRATION
Distance
from
origin
AND
METABOLISM
(cm)
FIG. 3. Separation of [Vlpyrethroid metabolites. Insects were dosed with 5.0 pg of cis, trans-permethrin (A) or 2.5 kg of cis, trans-cypermethrin (II) and the radioactive metabolites separated by TLC in formic acid-saturated toluene:diethyi ether (IOJ, by vol).
chromatographically separated from the bodies of larvae dosed with 2.5 ,ug of cis, truns-[14C]cypermethrin (Fig. 3B). Bands 1 and 2 cochromatographed in five solvent systems with cypermethrin and DCVA, respectively. Bands 3 -5 were qualitatively identical with permethrin metabolite bands 3-5.
Effects of Dose Size and Composition Metabolism
on
The distribution of radioactivity among the five main metabolite bands was analyzed at time intervals up to 24 hr after dosing for 0.5- and 5-pg doses of cis-, trans-,
Amount
of Unmetabolized
Pyrethroid
IN
PORINA
and l:l, cis, trans-permethrin and for 0.5 and 2.5~pg doses of cis-, tram-, and l:l, cis, trans-cypermethrin. Results for the proportion of unmetabolized pyrethroid 3 and 24 hr after dosing are summarized in Table 2 and results for 2.5 pg ciscypermethrin are presented in Fig. 4. The amounts of unchanged pyrethroids in the larvae were calculated from the penetration data and from the results of chromatographic separation of unchanged pyrethroids and metabolites. For permethrin greatest differences occurred 3 hr after a 5-pg dose (Table 2). trans-Permethrin accumulated in larger amounts than the more slowly penetrating cis isomer and cis, tram mixture suggesting that under these conditions metabolism was the rate-limiting process regulating body levels of unchanged permethrin. Highest body levels of cypermethrin 3 hr after a 2.5~pg dose were achieved with the cis isomer. As penetration rates of the cis and trans isomers were similar (Fig. 2) decreased hydrolysis of cis-cypermethrin may account for the greater body levels of the cis compound (Table 2). At longer time intervals (Fig. 4) decreasing body levels of cis-cypermethrin were accompanied by increased amounts of DCVA and other metabolites. When piperonyl butoxide or carbaryl dissolved in 1 ~1 n-heptane was topically applied to larvae 10 min before topical administration of 0.5 pg of cis, trans-permethrin the body levels of permethrin were significantly higher at 5 and 24 hr after dosing compared with control larvae only
TABLE 2 in the Body Amount
at 3 and 24 hr after of pyrethroid”
Permethrin Permethrin Cypermethrin Cypermethrin o Results
(0.5 cc.g) (5.0 pg) (0.5 pg) (2.5 pg) are means
cis 0.08 0.61 0.09 0.51
-rr -+ 2
2 SD for groups
0.07 0.89 0.13 0.35
+ + 2 ‘-
of five
Dosing
24 hr
trans 0.01 0.11 0.01 0.09
Topical
(pg)
3 hr Compound
201
CATERPILLARS
cis, trans 0.02 0.08 0.02 0.06 insects.
0.10 0.68 0.13 0.45
k f % 2
0.02 0.08 0.02 0.08
cis 0.02 0.59 0.08 0.48
+ k 2 k
trans 0.01 0.19 0.02 0.10
0.03 0.69 0.07 0.32
k f k 2
cis. tran.7 0.01 0.18 0.02 0.12
0.03 0.56 0.07 0.36
k 2 k -t
0.01 0.16 0.02 0.11
CHANG
AND
JORDAN
12 Time
24 (hr)
FIG. 4. Penetration and metabolism of 2.5 pg cis-cypermethrin. Vertical bars represent standard deviations for groups offive insects. Total body pyrethroid and metabolites, R body cypermethrin, A; DCVA, 0.
administered the pyrethroid (Table 3). Piperonyl butoxide was used as an inhibitor of pyrethroid oxidation and carbaryl as an inhibitor of pyrethroid hydrolysis. Pretreatment with either inhibitor significantly increased the amount of unchanged permethrin at 5 and 24 hr. Neither compound altered the penetration of cis, truns-perme&in. Greatest effects on prolonging the lifetime of permethrin in the larvae were given by carbaryl which substantially inhibited hydrolysis of permethrin to DCVA. DISCUSSION
Our approach in this study has been to characterize the relative roles of penetration, excretion, and metabolism in the
maintenance of body levels of pyrethroids in porina caterpillars. Permethrin and cypermethrin were metabolized by similar pathways in porina larvae to those reported in other insects (4-9). The partial inhibition of metabolism by piperonyl butoxide indicates that oxidation of pyrethroids occurred in this species. However, hydrolysis, which was partially inhibited by carbaryl, was the major metabolic route. Water-soluble products were important metabolites at the longer time intervals after dosing (Fig. 3, bands 3-5). The behavior of compounds in bands 3 and 4 was consistent with that of amino acid conjugates of DCVA and possibly its 2-methyl-hydroxylated derivatives. Band 5 appears to contain conjugates of 4’-
PYRETHROID
PENETRATION
AND
METABOLISM
TABLE3 Effects of Inhibitors on Permethrin Metabolism Percentage
of dose
present
Permethrin Dose Pamethrinb Pemethrin” + 2.5 pg piperonyl butoxide PemetIuinb + 1 pg carbaryl
shr
aso
DCVA 24 hr
5 hr
24 hr
2023
11~1
2022
2121
33+2
14+1
15+1
23+1
3823
2022
1221
1221
(L Results are means + SD for groups b 0.5 wg cis. truns-pemethrin.
of 10 insects.
hydroxypermethrin and roughly equal amounts of amino acid and sulfate/glycoside conjugates of DCVA and its hydroxylation products. On the basis of our postulated identification we calculate that 20% of the body radioactivity 24 hr after dosing with 5 pg of permethrin contained hydroxylated metabolites and that 80-90% of the body permethrin had been hydrolyzed to DCVA and its oxidation products. Cypermethrin was more resistant than permethrin to hydrolysis (Table 2, Fig. 3). We calculate that approximately half of a 2.5pug cypermethrin dose was converted to DCVA and an additional 20% of the dose was metabolized to hydroxylated DCVA derivatives. Less than 2% was found as cypermethrin metabolites hydroxylated in the aromatic rings. cis-Cypermethrin was the most stable compound examined. Relative resistance of cis-cypermethrin to hydrolysis has also been reported in other insects (4, 5, 8, 9). The relative toxicity of cypermethrin compared with permethrin in porina larvae (2) may in part be attributed to the slower metabolism of cypermethrin. Variations in penetration rates were also apparent. Small doses (0.5 pg) of transpermethrin and trans-cypermethrin were consistently absorbed faster than the cis isomers but at higher doses (2.5 pg cypermethrin or 5 pg permethrin) no general trend was apparent. The interaction of penetration rates with rates of hydrolysis and oxidation appeared to be important in the regulation of body levels of unchanged py-
IN
PORINA
CATERPILLARS
203
rethroid. The relatively high levels of ciscypermethrin 3 and 24 hr after a 2.5~pg dose (Table 2) may be due to decreased metabolism of that isomer while enhanced accumulation of trans-permethrin 3 and 24 hr after a 5-pg dose appeared to be related to the fast rate of truns-permethrin penetration. After 24 hr when penetration of most isomers was slow metabolism was the major factor affecting body levels of unchanged pyrethroids (Fig. 4). Rapid penetration of topically applied pyrethroids is common in a wide range of insects (4, 5, g-10). Little excretion of pyrethroids by porina caterpillars was observed in our studies. In all cases less than 5% of the dose was voided in feces in the 24-hr experimental period. In an attempt to distinguish between unabsorbed and excreted pyrethroid we have suspended larvae by cotton threads above beakers and collected feces voided into the beakers. Porina larvae produced relatively little solid or liquid feces under these conditions and the low rate of general and pyrethroid excretion may be due to lack of feeding during, and in the period prior to, the experiment (11). However, similar experiments with another New Zealand Hepialid, the larvae of the puriri moth, Aenetus virescens, which excreted appreciable amounts of solid feces showed that topically applied permethrin and cypermethrin were not voided and were absorbed and metabolized in a manner similar to that demonstrated for porina caterpillars (C. K. Chang and T. W. Jordan, unpublished). Our studies indicate that considerable quantities of unchanged pyrethroids accumulate in the bodies of porina larvae which, however, resist poisoning by permethrin and cypermethrin. Approximately 0.5 pg of cypermethrin is found in the body 3-24 hr after dosing 2.5 pg of the cis isomer but larvae are not killed. Under these circumstances it appears that storage of pyrethroid in the body or resistance at the level of the nervous system must account for porina’s tolerance to the synthetic p’yrethroids.
204
CHANG
AND
JORDAN
ACKNOWLEDGMENTS
This work was supported by the New Zealand University Grants Committee, Lottery Profits Distribution Committee, Internal Research Committee of Victoria University, and by gifts of chemicals from Shell, ICI, FMC Corporation, and Sumitomo Chemical ComPanY. REFERENCES
1. D. N. Ferro, Pasture pests, in “New Zealand Insect Pests” (D. N. Ferro, Ed.), p. 105, Caxton Press, Christchurch, 1976. 2. G. D. G. Du Toit, R. J. Townsend, and S. J. Armstrong, Lack of response in porina (Wiseana sp: Hepialidae) caterpillar to treatment with pyrethroid insecticides, N.Z.J. Exp. Agr. 6, 175 (1978). 3. T. M. Priester and G. P. Georghiou, Crossresistance spectrum in pyrethroid-resistant Culex quinqefasciatus, Pestic. Sci. 11, 617 (1980). 4. J. S. Holden, Absorption and metabolism of permethrin and cypermethrin in the cockroach and the cotton-leafworm larvae, Pestic. Sci. 10,295 (1979). 5. W. S. Bigley and F. W. Plapp, Jr., Metabolism of
6. 7. 8.
9.
10.
cis and trans-[l*C]permethrin by the tobacco budworm and the bollworm, J. Agr. Food Chem. 26, 1128 (1978). I. Yamamoto, E. C. Kimmel, and J. E. Casida, Oxidative metabolism of pyrethroids in housetlies, J. Agr. Food Chem. 17, 1227 (1969). J. Miyamoto and T. Suzuki, Metabolism of tetramethrin in houseflies in vivo, Pestic. Biochem. Physiol. 3, 30 (1973). T. Shono, T. Unai, and J. E. Casida, Metabolism of permethrin isomers in American cockroach adults, housefly adults, and cabbage looper larvae, Pestic. Biochem. Physiol. 9, 96 (1978). D. M. Soderlund, Pharmacokinetic behaviour of enantiomeric pyrethroid esters in the cockroach, Periplaneta americana L., Pestic. Biothem. Physiol. 12, 38 (1979). F. P. W. Winteringham, A. Harrison, and P. Bridges, Absorption and metabolism of [14C]pyrethroids by the adult housefly, Musca domestica L., in vivo, Biochem. .I. 61, 359 (1955).
11.
T. W. Jordan, C. K. Chang, and J. N. Smith, Factors affecting the metabolism, distribution and excretion of aromatic acids in Periplaneta americana and Acanthoxyla intermedia, Insect Biothem. 10, 265 (1980).
BIOCHEMISTRY
AND
17, 196-204
PHYSIOLOGY
(1982)
Penetration and Metabolism of Topically Applied Permethrin and Cypermethrin in Pyrethroid-Tolerant Wiseana cerwinata Larvae C.K. Biochemistry
CHANGAND
Department,
T.W.
JORDAN
Victoria University, Wellington, New Zealand
Received October 6, 1981; accepted January 4, 1982 The penetration, excretion, and metabolism of topically applied [Wlpermethrin and rV]cypermethrin have been examined in larvae of the porina moth Wiseana cervinata to determine the factors which affect body levels of unchanged pyrethroids. Metabolism was by hydrolysis and to a lesser extent oxidation and the primary metabolites were quickly conjugated to water-soluble products. Little excretion occurred and body levels of unchanged pyrethroids were dependent on the interaction of penetration and metabolism. cis-Cypermethrin was more resistant to metabolism than trans-cypermethrin and cis- and trans-permethrin. trans-Permethrin most readily penetrated into larvae. The body levels of unchanged pennethrin were enhanced by pretreatment of larvae with the metabolic inhibitors carbaryl or piperonyl butoxide. Tolerance of the pasture pest porina to the synthetic pyrethroids is discussed in relation to these findings. INTRODUCTION
Larvae of the Hepialid moth porina, Wiseana cervinata (Walker), are a major New Zealand insect pest competing effectively with livestock for pasture feed (1). The larvae live in subterranean burrows and emerge at night to feed on grasses, clovers, lucerne, tussocks, and pampas. Porina damage is characterized by open patches of sward and may result in permanent changes in the composition of pasture plants. Good control can be achieved with diazinon or fenitrothion but the synthetic pyrethroids are not effective in economical quantities. Porina caterpillars show tolerance to the synthetic pyrethroids, LD,, values being approximately 6 pg/insect for cypermethrin and greater than 20 pg/insect for permethrin (2). Resistance to the synthetic pyrethroids is now well documented in a number of insect strains. Several-hundredfold resistances to permethrin and other pyrethroids in Cufex quinqefusciutus are accompanied by cross-resistance to a range of insecticides and a Kdr-type mechanism has been suggested as an important component of resistance in this species (3). Reduced penetration or increased metabolism of topically 196 0048-3575/82/020196-09$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
applied permethrin did not appear to contribute significantly to the fourfold resistance in a permethrin-resistant strain of Egyptian cotton-leafworm (Spodopteru littoralis) larvae (4) but the greater tolerance of tobacco budworm (Heliothis virestens) compared with the bollworm (Heliothis zeu) to permethrin has been attributed to the greater rate of metabolism in budworm larvae (5). The widespread occurrence of oxidative and hydrolytic pyrethroid metabolism in intact insects (4-9) suggests that metabolic resistances to the synthetic pyrethroids may be expected. We have studied the fate of topically applied pyrethroids in late-instar porina caterpillars and here report the penetration, distribution, and metabolism of permethrin and cypermethrin in these insects. MATERIALS
AND
METHODS
Insects. Porina caterpillars weighing 0.4-0.8 g were collected from infested patures and kept individually at 4°C for up to 12 weeks before use. Experiments carried out with freshly collected larvae gave results identical to those obtained with insects previously stored at 4°C.
PYRETHROID
PENETRATION
AND METABOLISM
IRS-cis, trans-Permethrin (93.6%, w/w), IRS- cis, trans-cypermethrin (98.5%, w/w), and cis, trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (DCVA) (99.6%, w/w) were gifts from Shell, Sittingboume, Kent, United Kingdom. 2’-Hydroxypermethrin (3-[2’-hydroxyphenoxylbenzyl-cis, trans-3-[2,2-dichlorovinyl]-2,2-dimethylcyclopropanecarboxylate) and 4’-hydroxypermethrin (3-[4’-hydroxyphenoxylbenzyl-trans-3-[2,2dichlorovinyl] - 2,2 - dimethylcyclopropanecarboxylate) were provided by FMC Corporation, Middleport, New York. Cyclopropane-I-14C-labeled cis- and rrans-permethrin and cis- and trans-cypermethrin with specifc radioactives 50 and 54 mCi/ mmol, respectively, were gifts from ICI, Jealott’s Hill, Berkshire, United Kingdom, and were greater than 97% pure when chromatographed in three separate thin-layer chromatography (TLC) systems. cis and trans isomers of permethrin were separated from cis, trans-permethrin by TLC on activated silica gel GF,,, (MERCK) using n-hexane:diethyl ether (lO:l, by vol) as the developing solvent. cis and trans isomers were similarly separated from cis, trans-cypermethrin by TLC using benzene:carbon tetrachloride (l:l, by vol) as the developing solvent. Purities of sepaChemicals.
of Pyrethroids
and Metabolites
Insect dosing and analysis of penetration and excretion. [14C]Pyrethroids (approxi-
mately 105 dpm) were topically applied, dissolved in 2 ~1 of n-heptane, to the dorsal abdominal surface of insects suspended by cotton threads above lOO-ml beakers. At intervals up to 24 hr after dosing groups of five insects were individually rinsed with 2 x 1 ml of n-heptane. The n-heptane extracts were transferred to 20-ml scintillation vials and 10 ml of a scintillation cocktail containing 0.5% 2,5-diphenyl oxazoie and 0.03% 1,4-bis-(4-methyl-5-phenoxyloxazol-2-yl)benzene in toluene was added. After storage for 24 hr in the dark, radioactivity was measured in a Beckman LS3100 liquid scintillation counter at ambient temperature. Rates of penetration were thus measured as loss of n-heptane-soluble radioactivity from porina surfaces. TLC of the n-heptane extracts showed that greater than 97% of the surface radioactivity was identical with the applied pyrethroid. Heptane washings of the beakers over which larvae had been suspended were also ana-
1 after
TLC on Activated
Silica
Gel GFps
&
Compound cis-Pennethrin rrans-Permethrin cis-Cypermethrin trans-Cypermethrin cis-DCVA trans-DCVA 2’-Hydroxypermethrin 4’-Hydroxypermethrin
Solvent system:
197
rated isomers, checked by TLC and by NMR using a Varian FT 80A NMR spectrometer, were greater than 97%. The Rf values of reference compounds in several TLC systems are reported in Table 1.
TABLE RI Values
IN PORINA CATERPILLARS
1
2
3
4
5
0.37 0.28 0.07 0.05 -
0.68 0.61 -
0.82 0.82 0.80 0.80 0.65 0.59 0.71 0.58
0.87 0.87 0.80 0.80 0.57 0.47 0.80 0.77
0.80 0.80 0.75 0.75 0.60 0.57 0.73 0.61
:-
-
-h -0.77 0.77 0.73 0.73 0.40 0.30 0.67 0.57
Note. Solvent systems were: (I) n-hexane:diethyl ether (ilkl, by vol); (2) benzene:carbon tetrachloride (1: 1, by vol); (3) toluene saturated with 98% formic acid:diethyl ether (10:3, by vol); (4) benzene:ethyl acetate:methanol (15:5:1, by vol); (5) formic acid-saturated benzene:diethyl ether (10:3, by vol); (6) benzene:ethyl acetate (6:1, by vol).
198
CHANG
AND
lyzed by liquid scintillation counting to detect pyrethroids or metabolites excreted in feces. Analysis of body radioactivity for unchanged pyrethroids and metabolites. Groups of heptane-rinsed larvae were added to 10 ml methanol and immediately homogenized in a Polytron PT-10 homogenizer (Kinematica, GmbH, Lucerne, Switzerland). Homogenized extracts were centrifuged at 5OOOg for 10 min and the supernatant was concentrated to a small volume by rotary evaporation at 50°C. Aliquots were separated by TLC on activated silica gel GFZs4 plates using toluene saturated with 98% formic acid:diethyl ether (10:3, by vol) as the developing solvent. Up to five major bands of radioactivity (bands l-5) from permethrinor cypermethrindosed caterpillars were detected either by radioautography or by liquid scintillation counting of OS-cm zones successively scraped from the origin to the solvent front of chromatograms developed for 10 cm. For radioactivity determination of the silica gel scrapings the scintillation cocktail contained 0.5% 2,5-diphenyloxazole and 0.03% 1,4-bis-(4-methyl-5-phenoxyloxazol-2-yl)benzene dissolved in toluene:Triton X-100 (2:2, by vol). In some experiments radioactivity retained in the body after heptane washing was measured following bleaching and solubilization. Bodies from heptane-washed insects were homogenized in methanol. Aliquots of the homogenates were concentrated to dryness and heated at 50°C for 1 hr in stoppered vials with 1 ml of 30% (w/v) H,Oz. Hyamine 10-X hydroxide (lo%, w/v, 2 ml) was added and the stoppered vials were incubated at 50°C for 2 hr. Under these conditions the total recovery of radioactivity in the methanol extracts used for quantitative metabolite analysis was 90-100% of the radioactivity measured after bleaching and solubilization. Identification of metabolites in body extracts. The 24-hr methanol extracts of heptane-rinsed bodies from larvae dosed with known amounts of either cypermethrin
JORDAN
or permethrin were pooled and concentrated by rotary evaporation. The five major metabolite bands were separated by preparative TLC on l-mm-thick layers of silica gel GF,, using formic acid-saturated toluene:diethyl ether (10:3) as the developing solvent. The separated bands were eluted and analyzed by TLC and by acid, base, and enzymatic hydrolysis. Bands were acid or base hydrolyzed by heating in either 0.2 M NaOH, 1.O M NaOH, or 0.5 M HCl in sealed tubes in a boiling water bath for 10 min or by reflux in 6 M HCl for 1 hr. Under these conditions the hydrolysis of [14C]permethrin to DCVA as measured by TLC and radioactive zone counting was 69% in 0.2 M NaOH, 80% in 1 M NaOH, 5% in 0.5 M HCl, and 17% in 6 M HCl. Enzymatic hydrolyses were carried out by incubating separated metabolite bands with 2 mg each of P-glucuronidaselaryl sulfatase (BHD Chemicals, Poole, Dorset, U.K.) and P-glucosidase (Koch-Light, Colnbrook, Bucks., U.K.) in 1 ml of 0.1 M sodium acetate-acetic acid buffer, pH 4.6, at 37°C for 6-24 hr in a rocking water bath. After acid, base, or enzyme hydrolysis incubation mixtures were adjusted to pH 2 and extracted with 3 x 3 vol of diethyl ether. Ether extracts were analyzed by TLC and radioactive zone counting. RESULTS
Penetration and Excretion Rates of penetration of topically applied permethrin and cypermethrin are shown in Figs. 1 and 2. Doses used were less than the LDso but at the higher doses some insects became moribund and showed water loss. Porina larvae did not produce substantial quantities of solid feces and in all cases less than 5% of the applied pyrethroid was found in the beakers containing excreted feces. trans-Permethrin was absorbed faster than cis-permethrin and 0.5~pg doses were absorbed faster than 5 .O-pg doses. cis :trans (1: 1, by, weight) mixtures of permethrin were absorbed at the slower rate of the cis isomer for both 0.5- and 5.0-pg doses. For
PYRETHROID
PENETRATION
AND
METABOLISM
12 Time (hr)
IN
PORINA
199
CATERPILLARS
24
FIG. 1. Penetration of topically applied permethrin in porina larvae. Groups offive insects were analyzed at each time interval. Vertical bars represent standard deviations. cis-Permethrin, 0.5 pg. l ; trans-permethrin, 0.5 gg, 0; cis-permethrin, 5.0 pg, m; trans-permethrin, 5.0 pg. 0.
cypermethrin 0.5 pg of the tram isomer was absorbed faster than 0.5 pg of the cis isomer but 0.5 pg of a 1:l (by weight), cis:truns mixture was absorbed at an intermediate rate. No apparent differences between rates of penetration of 2.5 pg of cis-; truns-, or cis, trans-cypermethrin were observed. Separation and Partial Characterization of Pyrethroid Metabolites
The five major metabolite bands separated from the heptane-washed bodies of larvae dosed with 5 pg of cis, trans[14C]permethrin (Fig. 3A) were partially characterized by their TLC behavior, sol-
vent solubility, and susceptibility to hydrolysis. Band 1 contained 10% of the body radioactivity and was identified as unchanged permethrin by cochromatography with permethrin in solvent systems 1 and 3-6 (Table 1) and by acid and base hydrolysis to DCVA. Band 2 contained approximately 30% of the body radioactivity and was identified as more than 95% DCVA by cochromatography in solvent systems 1 and 3-6 and by resistance to acid, base, and enzymatic hydrolysis. Band 3 (15% of the body radioactivity) contained at least four compounds distinguishable by radioautography of extracts chromatographed in solvent systems 1-6. Band 3 was partially
CHANG
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24
12 Time
(hrsj
FIG. 2. Penetration of topically applied cypermethrin in porina larvae. Groups ofJve insects were analyzed at each time interval. Vertical bars represent srandard deviations. cis-Cypermethrin, 0.5 +g, 0; trans-cypermefhrin, 5.O’kg, 0; ck-cypermethrin, 2.5 pg, n ; trans-cypermethrin, 2.5 pg, 0.
cleaved to radioactive compounds of higher & only by reffuxing in 6 M HCl for 1 hour, Band 4 contained 5% of the radioactivity and was hydrolyzed, only in 6 h4 HCl, to a radioactive compound migrating with DCVA in TLC systems 1-6. Band 5 (40% of the body radioactivity) contained water-soluble metabolites which could not be extracted from acidic, basic, or neutrai solution into diethyl ether and which remained at the origin after TLC in formic acid-saturated toluene:diethyl ether (10:3). After overnight enzymatic hydrolysis of band 5, 7% of the radioactivity (band HI) migrated in the permethrin zone, 34% (band H2) with DCVA, 26% (band H3)
slightly behind DCVA, and 32% remained at the origin (band H4). Small amounts of radioactive material migrating with 4’hydroxypertnethrin in solvent systems 3 -6 were also present in band H2. Band H3 could not be further cleaved by acid or base hydrolysis and could only be extracted into ether from acidic solutions. Band H4 was refluxed in 6 M HCI for 1 hr, the extract cooled, and the radioactivity extracted into diethyl ether. About 75% of the extracted radioactivity migrated with DCVA after TLC. The rest of the ether extracted radioactivity from refluxed band H4 had an Rf similar to that of band H3. Five major metabolite bands were also
PYRETHROID
PENETRATION
Distance
from
origin
AND
METABOLISM
(cm)
FIG. 3. Separation of [Vlpyrethroid metabolites. Insects were dosed with 5.0 pg of cis, trans-permethrin (A) or 2.5 kg of cis, trans-cypermethrin (II) and the radioactive metabolites separated by TLC in formic acid-saturated toluene:diethyi ether (IOJ, by vol).
chromatographically separated from the bodies of larvae dosed with 2.5 ,ug of cis, truns-[14C]cypermethrin (Fig. 3B). Bands 1 and 2 cochromatographed in five solvent systems with cypermethrin and DCVA, respectively. Bands 3 -5 were qualitatively identical with permethrin metabolite bands 3-5.
Effects of Dose Size and Composition Metabolism
on
The distribution of radioactivity among the five main metabolite bands was analyzed at time intervals up to 24 hr after dosing for 0.5- and 5-pg doses of cis-, trans-,
Amount
of Unmetabolized
Pyrethroid
IN
PORINA
and l:l, cis, trans-permethrin and for 0.5 and 2.5~pg doses of cis-, tram-, and l:l, cis, trans-cypermethrin. Results for the proportion of unmetabolized pyrethroid 3 and 24 hr after dosing are summarized in Table 2 and results for 2.5 pg ciscypermethrin are presented in Fig. 4. The amounts of unchanged pyrethroids in the larvae were calculated from the penetration data and from the results of chromatographic separation of unchanged pyrethroids and metabolites. For permethrin greatest differences occurred 3 hr after a 5-pg dose (Table 2). trans-Permethrin accumulated in larger amounts than the more slowly penetrating cis isomer and cis, tram mixture suggesting that under these conditions metabolism was the rate-limiting process regulating body levels of unchanged permethrin. Highest body levels of cypermethrin 3 hr after a 2.5~pg dose were achieved with the cis isomer. As penetration rates of the cis and trans isomers were similar (Fig. 2) decreased hydrolysis of cis-cypermethrin may account for the greater body levels of the cis compound (Table 2). At longer time intervals (Fig. 4) decreasing body levels of cis-cypermethrin were accompanied by increased amounts of DCVA and other metabolites. When piperonyl butoxide or carbaryl dissolved in 1 ~1 n-heptane was topically applied to larvae 10 min before topical administration of 0.5 pg of cis, trans-permethrin the body levels of permethrin were significantly higher at 5 and 24 hr after dosing compared with control larvae only
TABLE 2 in the Body Amount
at 3 and 24 hr after of pyrethroid”
Permethrin Permethrin Cypermethrin Cypermethrin o Results
(0.5 cc.g) (5.0 pg) (0.5 pg) (2.5 pg) are means
cis 0.08 0.61 0.09 0.51
-rr -+ 2
2 SD for groups
0.07 0.89 0.13 0.35
+ + 2 ‘-
of five
Dosing
24 hr
trans 0.01 0.11 0.01 0.09
Topical
(pg)
3 hr Compound
201
CATERPILLARS
cis, trans 0.02 0.08 0.02 0.06 insects.
0.10 0.68 0.13 0.45
k f % 2
0.02 0.08 0.02 0.08
cis 0.02 0.59 0.08 0.48
+ k 2 k
trans 0.01 0.19 0.02 0.10
0.03 0.69 0.07 0.32
k f k 2
cis. tran.7 0.01 0.18 0.02 0.12
0.03 0.56 0.07 0.36
k 2 k -t
0.01 0.16 0.02 0.11
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12 Time
24 (hr)
FIG. 4. Penetration and metabolism of 2.5 pg cis-cypermethrin. Vertical bars represent standard deviations for groups offive insects. Total body pyrethroid and metabolites, R body cypermethrin, A; DCVA, 0.
administered the pyrethroid (Table 3). Piperonyl butoxide was used as an inhibitor of pyrethroid oxidation and carbaryl as an inhibitor of pyrethroid hydrolysis. Pretreatment with either inhibitor significantly increased the amount of unchanged permethrin at 5 and 24 hr. Neither compound altered the penetration of cis, truns-perme&in. Greatest effects on prolonging the lifetime of permethrin in the larvae were given by carbaryl which substantially inhibited hydrolysis of permethrin to DCVA. DISCUSSION
Our approach in this study has been to characterize the relative roles of penetration, excretion, and metabolism in the
maintenance of body levels of pyrethroids in porina caterpillars. Permethrin and cypermethrin were metabolized by similar pathways in porina larvae to those reported in other insects (4-9). The partial inhibition of metabolism by piperonyl butoxide indicates that oxidation of pyrethroids occurred in this species. However, hydrolysis, which was partially inhibited by carbaryl, was the major metabolic route. Water-soluble products were important metabolites at the longer time intervals after dosing (Fig. 3, bands 3-5). The behavior of compounds in bands 3 and 4 was consistent with that of amino acid conjugates of DCVA and possibly its 2-methyl-hydroxylated derivatives. Band 5 appears to contain conjugates of 4’-
PYRETHROID
PENETRATION
AND
METABOLISM
TABLE3 Effects of Inhibitors on Permethrin Metabolism Percentage
of dose
present
Permethrin Dose Pamethrinb Pemethrin” + 2.5 pg piperonyl butoxide PemetIuinb + 1 pg carbaryl
shr
aso
DCVA 24 hr
5 hr
24 hr
2023
11~1
2022
2121
33+2
14+1
15+1
23+1
3823
2022
1221
1221
(L Results are means + SD for groups b 0.5 wg cis. truns-pemethrin.
of 10 insects.
hydroxypermethrin and roughly equal amounts of amino acid and sulfate/glycoside conjugates of DCVA and its hydroxylation products. On the basis of our postulated identification we calculate that 20% of the body radioactivity 24 hr after dosing with 5 pg of permethrin contained hydroxylated metabolites and that 80-90% of the body permethrin had been hydrolyzed to DCVA and its oxidation products. Cypermethrin was more resistant than permethrin to hydrolysis (Table 2, Fig. 3). We calculate that approximately half of a 2.5pug cypermethrin dose was converted to DCVA and an additional 20% of the dose was metabolized to hydroxylated DCVA derivatives. Less than 2% was found as cypermethrin metabolites hydroxylated in the aromatic rings. cis-Cypermethrin was the most stable compound examined. Relative resistance of cis-cypermethrin to hydrolysis has also been reported in other insects (4, 5, 8, 9). The relative toxicity of cypermethrin compared with permethrin in porina larvae (2) may in part be attributed to the slower metabolism of cypermethrin. Variations in penetration rates were also apparent. Small doses (0.5 pg) of transpermethrin and trans-cypermethrin were consistently absorbed faster than the cis isomers but at higher doses (2.5 pg cypermethrin or 5 pg permethrin) no general trend was apparent. The interaction of penetration rates with rates of hydrolysis and oxidation appeared to be important in the regulation of body levels of unchanged py-
IN
PORINA
CATERPILLARS
203
rethroid. The relatively high levels of ciscypermethrin 3 and 24 hr after a 2.5~pg dose (Table 2) may be due to decreased metabolism of that isomer while enhanced accumulation of trans-permethrin 3 and 24 hr after a 5-pg dose appeared to be related to the fast rate of truns-permethrin penetration. After 24 hr when penetration of most isomers was slow metabolism was the major factor affecting body levels of unchanged pyrethroids (Fig. 4). Rapid penetration of topically applied pyrethroids is common in a wide range of insects (4, 5, g-10). Little excretion of pyrethroids by porina caterpillars was observed in our studies. In all cases less than 5% of the dose was voided in feces in the 24-hr experimental period. In an attempt to distinguish between unabsorbed and excreted pyrethroid we have suspended larvae by cotton threads above beakers and collected feces voided into the beakers. Porina larvae produced relatively little solid or liquid feces under these conditions and the low rate of general and pyrethroid excretion may be due to lack of feeding during, and in the period prior to, the experiment (11). However, similar experiments with another New Zealand Hepialid, the larvae of the puriri moth, Aenetus virescens, which excreted appreciable amounts of solid feces showed that topically applied permethrin and cypermethrin were not voided and were absorbed and metabolized in a manner similar to that demonstrated for porina caterpillars (C. K. Chang and T. W. Jordan, unpublished). Our studies indicate that considerable quantities of unchanged pyrethroids accumulate in the bodies of porina larvae which, however, resist poisoning by permethrin and cypermethrin. Approximately 0.5 pg of cypermethrin is found in the body 3-24 hr after dosing 2.5 pg of the cis isomer but larvae are not killed. Under these circumstances it appears that storage of pyrethroid in the body or resistance at the level of the nervous system must account for porina’s tolerance to the synthetic p’yrethroids.
204
CHANG
AND
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ACKNOWLEDGMENTS
This work was supported by the New Zealand University Grants Committee, Lottery Profits Distribution Committee, Internal Research Committee of Victoria University, and by gifts of chemicals from Shell, ICI, FMC Corporation, and Sumitomo Chemical ComPanY. REFERENCES
1. D. N. Ferro, Pasture pests, in “New Zealand Insect Pests” (D. N. Ferro, Ed.), p. 105, Caxton Press, Christchurch, 1976. 2. G. D. G. Du Toit, R. J. Townsend, and S. J. Armstrong, Lack of response in porina (Wiseana sp: Hepialidae) caterpillar to treatment with pyrethroid insecticides, N.Z.J. Exp. Agr. 6, 175 (1978). 3. T. M. Priester and G. P. Georghiou, Crossresistance spectrum in pyrethroid-resistant Culex quinqefasciatus, Pestic. Sci. 11, 617 (1980). 4. J. S. Holden, Absorption and metabolism of permethrin and cypermethrin in the cockroach and the cotton-leafworm larvae, Pestic. Sci. 10,295 (1979). 5. W. S. Bigley and F. W. Plapp, Jr., Metabolism of
6. 7. 8.
9.
10.
cis and trans-[l*C]permethrin by the tobacco budworm and the bollworm, J. Agr. Food Chem. 26, 1128 (1978). I. Yamamoto, E. C. Kimmel, and J. E. Casida, Oxidative metabolism of pyrethroids in housetlies, J. Agr. Food Chem. 17, 1227 (1969). J. Miyamoto and T. Suzuki, Metabolism of tetramethrin in houseflies in vivo, Pestic. Biochem. Physiol. 3, 30 (1973). T. Shono, T. Unai, and J. E. Casida, Metabolism of permethrin isomers in American cockroach adults, housefly adults, and cabbage looper larvae, Pestic. Biochem. Physiol. 9, 96 (1978). D. M. Soderlund, Pharmacokinetic behaviour of enantiomeric pyrethroid esters in the cockroach, Periplaneta americana L., Pestic. Biothem. Physiol. 12, 38 (1979). F. P. W. Winteringham, A. Harrison, and P. Bridges, Absorption and metabolism of [14C]pyrethroids by the adult housefly, Musca domestica L., in vivo, Biochem. .I. 61, 359 (1955).
11.
T. W. Jordan, C. K. Chang, and J. N. Smith, Factors affecting the metabolism, distribution and excretion of aromatic acids in Periplaneta americana and Acanthoxyla intermedia, Insect Biothem. 10, 265 (1980).