METABOLISM OF PERMETHRIN BY THE COMMON GREEN LACEWING, CHRYSOPA CARNEA STEPHENS.

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Bashir, Nabil Hamid Hassan

METABOLISM OF PERMETHRIN BY THE COMMON GREEN LACEWING, CHRYSOPA CARNEA STEPHENS

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University Microfilms International

METABOLISM OF PERMETHRIN BY THE

COMMON GREEN LACEWING, CHRYSOPA

CARNEA STEPHENS

by

Nabil Hammid Hassan Bashir

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF ENTOMOLOGY

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1 9 8 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have read

the dissertation prepared by Nabil Hamid Hassan Bashir

entitled Metabolism of Permethrin by the Common Green Lacewing, Chrysopa carnea Stephens

and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy

Date

Date

Date 'i" , (~/:-. i- I, _,( (L. ( _ Cfi L ( L /J. /,j J) Date

Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

Date STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to bor­ rowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or re­ production of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the in­ terests of scholarship. In all other instances, however, permission must be obtained from the author.

r7~---:r<, SIGNED: J\~ ------~.~~~~~------

... --.-.. ,~~ . ACKNOWLEDGMENTS

It is gratifying for me to have a chance to work under the

supervision of Professor L. A. Crowder. In particular, I would like

to thank him for his patience, understanding, reviewing the manuscript,

discussing the data which were largely contributed to the integrity

and achievement of this work.

Thanks are also due to Professor Dean Carter and Dr. G. Sipes

of the Department of Toxicology for their suggestions and support.

For Professor G. W. Ware, the Head of the Entomology Department, and

Professor W. L. Nutting all the gratitude is due for serving on my

committee.

Above all, I wish to express my gratitude to the People of the

Democratic Republic of the Sudan and the University of Gezira, Wad

Medani, for their financial support throughout this work despite the

difficulties that are confronting the country's economy. I hope that

one day I will be able to return the favor.

Thanks are due to Betty Estesen and Norman Buck of the Depart­

ment of Entomology for their invaluable technical and emotional support.

I wish also to express my gratitude to every member in the Department

of Entomology, staff, secretaries, and graduate students for making me

feel at home.

For my parents, my wife Dr. Nafisa Khalid Musa, and all

brothers and sisters; I cannot say enough, at least that is what

iii iv families are for. Thank you all and thank God for making you a very important part of my life, BIOGRAPHICAL SKETCH

Nabil H. H. Bashir is the first born of eight children for Mr.

Hamid Hassan Bashir (Sudanese) and Mrs. Olfat R. A. Moharam (Egyptian).

Nabil was born on March 19, 1949 in EI-Obied, Kordofan Province.

He obtained his High School Diploma from Khartoum Egyptian

Secondary School in 1967. He finished his undergraduate studies at the University of Alexandria, College of Agric., Dept. of Entomology in 1971. In 1971/72 he joined the Plant Protection Dept., Ministry of Agric., in the Sudan. He obtained his M.Sc. in 1974 from the Dept. of Entomology, College of Agric., University of Alexandria under the supervision of Professor A. Y. EI-Shazli, and his topic was "Studies on Esterases of the Cotton Leafworm, Spodoptera littoralis (Bois.)

After working for the Agric. Res. Corp. in the Sudan, as an

Entomologist for 18 months, Nabil joined the University of the Gezira,

Wad Medani, Sudan as a Teaching Assistant in October, 1977 and granted a scholarship to finish his Ph.D. at the University of Arizona,

Tucson, to qualify him as an Assistant Professor in Insect Physiology and Toxicology.

He married Dr. Nafisa Khalid Musa (Physician) on March 19,

1981. No children yet.

v TABLE OF CONTENTS

Page

LIST OF TABLES . . • •

LIST OF ILLUSTRATIONS . . -xi

ABSTRACT. ·xii

LITERATURE REVIEW 1

Green Lacewing 1 Description 1 Life History • 2 Mass Rearing 3 Insecticide Tolerance 4 Pyrethroids .•••••. · 11 General • • . . • • . .11 Synthetic Pyrethroids 12 Mode of Action of Pyrethroids .• 14 Structure-Activity Relationships 18 Metabolism of Pyrethroids .21 Resistance to Pyrethroids 30 Summary and Statement of Problem • 34

MATERIALS AND METHODS 36

Insect Rearing • . 36 Metabolism of Permethrin Studied ---in vivo . 36 Metabolism of Permethrin Studied in vitro • 43 Comparison of C. carnea and H. viresens 43 Metabolism by ~. carnea 45

RESULTS 47

Metabolism of Permethrin by Larvae of C. carnea Studied in vivo ...... 47 ---Recovery Efficiency 47 TLC of Standards . . 49 Metabolism of Permethrin by Chrysopa carnea Studied in vivo • 56 CiT •••••• 56 Cis-Permethrin . . • • . . . · 61 ---Trans-Permethrin • • . • . . 65 Metabolism of Permethrin Studied in vitro: Comparison of Chrysopa carnea and Heliothis vfrescens •• 69

vi vii

TABLE OF CONTENTS--Continued

Page

Esterases plus Oxidases • • • • • 69 Oxidases Alone . • • • • 71 Esterases Alone • • • • • . 71 Metabolism of Permethrin by ChrYSOya carnea Studied in vitro 76 Metabolites of Permethrin (CIT • • • • 76 Summary of CiT 81 Metabolites of cis 81 Summary of cis 84 Metabolites-oi trans 84 Summary of Trans • • • • 90 Comparison of the Two Enzyme Systems in vitro • 90 Comparison of the in vivo and in vitrO-Metabolism of Permethrin by ~hr~ carnea TLC • 90 Permethrin (CIT) •• • • •• 90 Cis • 91 Trans • 91

DISCUSSION 93

Methodology 93 Gas-Liquid Chromatography/Electron-Capture Detector 93 Efficiency of Recovery (ER) . . • . . • . • . • 93 Conjugated Compounds •.••...••.•.. 94 Metabolism of Permethrin by Chrysopa carnea Studied in vivo 94 GLC Surface Fraction 94 Cup Fraction . • • . 95 Internal Fraction .. 96 Comparison of Permethrin Metabolism by Chrysopa carnea and Heliothis virescens Studied in vitro . . • .• 98 Metabolism of Permethrin by Chrysopa carnea Studied in vivo • 102 Permethrin (C/T) • 102 Cis ...... 105 Trans . . • . • . 106 Metabolism of Permethrin by Chrysopa carnea Studied in vitro: TLC •• 107

SUMMARY •. 112

APPENDIX A: TYPICAL THIN-LAYER CHROMATOGRAMS SHOWING METABOLITES OF PERMETHRIN ...... •... 116 TABLE OF CONTENTS--Continued

Page

LITERATURE CITED . • . • ...... • . . . . . • . • ...... 124 LIST OF TABLES

Table Page

1. Extraction efficiency of permethrin isomers in the presence of Chrysopa carnea tissue • . . . • • • • • • . . • • • 48

2. Metabolism of permethrin (CiT) by larvae of Chrysopa carnea following topical application (25 larvae per repli- cate, three replicates) .•.•...•.••....•.•• 51

3.· Surface-fraction of permethrin (CiT) after dosing larvae of Chrysopa carnea with 100 ~g per larva . . • • . 52

4. Cup-fraction of p~rmethrin (CiT) after dosing larvae of Chrysopa carnea with 100 ~g per larva . . . .. • 53

5. Rf values for some standard compounds and permethrin meta­ bolites using four different solvent systems and thin-layer chromatograph • . . • . . . • . • . . . . • • . . . . 55

6. Metabolites of permethrin in the chloroform extract of Chrysopa carnea larvae within 24 and 48 hours of dosing in vivo •. · 57 7. Metabolites of permethrin in methanol and water extract of Chrysopa carnea larvae within 24 and 48 hours of dosing, in vivo • . . · 60

8. Metabolism of cis-permethrin in chloroform extract of Chrysopa carnea larva within 24 and 48 hours of dosing, in vivo •. · 62

9. Metabolites of cis-permthrin in methanol and water extracts of Chrysopa car~ larvae within 24 and 48 hours, in vivo •• 64

10. Metabolites of trans-permethrin in chloroform extracts of Chrysopa carnea larvae within 24 and 48 hours of dosing, in vivo • • ...... •• 66

11. Metabolites of ~-permethrin in methanol and water ex­ tract of Chrysopa carnea larvae within 24 and 48 hours of dosing, in vivo ...... •..• . .. .. 68

12. Metabolism of permethrin studied in vitro; comparison of Chrysopa carnea and Heliothis vir~c~30 mg tissue per test tube) ...... • ....•....•. . . 75

ix x

LIST OF TABLES--Continued

Table Page

13. Metabolites of permethrin (CiT) by Chrysopa carnea homogenates studied in vitro: esterases plus oxidases ••• 77

14. Metabolites of permethrin (CiT) by Chrysopa carnea homogenates studied in vitro: est erases alone •••••.• 79

15. Metabolites of permethrin (CiT) by Chrysopa carnea homogenates studied in vitro: oxidases alone •••.• 80

16. Metabolites of cis-permethrin from Chrysopa carnea homogenates studied in vitro: ester&ses plus oxidases . 82

17. Metabolites of cis-permethrin by Chrysopa carnea homogen- ates studied in vitro: esterases alone ••.•••••.. 83

18. Metabolites of cis-permethrin by Chrysopa carnea homogen- ates studied in-vItro: oxidases alone • • • •. ••.• 85

19. Metabolites of trans-permethrin by Chrysopa carnea homogen­ ates studied in vitro: esterases plus oxidases •..... 86

20. Metabolites of trans-permethrin by Chrysopa carne a homogen­ ates studied in vitro: esterases alone ••...••.•• 88

21. Metabolites of trans-permethrin by Chrysopa carnea homogen­ ates studied in vitro: oxidases alone •••..•••..• 89 -LIST OF ILLUSTRATIONS

Figure Page

1. Structure of standards of permethrin and its alcoholic moiety metabolites taken from a diagram by Shono et ale (1978) ...... " . . 39

2. The acidic moiety metabolites as depicted by Gaughan et ale (1977) and Shono et ale (1978) • • • . . • . . . . 40

3. Metabolism of permethrin by larvae of Chrysopa carnea studied in vivo 50

4. Typical chromatograms obtained from GLC analysis of extracts from permethrin metabolism studied in vivo 54

5. Metabolism of cis- and trans- by Chrysopa carnea and Heliothis virescens: effect of esterases plus oxidases studied in vitro • 70

6. Metabolism of cis- by Chrysopa carnea and Heliothis virescens: effect of oxidases alone studied in vitro 72

7. Metabolism of cis- and trans- by Chrysopa carnea and Heliothis virescens: effect of est erases alone, studied in vitro ...... 73

8. Typical chromatograms obtained from GLC analysis of permethrin metabolism by Chrysopa carnea and Heliothis ' virescens, studied in vitro • . • • . . • • . • . . • •• 74

xi ABSTRACT

Larvae of the common green lacewing (GLW), Chrysopa carnea

Stephens, have been reported tolerant to synthetic pyrethroid insecti- cides including permethrin (CiT) (3-phenoxybenzyl-3-(2,2-dichlorovinyl)-

2,2-dimethy1-cyclopropanecarboxylate), the trans isomer being more toxic t han cis. An investigation was performed to determine the pos- sible role of metabolism in this tolerance.

Following topical application, GLW metabolized 80% of cis and

71% of trans within two hrs. About 95% of both cis and trans were metabolized by 50 hrs.

Metabolism of CiT in vitro was compared to a susceptible in- sect, the tobacco budworm (TBW), Heliothis virescens (F.). GLW degraded cis 1.7-fo1d faster than trans, while TBW metabolized trans at a slightly higher rate than cis. When esterases and oxidases were active together or alone, cis and ~ were metabolized faster by GLW than

TBW. Metabolism of CiT by GLW was primarily oxidative with hydrolysis as a secondary mechanism. Trans is more toxic to GLW apparently be- cause of this isomer's slower rate of detoxication.

Several metabolites of CiT, cis-, and trans-permethrin were identified in studies with GLW in vivo and in vitro. It appeared that cis was metabolized more intensively than trans in vivo.

The roles of esterases and oxidases in metabolizing CIT, cis-, and ~rans-isomers were studied in vitro and the following number of

xii xiii metabolites were identified: with CiT five with esterases plus oxidases, six with esterases, and seven with oxidases alone. With cis - six metabolites were produced when esterases plus oxidases were active, five with esterases, and four with oxidases alone. With trans - est erases plus oxidases produced four metabolites, three with est erases alone, and seven with the oxidases alone. A few unknowns were exhibited in each case.

Hydroxylation at the 2'-position of the phenoxybenzyl group seems to be important for GLW tolerance to CiT. Hydroxylation could be the first step in detoxifying CiT and its isomers. The toxicity of trans to GLW could be explained by the limited routes by which ester­ ases acting alone can degrade this isomer; only three metabolites were produced with esterases while seven were produced with oxidases. LITERATURE REVIEW

Green Lacewing

Description

Although many members of the order Neuroptera are predacious, some of the most important of these beneficial insects belong to the genus Chrysopa (Neuroptera: Chrysopidae). The predominant green lace­ wing (GLW) associated with alfalfa and cotton during the summer months in Arizona is Chrysopa carnea Stephens, the COllmon California green lacewing (Werner, Moore, and Watson, 1979).

Adults of GLW are easily recognized by their fairly small size, green to yellowish-green color in summer and reddish color in winter, with longitudinal yellow stripe, and red cheek markings. The genitalia of this species are also distinctive and have been described

(Killington, 1937; Neumark, 1952; Adams, 1953; Bram and Bickley, 1963).

This species has been referred to as ~. californica Coquilett,

C. plorabunda Fitch, and ~. pedunculata var. Californica. However,~. californica was considered a synonym of~. plorabunda (Smith, 1932).

C. plorabunda was later reported in synonymy with the old world species

C. carnea (Tjeder, 1960) so that presently the names are synonymous mthGW.

GLW eggs are 0.89 mm in length, 0.39 mm in width, and stalk length 3.5 mm (Smith, 1932; Killington, 1937; Neumark, 1952). As in larvae of many species of chrysopids, coloration is frequently

1 2 diagnostic (Smith, 1922; Killington, 1937; and Neumark, 1952). The aforementioned authors also reported the "cocoon as white, almost spherical complete on all sides, thin walled and somewhat transparent,

3.29 rom in length and 2.64 rom in width."

Life History

N~~umark (1952) reported that the minimum time for the preovi­ position pe~iod was four days under his laboratory conditions. Rarely was more than one egg found on a leaf in nature. The incubation period in the laboratory under varying conditions was given by Toschi (1965).

Hatching is aided by means of an egg burster; the process was described by Smith (1922). After descending from the stalk, the larvae feed im­ mediately. If some food was not obtained within 24 hours, the larva would not survive. Larvae were also seen to attack each other. Larvae can feed on aphids, psocids, mites, scale insects and first and second instars of some lepidopterous insects (Toschi, 1965; Finney, 1950;

Wildermuth,1916). The average length of time between ecdyses for GLW in the laboratory under varying conditions of light, temperature, and humidity was six days for the first, four days for the second, and five days for the third stage until spinning (Toschi, 1965). On the average, four days after the cocoon was spun, the last larval exuviae were shed.

The adult emerges after an average of ten days through a circular split in one end of th~ cocoon. Emergence occurs in the evening and in the morning. The pharate adult crawls around for a short period before undergOing ecdyses to the imago (Toschi, 1965). The final ecdyses was one of the most critical stages in laboratory rearing of lacewings

(Smith, 1922). 3

GLW overwinters in the adult stage. About the first of October the adults assume a yellowish-brown color. The abdomen becomes promin­ ently marked with median red spots (Toschi, 1965). Tauber and Tauber

(1972) studied two strains of GLW, one originating from New York and the other from Arizona. The strains displayed marked differences in both initiation and intensity of the facultative reproductive diapause.

The northern stock had a critical photoperiod one hour longer than the southern one. The longer critical photoperiod of the northern popu­ lation was concluded by these authors to allow entering diapause earlier in the season and adapting to the long day lengths of late summer and early-occurring winter conditions at higher latitudes.

Tauber and Tauber (1972) stated also that GLW's ability to adjust to both: 1) critical photoperiod and diapause depth, and 2) to different photoperiods and conditions found at different latitudes, probably promoted the species broad latitudinal distribution.

Mass Rearing

Finney (1948), who was the first to develop methods for the mass rearing of GLW for biological control, found that a diet consist­ ing solely of honey resulted in a very low level of oviposition, but when this food was supplemented with a coccid honeydew a much higher level was achieved. Neumark (1952) was able to obtain high levels of oviposition using aphid honeydew as the sole food, while Sundby

(1966, 1967) was successful in maintaining adults and securing reasonable numbers of eggs from females which were fed upon diet of honey and pollen. 4

Tulisalo, Tuovinen and Kurppa (1977) reported on larvae of GLW being fed with adult Sitotroga cerealella Olivo ~ The average number of eggs per female was about 900 with about 84% egg hatch. In mass rearing 12 mg of food were used per larva daily, i.e., three times the quantity required in individual rearing.

Morrison, House and Ridgway (197S) designed an improved SOO cell unit for production of GLW. Results of 839 units tested over a

2S-week period showed an average 62% of the cells produced adults.when frozen eggs of ~. cerealella were used as the larval diet.

An improved procedure for rearing GLW was developed by Ridgway, R Morrison and Dadgley (1970). A paper, a Hexcel , was used in fabric- ating a larval rearing unit provided an inexpensive method for isolat- ing larvae in individual compartments to prevent cannibalism. The technique required very little mechanization and provided a method by which three technicians could rear enough adults to produce as many as

7S0,000 per day.

Insecticide Tolerance

~. Bartlett (1964) tested 60 commercial pesticide formula- tions in the laboratory for toxicity to GLW at dosages similar to those used in orchards. Eggs were treated directly with a spray of the pesticide. Egg hatch was delayed by parathion (O,O-dimethyl o-p- nitrophenyl phosphorothioate); other materials had little effect.

Parathion caused a full day's delay in egg development even though it did not cause death. Helgesen and Tauber (1974) studied the effect of pirimicarb (S,6-dimethyl amino-4-pirimi-dinyl dimethyl carbamate), and 5 its selectivity against the green peach aphid, Myzus persicae (Sulzer), and one of its predators, GLW; there was good egg hatch in GLW.

Larvae. Ahmed et al. (1954) determined that all stages of larvae of C. oculata and C. rufilabris were practically immune to systox (O,O-diethyl-O-2-ethyl-mercaptoethyl thiophosphate) when fed cotton aphids pOisoned by the material. Later, Ahmed et al. (1955), mentioned that larvae and adults of all tested species (four predatory species) except~. vulgaris were highly susceptible to systox when fed on aphids treated by a dipping method. On the other hand, aphi.ds killed by schradan (octamethyl pyrophophoramidate) were generally not as poisonous as those killed by systox.

Larvae of GLW are highly resistant to DDT (1,1, l-trichloro-2,

2-bis-(1-chlorophenyl) ethane) (Putman, 1956; Van den Bosch et al.,1956;

Fleschner and Scriven, 1957; Bartlett, 1964; Herne and Putman, 1966),

Methoxychlor (1,1,1-trichloro-2,2-bis (P-methoxyphenyl) ethane)

(Bartlett, 1964). Larvae were relatively tolerant to a wide variety of insecticides tested: Endrin (1,2,3,4,lO,lO-hexachloro-6,7-epoxy-1,

4,4a,5,6,7,8,8a-octahydro-1,4-endo-endo-5, 8-dimethanonaphtha1ene)

(Van den Bosch et a1., 1956), Toxaphene (67-69% chlorinated camphene)

(Van den Bosch et a1., 1956; Bartlett, 1964; Wilkinson, Biever and

Ignoffo, 1975), sulfur (Van den Bosch et al., 1956), DDT and sulfur

(Van den Bosch et a1., 1956), Malathion (O,O-dimethyl S-(1,2- dicarbethoxyethyl) phosphoro-dithioate) (Van den Bosch et al., 1956;

Hamilton and Kieckhefer, 1969; Lingren and Ridgeway, 1967), Parathion

(O,O-dimethyl S-(methyl carbamoylmethyl) (Bartlett, 1964), Dioxathion,

Naled (1,2-dibromo-2,2-dichloroethyl dimethyl phosphate) (Bartlett, 6

1964), Trichlorfon (dfmethy1 (2,2,2-trich1oro-1-hydroxyethy1) phosphate)

(Bartlett, 1964), Nicotine sulfate solution (Bartlett, 1964).

Lingren and Ridgeway (1967) demonstrated that Bordeaux among the fungicides showed no toxicity to larvae as did calcium arsenate and the botanical rotenones; toxaphene almost completely ~topped further growth of the larvae surviving treatment. Methyl parathion (0,0- dimethyl o-p-nitropheny1 phosphorothioate) and Bidrin (Dimethyl cis-

2-dimethy1-carbamoyl-1-methylviny1 phosphate) were more toxic than the other materials to all predators except to larvae of GLW; Phosphamidon

(0, O-dimet hy1-0-(2-ch1oro-2-dimethy1 carbamoy1-1-methy1-viny1) phos­ phate) was more toxic than Bidrin.

In a study by Bartlett (1964) 'with 60 commercial pesti- cide formulations previously mentioned, high larval tOXicity was shown by Aldrin (1,23,4,10,10-hexach1oro-1,4,4a,5,8,8a-dimethanonaphtha1ene),

Chlordane (1,2,4,5,6,7,8, 8-oct ach1or-2 ,3, 3a, 4,7 ,7 a-hexahydro-4 ,7- methanoindane), Dieldrin (hexach1oro-epoxy-octahydro-endo-exo­ dimethanora phthalene), Endosu1fan (6,7,8,9,10,10-hexach1oro-1,5,5a,

6,9,9a-hexahydro-6,9-methano-2,4,3-benzo(e)-dioxathiepin-3-oxide),

Endrin (hexach1oroepoxyoctahydro-endo-endo-dimethanonaphtha1ene), and

Heptachlor (1,4,5,6,7,a,8-heptach1oro-3a,4,7,7a-tetrahydro-4,7- R methanoindene). Di1ane (mixture of one part 1,1-bis (p-1,1-bis

(p-ch1oropheny1)-2-nitrobutane) and Lindane (1,2,3,4,5,6,-hexach1oro­ cyc1ohexane) were immediate in their effects.

Lingren and Ridgway (1967) discovered that Trich1orofon was less toxic than any of the other materials tested (i.e., demeton, 7 phosphamidon, bidrin, and methyl parathion) to larvae of Chrysopa spp.

Phosphamidon (O,O-dimethyl)-O-(2-chloro-diethylcarbamoyl-l-methyl­ vinyl) phosphate) was more toxic than Bidrin. Helgesen and Tauber

(1974) studied the effect of pirimicarb and its selectivity against

~. persicae and GLW. These workers concluded that pirimicarb was highly toxic to the pest, but it was not toxic to GLW. Wilkinson et al. (1975) studied eight pesticides, methyl parathion, malathion,

toxaphene, carbaryl (l-naphthyl N-methyl carbamate), pyrethrin,

Bacillus thuringiensis, nuclear polyhydrosis virus (NPV), and 2,4-DEB

[(mixture of tris (2,4-dichlorophenoxyethyl) phosphite and bis (2,4-

dichloro-phenoxyethyl) phosphite)]. These compounds were evaluated

at the minimum recommended field dose and reduced dosages for contact

toxicity to many parasites and predators including GLW. The GLW was

the most tolerant species to the chemical treatments and larvae were more tolerant than adults. !. thuringiensis caused no mortality to

larvae, and the same was true for NPV.

Van Steenwyk et al. (1975) studied the effect of Monocrotophos

(dimethyl phosphate of 3-hydroxy-N-methyl-cis-crotonamide), Methomyl

(s-methyl-N-(methyl cardamoyl) oxy) thioacetimidate), Dicrotophos

( .. Bidrin), Chlorodim~form (N'-(4-chloro-o-tolyl)-N,N-dimethyl

formamidine), and AZinophosmethyl (O,O-dimethyl 5-4-oxo-l,2,3-benzo­

triazin-3(4H)-YL methyl phosphorodithioate) on Heliothis spp. in

cotton under various insecticidal treatments. They stated that GLW was least affected by increased insecticide usage. From their results

one can conclude that the larvae were more tolerant than the adults to

all treatments even by using combinations of different insecticides. 8

Summers, Coviello and Cothran (1975) determined Chrysopa spp. R were relatively tolerant to Pirimicarb, Methomyl, Vydate (5-methyl l-(dimethyl carbamoyl)-N-(methyl carbamoyl)oxy) thioformamidates) and

Demeton. These authors also stated they would not classify Pirimicarb as "non-toxic" to any of the Chrysopa spp. evaluated, but it was ob- viously less toxic than Methomyl and Vydate and generally less toxic than Demeton. R Diflubenzuron CDimilin , 1-(4-chlorophenyl) 3-(2,6-difluoro- benzoyl) urea) is an insect growth regulator demonstrated to inhibit chitin synthesis in a broad range of insect species (Jakob, 1973;

Post and Vinet, 1973; Granet and Dunbar, 1975; Ables, West~and Shepard,

1975; Schaefer, Wilder and Mulligan, 1975; Moore and Taft, 1975).

Keever, Bradley,and Ganyard (1975) studied the effect of diflubenzuron on field populations of various predators in cotton including Chrysopa spp. Their results showed no significant reduction of predator popu- lations in diflubenzuron-treated fields.

Representatives of different classes of insecticides were tested by Plapp and Bull (1978) for toxicity to larvae of GLW. Most organophosphate insecticides and the carbamate, Methomyl were highly toxic. Carbaryl, Chlorodimeform and several pyrethroids (20% pyre­

. R thrins; Pydr~n (cyano(3-phenoxyphenyl)methyl 4-chloro-alpha-(1- methylethyl) benzene acetate); permethrin «3-phenoxyphenyl)methyl

(+ cis, trans-3-(2,2-dichloroethenyl)-2,2-dimethyl cyclopropane carboxylate); and NRDC 161 (Decamethrin; (S)-a-cyano-3-phenoxybenzyl l-CR)-cis-3(2,2-dibromovinyl)2,2-dimethyl-cyclopropanecarboxylate) and several organochlorines were much less toxic. In comparison with 9 the tobacco budworm, (TBW) , Heliothis virescens (F.) phosphorothionate·

(paS) insecticides were highly selective against GLW. In contrast, synthetic pyrethroids, and endosulfan were highly selective against

TBW. Most other insectides were of similar toxicity to both species or slightly more toxic to GLW. Synthetic pyrethroids proved to be the most selective against TBW of any insecticide tested. Decamethrin, the most selective of these, was 70 times more toxic to TBW than to GLW at R the LC and 200 times as toxic at the LC • Pydrin and permethrin 50 90 were also less toxic to GLW than to TBW. These data supported the previous observation of Bartlett (1964b) and Lingren and Ridgway (1967) that trichlorofon is quite safe for Chrysopa. Bartlett (1964a) re­ ported cyclodiene insecticides, particularly endosulfan, quite low in toxicity to Chrysopa. Plapp and Bull (1978) did not have an explana- tion for this contradiction.

Tolerance to the synthetic pyrethroids was shown in the third instar larvae of GLW by Shour (1979). Range-finding studies revealed R R that larvae could tolerate a wide range of doses of Pydrin , Pounce ,

Cis-permethrin, Trans-Permethrin, and Ambush. Larvae exhibited marked tolerance to all pyrethroids over a 72 hr period when dosed topically with 250 ~g insecticide per insect. This investigator also observed larval survivorship and adult emergence, fecundity, and longevity. He showed that GLW were tolerant to pounce but not to

Pydrin at the 1000 ~g per g level through one generation. The ED50 value (paralysis, failure to pupate, Kd, and mortality) for Pydrin was

100 ~g per g whereas the ED50 for the other compounds was greater than 25,000 ~g per g. 10

Adults. Fleschner and Scriven (1957) proved that C. californica adults laid more eggs on citrus trees growing in the soil to which DDT had been added than they did on trees growing in untreated soil.

Bartlett (1964) tested adults of GLW one-day-old residues of 60 Com- mercial pesticide formulations. The pesticides were rated as having high, medium, low, or 0 toxicity. Among the fungicides Bordeaux showed an unexpected toxicity to adults but not to larvae, as did cal- cium arsenate among the stomach poisons and otenone among the botan- icals. In general, adults ·iere more susceptible than larvae to the various toxicants with no materials being moderately toxic to larvae without having a corresponding effect upon adults. This author stated that death of adults without severe effect upon larvae was partic­ R ularly evident with DDT, TOE, Methoxychlor, Dilane , dimethoate, and

Dioxathion, and this toxicity difference occurred to a lesser extent with many of the other materials tested. Few contact insecticides were completely harmless to all stages of this insect. Those most nearly approaching this ideal would seem to be Nicotine sulfate solu- tion and Trichlorfon. Lingren and Ridgway (1967) discovered that

Trichlorofon was less toxic than Demeton, Phosphamidon, Bidrin, and

Methyl parathion to adults of Chrysopa spp.

The Hamilton and Kieckefer (1969) results clearly demonstrated that with Malathion the immature stages of aphid predators had higher

LD50 values than adults. Helgesen and Tauber's (1974) studies with

Pirimicarb demonstrated the ability of treated GLW adults to emerge and survive through the preoviposition period. Wilkinson et al.

(1975) reported larvae of GLW were more tolerant to Methyl parathion, 11

Malathion, Toxaphene, Pyrethrin, l!. thuringiensis, NPV, and 2,4-DEB than' adults. l!. thuringiensis caused 3.3% mortality to adults; no mortality was noted with NPV nor 2,4-DEB. No significant difference in fecundity ,) was detected when Keever et al. (1977) used iflubenzuron against GLW adults. Bartlett (1966) concluded that adult predators are ordinarily more susceptible to pesticide poisoning than their immature stages.

Pyrethroids

General

Pyrethrins, Cinerins, and Jasmoline, collectively called pyre- thrum, are naturally occurring chemicals, extracted from African chrysanthemums. They have been known for over a century to be very potent insecticides (Coats, Coats and Ellis, 1979). Matsumura (1975) stated the following: "Pyrethrum is found in the flowers of plants belonging to the family Compositae and the genus Chrysanthemum. The

I species which possess a high enough toxic content to be used for manu- facture are C. cinerariaefolium and C. coccineum. Originally pyrethrum flowers came from Yugoslavia and Japan, but Kenya now supplies most of them. Kenya flowers contain an average of 1.3% pyrethrins, reaching

3% in selected strains; Japanese flowers contain 1% and Yugoslav flowers 0.7%."

The natural pyrethroids are valued for their lethal action against a wide range of insect species; in a number of respects they are superior to many insecticides in current use. Changes in the

structural constitution and stereochemical detail of the pyrethroids occur rapidly when they are exposed to light and heat, acid or base, 12 and microbial activity -(Otieno and Pattenden, 1980). These conditions are met when tha insacticides are used in the control of insects, and also during the refining process leading to the preparation of commer­ cial products. In spite of the superior environmental qualities, the general stability of the natural pyrethroids has considerably restricted their development as all purpose plant protection agents. During the past decade, a number of studies have been madE! of t.he, fundamental chem­ ical processes involved during the degradation of the natural pyreth­ roids. Bu1livant and Pattenden (1976) studied the solution photochemistry of naturally derived (IR)-trans-Chrysanthemic acid; Ueda and Matsui (1971) investigated ditert-butyl ester. Irradiation of thin films of rethrin was reported by Chen and Cas ida (1969). Rethrolones were studied by Bullivant and Pattenden (1973), Pattenden and Storer

(1974), Elliott (1965). Rethrins were investigated by Bullivant and

Pattenden (1973); Nadaka, Yura and Murayama (1971, 1972) dealt with chrysanthemic acids.

Synthetic Pyrethroids

Since the chemical structures of the insecticidal components of pyrethrum extract were elucidated by La Forge and Haller (1936), a num­ ber of workers have studied analogous ester of chrysanthemic acid, the so-called synthetic pyrethroids. The first successful development of allethrin by La Forge and BartheL (1947a) and La Forge and Soloway

(1947b) stimulated studies on synthetic pyrethroids. However, the initial studias were undertaken primarily to improve the chemical stab­ ility of natural pyrethrins and to reduce the cost of insecticides l3

'based on them, improvement of their activity was only a secondary goal

(Nishizawa,197l). Actually, in an oil-based formulation or in an aerosol, the knockdown (Kd) and kill effects of allethrin, even when synergized with piperonyl but oxide (PB) , were inferior to those of pyrethrins, even though allethrin was more stable to heat and light than were pyrethrins. However, this increased stability of allethrin made it superior to the natural pyrethrins in both kill and Kd effects for use in mosquito coils, and it has been widely used in this way in

Japan and south east Asia. Other synthetic pyrethroids are superior to the natural pyrethrins in both kill and Kd effects in aerosols and in mosquito coils. These include tetramethrin (1-cyclohexene-l,2- dicarboximidomethyl-2,2-dimethyl-3(2-methyl propenyl)-cyclopropane carboxylate) (Kato, 1965), resmethrin (5-benzyl-3-furyl) methyl-2,2- dimethyl-3-(2-methyl propenyl) cyclopropanecarboxylate) (Elliott,

1967) • prothrin, and proparthrin which have made a great contribution to progress in this field (Nishizawa, 1971). In a study by Nishizawa

(1971), tetramethrin showed a very high Kd effect and resmethrin showed a remarkable kill effect in oil-based formulations. He also stated: "The efficacy of a suitable formulation of a mixture of tetra­ methrin and resmethrin surpassed that of natural pyrethrins synergized with PB." The same author also reported that "optically active" synthetic pyrethroids always showed superior Kd activity and higher killing activity than optically inactive compounds.

Two highly insecticidal esters of 3-phenoxybenzyl alcohol, permethrin (CiT) (Elliott et al., 1973a) and phenothrin (Fujimoto et al., 1973), differ in the following respects: CiT contains a 14 dichlorovinyl group and phenothrin an isobutenyl group in the acid moiety; the CiT isomers are more potent and longer acting than the corresponding phenothrin isomers (Elliott et al., 1973b; Burt and

Goodchild, 1974; Gaughan, Unai and Casida, 1977).

Mode of Action of pyrethroids

When an insect is intoxicated with pyrethroids, it quickly de­ velops hyperexcitation and tremors, which are followed by paralysis.

These symptoms of poisoning imply that pyrethroids act primarily on the neuromuscular system (Narahashi, 1971). It has in fact been shown that, when applied directly onto the excised tissue, pyrethroids cause hyperexcitation and block of the nerve (Lowenstein, 1942; Welsh and

Gordon, 1974; Yamasaki and Ishii, 1952).

As will be described later, the nerve excitation is not direct­ ly dependent upon metabolic energy. Therefore, pyrethroids can be assumed to act directly on the nerve membrane where excitation takes place (Narahashi, 1971). As a matter of fact, no enzymatic inhibition by pyrethroids is known to account adequately for the neurotoxic action.

Since the change in membrane potential is the principal parameter that can be observed during nerve excitation, the mechanism of action of pyrethroids can best be studied by means of electrophysiological tech­ niques (Narabashi, 1971).

Narahashi (1962a,b) was the first to utilize electrophysiolog­ ical techniques to study the action of pyrethroids. Three major ac­ tions of allethrin on the giant axon of the cockroach, Periplaneta americana L., were revealed: 1) at a low concentration (l~M) 15 - allethrin augmented and prolonged the negative after-potential that followed the spike phase; 2) when the temperature was relatively high o (25 C or above), repetitive after-discharges were superimposed on the increased negative after-potential. The resting potential underwent little or no change under these conditions; 3) at slightly higher con- centrations (3 ~M), the negative after-potential was augmented and prolonged, the action potential was gradually suppressed in magnitude, and the nerve conduction was eventually blocked. These three actions of allethrin - i.e., the increase in the negative after-potential, repetitive after discharge, and conduction block-can adequately account for the hyperexcitation and paralysis of the poisoned insect

(Narahashi, 1971). Narahashi also reported that nerve excitation occurs as a result of changes in nerve membrane permeabilities to sodium and potassium ions, therefore the effect of pyrethroids can be interpreted in terms of such permeabilities. Detailed analysis of the action of allethrin on the giant axons of the cockroach, the cray- fish (Cambarus virilis), and the squid (10ligo pealei) performed by means of intracellular microelectrodes and voltage clamp methods have revealed the ionic bases of the action of allethrin on the nerve.

Burt and Goodchild (1971) suggested that the neurotoxic action of pyrethrin in P. americana was associated with synaptic transmission, manifesting itself through increased post-synaptic activity. Van der

Berken, Akkermans and Van der Zalm (1973) showed that allethrin in- duced repetitive activity in the nervous system of the clawed toad, but it was confined to sensory fibprs. Moreover, the authors con- tinued, these effects were post-synaptic in origin and were proposed 16

to be induced by an effect on sodium conductance on the membrane. Using

the toxic isomer of bioresumethrin, Evans (1976) demonstrated that the

muscle fiber membrane and the neuromuscular junction were not sites of

actions for pyreth;roids in vertebrates. He stated further that "This

compound did not affect the acetyl choline release from the motor nerve

terminals, nor did it affect the kinetics of acetyl choline interac-

tion with the end-plate receptors. It was strongly suggested that the motor axons of the frogs were affected by this pyrethroid."

Spontaneous discharges were initiated by pyrethrins applied at

0.1 to 1 ppm to the peripheral nerves of the crayfish {Welsh and

Gordon, 1974), and small concentrations of pyrethrum and allethrin affected the action of cockroach heart (Naidu, 1955, 1956; Sudershan and Naidu, 1967). Narahashi (1962a, b) revealed that allethrin at con­ -6 centrations greater than 10 M unstabilized axonic conduction in cock- roach giant fibers, but studies of the spread of just-lethal doses of topically applied pyrethrin I from the cuticle of the cockroach to the nervous system suggested that the concentration of the insecticide near the nervous system was usually too little to affect axonic con- duction (Burt and Goodchild, 1971). Possible alternative sites of action were the units within the ganglia, already shown to be a source of abnormal symptoms by Lowenstein (1942). Burt and Goodchild (1971) reported spontaneous nervous activity in the sixth abdominal ganglion of the cockroach increased by much smaller concentrations of Pyrethrin

I than were needed to affect conduction in giant fiber axons, suggest- ing that the fatal lesions caused by Pyrethrin I may be within the

ganglia rather than associated with axonic conduction. 17

Toxic actions of decamethrin in mammals were first described by Barnes and Verschoyle (1974) who found that it differed from other

Pyrethroids in that no fine tremors or increase in body temperature was seen, and that symptoms (rolling convulsions) appeared to be central rather than peripheral in origin. Ray and Cremer (1979) revealed that when administered by a variety of routes, decamethrin produced a clearly defined sequence of symptoms in the rat. These involved progressively, chewing, salivation, pawing, choreoathetosis, tonic seizures, and death. The choreoathetotic symptoms were largely rever­ sible. Animals maintained adequate blood-gas values and arterial pressure until choreoathetosis was well advanced. Electron cephalo­ graph records showed generalized spike and wave discharges during choreoathetosis and auditory or somatosensory-evoked spike discharges prior to choreoathetosis-symptoms were partially antagonized by atropine.

Camougis (1973) showed that the pyrethroids have been shown to be highly lipid-soluble and not electrically charged. From this and other st~dies examined, he concluded: "The pyrethroid molecules are bound or dissolved in the lipid layer of the nerve cell membrane and thus exert their blocking effects. The dissolution in the lipid por­ tion of the nerve membranes may account for poor reversibility after washing. If the number of molecules in the nerve membrane is suffic­ iently large, interference with cationic conductances finally leads to nerve block." The author reported further that "The typical symptoms of pyrethroid intoxication of insects, Kd and paralysis, may be caus ed by excitat ion. and nerve-block at the systemic level." 18

Takeno et ale (1977) suggested that the mode of action for Kd and paralysis is different from that of death. Allethrin and tetra­ methrin which were good Kd agents, had a marked nerve stimulatory ac­ tion and poor killing ability. Permethrin (CIT) on the other hand, exhibited poor Kd and nerve stimulatory action but was shown to have had the highest killing power. Working with a crayfish nerve cord,

Nishimura and Narahashi (1978) observed that nerve potency and insec­ ticidal potency did not run parallel with each other for many pyre­ throids. The best example cited was CiT: the (+) cis, (±) cis, trans, and (-) trans forms were very effective on the nerve, whereas

(+) trans had very little nerve potency but had very high insecticidal power.

Structure-Activity Relationships

A variety of invertebrate nerve preparations were used to study the relationship of pyrethroid structure to the events of poisoning

(Burt and Goodchild, 1971, 1974; Narahashi et al., 1977; Takeno et al.,

1977; Nishimura and Narahashi, 1978; Miller, Kenedy and Collins, 1979;

Narahashi, 1962a, 1976; Gammon and Holden, 1979; Gammon, 1980; Clements and May, 1977; Adams and Miller, 1979, 1980; Blum and Kearns, 1956;

Wang, Narahashi and Scuka, 1972; Gammon, 1977, 1978; Narahashi, 1980).

The responses of relatively large diameter axons in the above cited reference may not accurately reflect poisoning in vivo since when sub­ jected to pyrethroids, such nerves fired repetitively following stimula­ tion over only fairly narrow temperature ranges; examples included giant axons in the cockroach (Narahashi, 1962), the squid (Narahashi, 1976) 19

and axons in the central-nervous system of the cotton leafworm larvae,

Spodoptera littoral is (Boisd.) (Gammon and Holden, 1979; Gammon,

1980). Housefly, Musca domestica L., flight motor neurons also fit

into this category by showing repetitive firing over only a narrow

temperature range (Adams and Miller, 1979, 1980). Because these

temperature ranges were relatively high, the pronounced negative temp­

erature coefficient of toxicity (Blum and Kearns, 1956) was once con­

sidered to be associated not with this excitatory effect but instead

with the nerve blocking action of pyrethroids which was particularly marked at low temperatures in vitro (Wang et al., 1972). However,

the neurophysiological effects of allethrin on the cockroach in vivo were excitatory at both high and low temperatures with temperature­

dependent difference in the types of nerve directly affected, i.e.,

repetitive firing in cercal sensory and motor axons (peripheral nervous system) and in the abdominal nerve cord at 32 0 C but only in the peripheral nervous system at l50 C; discharges in the central ner­ vous system being a secondary effect reproducible by physical stress.

This indicated that the negative temperature coefficient of allethrin

toxicity could be a result of greater sensitivity of certain types of peripheral nerves to repetitive firing at lower temperatures of cer­

tain types of peripheral nerves to repetitive firing (Cammon, 1977,

1978). Thus, pyrethroid action on such peripheral nerves should re­ flect the whole insect response more closely than action on a central nervous system preparation (Gammon, 1980).

Gammon's (1980) investigation revealed that pyrethroids fall

into two classes based on their action on the cercal sensory nerves 20 of the American cockroach recorded in vivo and in vitro and, on the symptomatology they produced in dosed cockroaches. Type I compounds included pyrethrins, s-Bioa11ethrin, (IR, cis) Resmethrin, Kadethrin, the (IR, trans) and (IR. cis) isomers of tetramethrin, phenothrin and

CiT, and oxime B-phenoxybenzy1 ether. E1ectrophysio1ogica1 recordings from dosed individuals revealed trains of cereal sensory spikes and sometimes also spike trains from the cereal motor nerves and in the central nervous system. Low concentrations of these pyrethroids acted in vitro to induce repetitive firing in a cereal sensory nerve fo11ow- ing a single electrical stimulus. This in vitro measurement, standard- ized for evaluating structure-activity relationships, showed that IR, insecticidal isomers were highly effective neurotoxins. The most potent compounds on the isolated nerve were (IR, trans)- and (IR, cis) 3 tetramethrin) each active at 3 X 10- M. The symptomatology of type I compounds in cockroaches were restlessness, incoordination, hyperac- tivity, prostoration, and paralysis.

Type II compounds, according to Gammon et a1. (1981), included

(IR, cis, as) and (IR, trans as) Cypermethrin (X-cyano·-3-phenoxybenzy1

-2,2-dimethyl-3-(2,2-dich1croviny1) cyclopropane-carboxylate), Delta­ methrin, and (S,S) Fenva1erate (pydrinR). These alpha-cyanophenoxy- benzyl pyrethroids did not induce repetitive firing in the cereal sensory nerves either in vivo or in vitro; moreover, they caused dif- ferent symptoms, including a pronounced convulsive phase. Two other pyrethroids with an alpha-Cyano substituent, i.e., fenpropathrin and an oxime, 6-a1pha-cyanophenoxybenzyl ether were classified as type I 21

based on their action on a cercal sensory nerve, but the symptoms with

these compounds" resembled ~ype II.

Metabolism of Pyrethroids

General. The metabolism of pyrethroids in insects was investi­

gated by several workers. Acree, Roan and Babers (1954) and

Win teringham , Harrison and Bridges (1955) demonstrated that pyrethroid esters were readily absorbed by insects and metabolized into signifi­ cant amounts of relatively non-insecticidal substances in 24 hrs.

Insecticidal chrysanthemates were oxidized at a methyl group

in the isobutenyl substituent by rats and houseflies and by rat liver and housefly microsomal oxidases (Yamamoto and Casida, 1966; Miyamoto,

Suzuki and Nakae, 1975; Casida et al., 1975/1976; Miyamoto, 1976).

This detoxification site was no longer present in highly potent pyre­ throids with the isobutenyl group replaced by a dihalovinyl group

(e.g., CIT, cypermethrin and decamethrin). Despite this type of structural modification, mouse and rat micro somes ra~idly hydroxylated the CIT isomers (Soderlund and Casida, 1977a,b,c). Rats ,quickly meta­ bolized cypermethrin isomers (Soderlund and Casida, 1977c) decamethrin

(Soderlund and Casida, 1977b). Shono, Unai and Casida (1978) reported that housefly and!. ni larvae slowly degraded the CIT isomers, in almost all cases by hydroxylation at either of methyl groups and at the 4'-position (Soderlund and Casida, 1977c; Gaughan et al., 1977;

Ruzo, Unai and Casida, 1978; Shono at al., 1978). Additional sites of hydroxylation were the 6-position of CIT isomers in houseflies

(Soderlund and Casida, 1977c), the 2 '-position of cypermethrin in rats 22

(Gaughan et al., 1977) ~ and the 2'- and 5'-postions of decamethrin in rats (Ruzo et al., 1978).

Ester cleavage was an important step in the biological and en­ vironmental degradati,on of pyrethroid insecticides (Casida et al.,

1975/76; Miyamoto, 1976; Casida, Gaughan and Ruzo, 1979; Cas ida and

Ruzo,1980). In mammals, rapid hydrolysis appeared to be a key de­ toxication step contributing to the low acute toxicity of some pyre­ throids (Abernethy and Casida, 1973; Abernethy et al., 1973; Soderlund and Casida, 1977), and the substrate specificity of liver microsomal esterases was examined in SOme detail (Abernethy et al., 1973;

Soderlund and Casida, 1977a,b). Pyrethroid ester cleavage in insects was first demonstrated with (±)-trans-tetramethrin in houseflies

(Miyamoto and Suzuki, 1973). Jao and Casida (1974a) reported the ex­ istence of pyrethroid esterase activity in acetone powder preparations of whole homogenates of several insect species and demonstrated the involvement of esterase inhibition in the synergism of (+)-trans­ chrysanthemate esters by NPC (a-naphthyl N-propyl carbamate) (Jao and

Casida, 1974b). Recent studies on the metabolic fate of permethrin and related compounds in insects both in vivo and in vitro demonstrated hydrolytic pathways in several species (Shono et al., 1978; Bigley and

Plapp, 1978; Holden, 1979; Shono, Ohsawa and Casida, 1979; Soderlund,

1979; Abd-Aal and Soderlund, 1980). In particular, hydrolysis appeared to be the major'metabolic pathway for trans-substituted esters in the larvae of lepidopterous species examined to date (Shono et al., 1978;

Bigley and Plapp, 1978; Holden, 1979). 23

Voluminous literature has been cited concerning the metabolism of allethrin (Acree et al., 1954; Winteringham et al., 1955; Bridges,

1957; Yamamoto et al., 1969), pyrethrins and Cinerin I (Zeid et al.,

1953; Chang and Kearns, 1969), tetramethrin (Miyamoto and Suzuki,

1973, Jao and Casida, 1974a, Miyamoto et al., 1968), resmethrin (Jao and Casida, 1974a, Ueda et al., 1975), S-bioallethrin (Jao and Casida,

1974a), Phenothrin (Miyamoto et al., 1975), fenothrin (Miyamoto et al.,

1974), Cypermethrin (Gaughan et al., 1980; Ishaaya and Casida, 1980;

Gaughan et al., 1977), and Fenvalerate (Gaughan et al., 1980).

Permethrin Metabolism. The TBW is resistant to essentially all insecticides registered for use on cotton (Adkisson, 1968; Nemec and Adkisson, 1969; Plapp, 1971, 1972). Studies with resistant TBW indicated resistance was due primarily to increased detoxification mechanisms involving ali-esterases, mixed-function oxidases (MFO) , and conjugating enzymes (Whitten and Bull, 1970; Bull and Whitten,

1972; Williamson and Schecter, 1970; Plapp, 1973). Gaughan et al.

(1977) reported that cis and trans-isomers of CiT were readily meta- bolized by ester cleavage in rats and the ester linkage of cis- was more stable than that of trans. They also found that oxidation occurred but was secondary in importance to ester cleavage as a de- toxication mechanism. 14 Bigley and Plapp (1978) studied the metabolism of (C )-cis- 14 and (C )- trans-permethrin by the TBW and the bollworm, Heliothis zea

(Boddie). All CIT preparations were stable in Heliothis spp. larvae under in vivo conditions, but older larvae detoxified the insecticide more rapidly than younger larvae. CiT metabolism was both hydrolytic 24 and oxidative, and trans was metabolized more rapidly than cis. Meta- bolism was more rapid in TBW than the bollworm, a factor probably responsible for the two-fold greater tolerance of the former to CiT.

The same authors stated that "Hydrolysis was relatively more important in cis and oxidation was relatively more important in trans metabolism.

Synergist pretreatments greatly suppressed permethrin metabolism and usually had less effect in the bollworm than in TBW. Retention of ab- sorbed radioactivity was lower and excretion more rapid for trans than for cis." Most of the radioactivity obtained by Bigley and Plapp from all treatments was present as unmetabolized insecticide. Phen- oxybenzyl alcohol (PBalc) was the major ether-soluble metabolite.

Metabolites soluble in ether after acidification included (2,2- dichlorovinyl)-2,2-dimethylcyclopropane-carboxylic acid (C1 CA) and 2 14 its unknowns which were probably hydroxylated derivatives from (C )_ alcohol labelled CiT. Cl CA was a major metabolite of both cis and 2 trans. The lactones were derived from cis-hydroxy-trans (C-HO,t-) or cis-C1 CA (C-CI CA). The majority of the metabolites from the alcohol 2 2 labelled CiT were hydroxylated derivatives of PBalc and PBacid.

Esterase-activity hydrolyzing both isomers of CiT was investi- gated by Abd-Aal and Soderlund (1980) in crude homogenates of the following larval tissues of the southern armyworm (Spodoptera eridania

Cramer); cuticle, gut, fat body, head capsule, Malpighian tubules, and silk gland. Neither substrate was detectably hydrolyzed by hemolymph. The highest esterase activities per insect equivalent of tissue were found in cuticle, gut and fat body for trans- and in cuticle and gut for cis. Each preparation hydrolyzed trans more 25

'rapidly, but the degree of specificity varied greatly between tissues.

Different apparent K (Michaelis constant) and Vmax (maximum rate of m formation of products or destruction of substrate by an enzyme) values

between the three most active tissues were three-fold or less for trans

hydrolysis, but differences between tissues of up to lOa-fold were

found for Km and Vmax values for cis hydrolysis. Hydrolysis of trans-

in cuticle, gut, and fat body homogenates was only partially inhibited

by NPC. Concentrations of NPC giving maximal inhibition of trans

hydrolysis had little effect on the hydrolysis of cis. Abd-Aal and

Soderlund (1980) concluded that: "These results demonstrate that

pyrethroid-hydrolyzing activity is broadly distributed in insect tis-

sues and results from the combined activity of several esterases with

different properties. It is likely that the trans and cis isomers of

permethrin are hydrolyzed by separate enzymes in insects."

Ishaaya and Cas ida (1980) found that the integument enzymes

of T. ni were less active on a per larva basis than the gut enzyme(s) 14 in hydrolyzing the (C ) CIT isomers, but were similar in activity on 14 . . the (C ) cypermethr1n ~somers. Trans was hydrolyzed two- to six-fold

more extensively than cis with both CiT and cypermethrin (and both the

gut and the integument enzymes). Gut esterase(s) hydrolyzing trans

was much more sensitive to inhibition by profenofos (0-14-bromo-2-

chlorophenyl)-O-ethyl S-propylphosphorothioate) than DEF (S,8,8-

tributylphosphorotrithioate) or carbaryl (I-Naphthyl N-methyl car-

bamate) both in vitro and following ingestion of treated lettuce discs.

The authors stated that: "Difference in sensitivity to profenofos in-

hibition indicated. the presence of more than one pyrethroid esterase 26 in both the gut and integument, the gut enzymes were more sensitive to profenofos both in vivo and in vitro with cis, and the integument enzymes in vitro with trans- isomers as substrates."

Shono et al. (1978) studied the metaboli.sm of permethrin in the American co~kroach, housefly, and cabbage looper in vivo and re- ported that degradative pathways were initiated either by ester hydrolysis or hydroxylation. They found that the preferred site for hydroxylation for all insects was the 4'-position of the phenoxybenzyl group. The authors stated that: "Metabolic detoxication was a maj or limiting factor of insecticidal activity of permethrin. Any synergist employed would have to inhibit both the est erases and oxidases present because when one pathway is blocked, the other increases its degrading activity." Thus, from these studies it was apparent that various in- sect species responded differently to pyrethroid insecticides and that stereoisomerism was also important in determining which scheme is ac- tivated within a given insect. Additional site of in vivo hydroxy- lation was the 6-position of CiT isomers in housefly (Soderlund and

Casida, 1977c).

Shono and Cas ida (1978) examined the sites of in vitro oxida-- tion of CiT isomers by enzymes of mouse, rat, trout, and carp liver, housefly abdomens plus thoraces, and cabbage looper guts. Housefly microsomes hydroxylated only the trans-methyl group of trans-, and carp and cabbage looper enzymes preferentially hydroxylated the trans- methyl group over the cis-methyl group of cis. The major site of attack in the alcohol moiety of CiT isomers was the 4'-position with 27 all enzyme preparations. Mouse and housefly microsomes also hydroxy-

1ated the 6-position of both isomers, and mouse microsomes hydroxy1ated the 2'-position of cis.

Shono et al. (1978) identified the presence of 42 insect meta- bo1ites of trans and cis. The less insecticidal trans was generally metabolized more rapidly than the more insecticidal cis, particularly in cabbage looper larvae; metabolites retaining the ester linkage appeared in larger amounts with cis. The alcoholic and phenolic met- abo1ites were excreted as glucosides and amino acid conjugates

(glycine, glutamic acid, glutamines, and serine) with considerable species variation in the preferred conjugating moiety.

Holden (1979) indicated that CiT was a relatively stable mo1e- cu1e in P. americana. Metabolism was very slow during the first three hours of poisoning and became significant only after 17 hrs when the cockroach was totally paralyzed. Trans was much more labile than cis, and inclusion of an a-cyano group as in cypermethrin virtually e1im- inated all metabolism. This was accompanied by a significant reduc- tion in the amount of cuticular penetration. The major metabolites formed from alcohol-labelled and acid-labelled CiT were identified as

3-phenoxybenzyl alcohol (3-PBa1c) and 2,2-dich1orovinyl-2,2-dimethy1 cyclopropane carboxylic acid (C1 CA). Holden's results indicated 2 that the ester-bond in CiT was the site at which the pyrethroid under- went the initial metabolic attack. His in vivo studies indicated that the overall metabolism of trans (esterase plus oxidase activity) was the same as that occurring with only esterase enzyme (approximately 28

75%). Therefore, hydrolysis was the dominant component in the meta­ bolism of this isomer. However, cis showed that 28% was metabolized in vivo compared with only 6% when using the in vitro esterase prepar­ ation. Thus, according to Holden (1979), cis, although more resistant to metabolic attack, was only slowly hydrolyzed leaving oxidative de­ gradation as a major route of metabolism. Both cypermethrin isomers underwent only oxidative metabolism. Metabolism of these two pyre­ throids in insects can therefore be both oxidative and hydrolytic, and the predominant pathway will vary, depending upon the insect and the particular isomer.

A review of studies by Cas ida and Ruzo (1980) on twenty pyre­ throids with nine different acid moieties and ten different alcohol moieties revealed a diversity of functional groups undergoing meta­ bolism in mamma~a, insects, other organisms, and microsomal esterase and oxidase systems. Seventy-nine metabolites were identified from cis and trans but fewer from other pyrethroids examined in less de­ tail. The sites and rates of metabolic attack on each pyrethroid depended on the organism or system. Metabolism of pyrethroids by esterase and oxidase action usually limited their toxicity to mammals more than insects thereby conferring useful selective toxicity prop­ erties. 3-(2,2-Dichlorovinyl)2,2-dimethylcyclopropanecarboxylic acids) derived by ester cleavage were conjugated as glucosides and with a variety of amino acids (glutamines glutamic acid, glycine, serine and taurine). The 2-methyl propyl-l-enyl substituent of the chrysanthe­ mates was susceptible to oxidative reactions but was no longer present 29

'in the trans- and cis-dichloro compounds nor in the (IR)-cis-dibromo

compound (Soderlund and Cas ida, 1977). Oxidative attack was shifted to

the geminal dimethyl group. with the degree of stereospecificity de­

pending on the isomer and the organism or oxidase system involved. The

hydroxymethyl compounds were excreted as such or underwent three types

of reactions: 1) conjugation as glucosides and sulfates; 2) lactone

formation, possibly as an artifact of analysis; or 3) oxidation to alde­

hyde and acid (Casida and Ruzo, 1980).

Elliott at al. (1976) discovered that the organochlorine moiety

of trans- and cis- and (IR. trans)- dichlorovinyl acid was rapidly and

almost completely eliminated from the rat, and only traces remained in

the fat and liver four days after oral administration. The authors

stated that: "This ease of elimination was associated with increased

polarity of the products which resulted from rapid in vivo glucuronida­

tion of dichlorovinyl acids and, to a lesser extent, hydroxylation of

one of the geminal dimethyl groups. At least some of the hydroxylated

acids underwent minor degrees of conjugation." Their studies also in­

dicated that much less hydroxylated acid was formed from trans indi­

cating that CiT was hydroxylated to some extent before hydrolysis.

The predominant sites of hydroxylation, according to Elliott et al.

(1976), appeared to be the 2-cis position for trans and the 2-trans

position for cis; presumably these methyl groups were sterically favored

by microsomal oxidase systems. The largest persistence was for prod­

ucts derived from 3-PBalc in the fat, liver, and kidney. The ratio of

conjugated 4-HO-3-PBacid to free and conjugated 3-PBacid in the excreta 30 was greater from cis- than from either trans- or 3-PBa1c. This sug­

gested that hydroxylation of the phenoxy group in the 4'-position was more important for detoxifying the less easily hydrolyzed cis than for

the more readily cleaved trans or for 3-PBalc. The authors stated

that: "If there are other sites of hydroxylation in the phenoxybenzyl moiety (leading to some of the unidentified metabolites), they must

constitute minor metabolic pathways compared with hydroxylation at the

4' -pos it ion • "

Resistance to pyrethroids

Of the 364 arthropod species (Elliott et al., 1978) in which

resistance of any sort has been reported, only six resist pyrethroids

(Georghiou, 1976), probably reflecting the limited use of these com-'

pounds compared with the major established insecticide groups (Elliott

et al., 1978; Georghiou, 1976).

Strong resistance has developed in Danish and Swedish house­

flies, which have been frequently selected with pyrethroids in the

field (Keiding, 1976) or in the laboratory (Franham, 1971, 1973;

Franham and Sawicki, 1976); but resistance has not arisen in Japanese

and California housefly populations with multiple resistance to organ­

ophosphates. It was suggested by Elliott et al. (1978) that there was

little or no cross-resistance between pyrethroids and organophosphates

in houseflies; however, cross-resistance with DDT was definitely es­

tablished in housefly (Busvine, 1951), mosquito, Culex tarsalis

(P1app and Hoyer, 1968), and possibly stable fly, Stomoxys ca1citrans

(Stenerson, 1965). 31

Four resistance factors were isolated by Franham (1973) from the NPR strain of houseflies, which resisted natural pyrethrins.

Franham stated the following: "The four factors were: pen, which re­ duces the rate of penetration of insecticides through the cuticle;

Kdr-NPR, a general pyrethroid resistance mechanism unaffected by the synergist sesamex (2-(2-ethoxyethoxy)ethyl-3,4-(methylenedioxy)pheny­ lacetal of acetaldehyde); ~-ses, a mechanism of resistance to natural pyrethrins that can be suppressed by Sesamex; and ~-ex, a factor that gives strong resistance to synergized natural pyrethrins and to the new synthetic esters, e.g., resmethrin, but little or none to natural py­ rethrin alone."

In 1977, Nolan et al. detected resistance to CiT, cypermethrin, decamethrin, and fenvalerate in a strain of cattle tick, Boophilus microplus, strongly resistant to DDT. These cases of cross-resistance were probably associated with a Kd resistance (Kdr) mechanism (Milani and Travaglino, 1957). Kdr gave moderate resistance to all pyreth­ roids examined, to DDT, and its analogs (Franham, 1971, 1973).

Recently a strain of the Egyptian cotton leafworm, ~. littoralis, which was already resistant to several insecticides, showed cross-resistance to CiT. It was not, however tolerant to cyper­ methrin. Since biochemical studies failed to find a reason for this phenomenon (Holden, 1979) a neurophysiological basis was sought by

Gammon (1980). The latter author concluded that: "Resistance to per­ methrin but not to cypermethrin appears to be based on a qualitative difference between the pyrethroids; it is unlikely that nerve blockage plays a maj or role in this resistance." 32

Nine- to fourteen-fold cross-resistance to fenvalerate and CiT was observed in TBW (Crowder et al., 1979; Twine and Reynolds, 1980).

Priester and Georghiou (1978) induced > 4000-fold resistance to trans in a strain of Culex quinguefasciatus (Say). DeVries and Georghiou

(198la) reported the results of nonmetabolic mechanisms of resistance.

A fenthion-resistant strain of the housefly, selected with biores- methrin, resulted in a ca. 90-fold resistance to the selecting agent.

This strain was subsequently selected by the above authors with trans, producing ca. 140-fold resistance to CiT. The strain was cross- resistant to several other pyrethroids, and demonstrated resistance to

Kdr by these insecticides as well as by DDT. The strain was 2.S-fold less sensitive to trans-ethanoresmethrin than the susceptible strain, and > 43-fold and> 67-fold less sensitive to cis, and trans- tetra- methrin and trans, respectively. It also displayed decreased penetra- 14 tion of trans (C ) when compared to the susteptible strain. Lower nerve sensitivity and decreased cuticular penetration were potential mechanisms of resistance to pyrethroids in houseflies in the United

States (DeVries and Georghiou, 1981a). In a following paper, the same authors (198lb) described studies aimed at determining whether or not an enhanced ability to detoxify trans- was also present in the resistant strain. The resistant-strain demonstrated twice as much cytochrome p-4S0 and cytochrome b and double the rate of NADPH-cytochrome C re­ S ductase activity than the susceptible strain. No difference between strains in the overall rate of metabolism of (C14)-trans and cis were noted. In 1981, Priester and Georghiou revealed that penetration of trans and cis was faster in the resistant strain of C. quinquefasciatus 33 susceptible strains; this was attributed to the continued activity of the mosquitoes throughout the exposure period which enhanced the meta- bolic rate in the insect. These authors concluded that penetration was not a factor in resistance to CiT in the strains studied.

Shour (1979) pOinted out that cis was the least toxic of the compounds tested against GLW larvae. These compounds were: CiT, cis, R trans, Ambush and pydrin . The author suggested that tolerance of GLW to several pyrethroids might be explained by a penetration barrier, metabolism and excretion. Approximately 25% of the parent CIT was un- extractable in the presence of tissues, possibly due to cuticular ab- sorption. However, an additional fraction (ca-60%) of the parffilt material was missing from insects dosed with the pyrethroid 48 hrs prior to homogenization. Therefore, although the penetration barrier factor cannot be totally eliminated, metabolism and excretion were ad- vanced to best explain the exhibited tolerance. Shour further referred to the 60% metabolism/excretion observed in GLW to be similar to the metabolism rate occurring in resistant TBW. He concluded that the iso- mers were metabolized/excreted at equal rates. These results were con- tradictory to the reports of several authors (Shono et al., 1978;

Bigley and Plapp, 1978; Gaughan et al., 1977). Metabolism of CiT in vivo showed that GLW degraded 63% of cis and 59% of trans within 48 hrs (Shour and Crowder, 1980).

In a recent study by Ishaaya and Casida (l98l) the tolerance of

GLW was attributed to detoxication by pyrethroid esterase(s). They demonstrated that the larval enzyme(s) has unusually high activity and 34 - a unique specificity for hydrolyzing cis (CiT) and -cypermethrin two- to three-fold faster than the corresponding trans-isomer. Deltameth- rin was also hydrolyzed rapidly. They stated that:

"Larval pyrethroid esterase(s) increases in activity for hydro­ lyzing trans- permethrin on larval growth in agreement with the increased tolerance to trans-permethrin poisoning. The rela­ tive rates of hydrolysis of deltamethrin and the cis- and trans­ isomers of permethrin and cypermethrin by the larval pyrethroid esterase(s) generally coincide with the tolerance of the larvae to these pyrethroids.

Ishaaya and Cas ida revealed that the potent inhibitor of larval pyre- throid esterase, phenyl saligenin cyclic phosphonate, synergized trans tOxicity by 68-fold from an LDSO of 17,000 ~g/g to one of 250

~g/g. The authors stated that:

"Although the involvement of penetration rates, nerve sensitiv'­ ity, and oxidative detoxification has not been evaluated, it is clear that pyrethraid esterase(s) is a major factor contributing to the natural pyrethroid tolerance of lacewing larvae."

Summary and Statement of Problem

GLW are important biological control agents. Their highly- rated searching capacity, high feeding rate, and wide predatory range make GLW excellent general predators. GLW have proved to be tolerant to a wide range of insecticides including chlorinated hydrocarbons, cyclodienes, organophosphates, and carbamates. Recently they showed tolerance to the synthetic pyrethroids.

A recent study attributed GLW pyrethroid tolerance to high esterase(s) activity as an important but probably not the only factor.

Other factors such as high MFa activity, slow penetration and insen- sitive nerve target could combine with esterase(s) to provide GLW with their high tolerance to pyrethroids. 35

Therefore, the purpose of the present investigation was to study

the metabolism of permethrin by third tnstar larvae of GLW. The fol­ lowing was to be determined; (1) The rate of metabolizing isomers in vivo; (2) the identification of metabolites, (3) the role of ester­ ase(s) and oxidases, and (4) to compare permethrin metabolism in GLW with a susceptible species, TBW, in vitro. This may aid in determin­ ing the reason(s) for tolerance of GLW to the synthetic pyrethroids. MATERIALS AND METHODS

Insect Rearing

A culture of GLW was established by collecting adults from alf-

alfa fields at the University of Arizona Agricultural Experiment Sta-

tion, Tucson, at several times of the year starting in September,

1979. The adults were immediately brought back to the laboratory,

identified, separated into groups of 50, and placed in breeding cham-

bers. The rearing procedures followed Shour (1979) with one exception

- the light:dark. cycle used was 16:8 for all stages.

For tests, third-instar larvae were collected after dismantling

the units. Larvae were kept separately in plastic cups because·of

their cannibalistic behavior, then provided with cabbage looper eggs,

!. ni, provided by USDA, Cotton Research Center, Phoenix, Arizona.

Metabolism of Permethrin Studied in vivo

Larvae were placed individually in plastic cups and held in a

refrigerator at 4°C for 45 min to reduce their activity (Shour, 1979). o The cups were removed and placed on crushed ice (6 C inside the cup)

immediately prior to dosing. The method of Hopkins et al. (1975) was

followed during dosing. The larval weight was determined with three replicates, each consisting of 100 third-instar larvae.

R Stock solutions (100 ~g per ~l) of permethrin [(Pounce, 40:60

cis/trans; (3-phenoxy-pheny1) methyl (± cis, trans-3-(2,2-dichloro-

ethenyl)-2,2-dimethylcyclopropanecarboxy1ate)], cis, and

36 37

trans-permethrin were prepared in acetone. Standard metabolites of the alcoholic moiety of permethrin were also provided by FMC Corporation

(Agric. Chern. Div., Middleport, New York, 14105). These compounds were: FMC 30062 (3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane car­ boxylic acid); FMC 30952 (m-phenoxybenzoic acid); FMC 30953 (m­ phenoxybenzoic alcohol); FMC 53813 (2'-hydroxy-phenoxybenzoic aci.d);

FMC53814 (2'-hydroxy-phenoxybenzoic alcohol)' FMC 53808 (4'-hydroxy­ phenoxybenzoic acid); FMC 53809 (4'-hydroxy-phenoxybenzoic alcohol);

FMC 53824 (4'-hydroxy, trans-permethrin); and FMC 53824 (2'-hydroxy­ permethrin).

One ~l of a stock solution was applied topically to the dorsal thoracic region of each larva and one ~l of acetone applied to each of the controls. Applications were made using a motor driven microap­ plicator (Instrumentation Specialties Co.) mounted with 1/4 cc syringe fitted with a 27-gauge needle. Four replicates (40 larvae per repli­ cate) were included for each treatment. Twenty-five out of the 40 larvae were later used for the extraction procedure. Insects were re­ turned individually to the plastic cup immediately after dosing and provided with a 2X2 em sheet of viable pink bollworm eggs, Pectinophora gossypiella (Saunders), or cabbage looper eggs. Cups were returned to the temperature cabinet for later use according to the designated treatment times: 2,3,4,5,10,15,20,25,30,35,40,45 and 50 hours after dosing.

Extraction consisted of homogenizing the treatments with a

Potter Elvehjem homogenizer mounted on a 1/50 HP motor. This was followed by counter-top centrifugation for 10 min at 2900 rpm (1019 g) 38

I. ~\ ~~ D CI,· 0 I I =,t.~6 t.¥r,/ ~ 0 ~ CI/ 3 . cis-permet:hrin (FMC 35171)

II. CI> ~ 0 0 CI ~J4.l /o~o~ oII Trans-permeth~n (FMC 30980)

lit 0t ~ ... OH HO~N II 0 o . " 4'-Hydroxy-phenoxybenzoic 4'-Hydroxy-ph~noxy benzyl acid (=4'-Ho-pBacid) "alcohol (a4'-Ho-pBalc) FMC 53808 FMC 53809

v. --.QoD

m-phenoxybenzyl alcohol (m-pBalc) FM 30952

V\' ~ 0 HO~o~ "o m-phenoxybenz-oic .'l.cid (m-pBacid) FMC 30953

Figure 1. Structure of standards of permethrin and its alcoholic moiety metabolites taken from a diagram by Shono et al. (1978). 39

VII. ~ HoAo)) o 2'-Hydroxy-phenoxy benzoic acid (2'-Ho­ pBacid) FMC 53813

Vlll.~ 0 HO I o~"'OH

2'-Hydroxy-phenoxybenzyl alcohol (2' -Ho-pBalc) FMC 53814

IX. ~~ ~ 01. e~_ j)·I ... - 0 el;' 1111. ,,/ # 0 ~ II . o 4'-Hydroxy, trans-permethrin (4'-Ho, t-permr-- FMC 53824

x ~~ ~ • el,= o~ ~"OH0 ell' Itll ,,/ 0 1\o ~'-Hydroxy-permethrin (2' -Ho-perm) FMC 53824

Figure 1., Continued. 40

0H ~"

CI, =\'~6 IIII-OH CI/ II o cis-Hydroxy, cis-dichlorovinyl Trans-Hydroxy, Dichloro- dimethylcyclopropane carboxylic . vinyl dimethyl cyclopropane acid (C-Ho, C-C12 CA) carboxylic acid (t-Ho,cl CA) 2

CI,_:\\\\6 II',~OH CI/- II o Trans-Hydroxy, dichlo~o­ cis-Dichlorovinyl dimethyl cyclo­ vinyl dimethyl-cyclopropane propane carboxylic acid (C-C1 CA) 2 carboxylic acid (t-Ho,C-Cl2CA)

:\\OH CI, CI/-- ~III,-OH II o cis-Hydroxy, cis-dichlorovinyl cis-Hydroxy,. .­ dimethylcyclopropane carboxylic Trans-dichlorovinyldimethyl acid (C-Ho, C-C12 CA) cyclopropane carboxylic acid (C-Ho, t-C1 CA). 2 Figure 2. The acidic moiety metabolites as depicted by Gaughan et al. (1977) and Shono et al. (1978). These compound were not available for this work. 41

to recover organosoluble metabolites. The precipitates were re-

extracted twice, each in 5 ml of methanol. The combined supernatants were added to 15 ml of hexane in a separatory funnel and shaken for 2 min, then 90 ml distilled water and 5 ml saturated sodium chloride solution were added and the mixture was shaken for another 2 min. The hexane layer was separated, dried through sodium sulfate, and con-

taminants removed by passing through a standard 1" (l.54 em) florisil

column. The resulting extract was then concentrated under nitrogen

and brought up to volume (10 ml) with hexane. The method modified by

Shour and Crowder (1980) was used so that gas-liquid chromatography

(GLC) could be employed for analysis instead of thin-layer chromato- graphy (TLC).

Analyses of the hexane extracts were performed on a Tracor MT 63 220 GLC using an electron-capture Nl detector (ECD) and 3'5" (43.18

em) pyrex column (ID 4mm) packed with 3% sp 2330 on 100/120 mesh chromosorb W (27% A.W., 70% H.P.) Inlet, column and detector temper­ atures were 200, 195, and 265 0 C, respectively; N2 carrier flow rate was 92 ml/min.

Quantitation was by peak height. Injection of 5 ~l analytical standards in hexane were used to prepare a standard curve before every use of the GLC. Each replicate was sampled at least twice and then averaged. The results were corrected by using the recovery efficiency coefficient (REC).

For REC determination 2500 ~g of the stock solution were added

to 15 ml hexane, homogenized, centrifuged and extracted. The forti- fied control consisted of spiking the tissues of 25 larvae with 42

2500 ~g of the stock solution in methanol upon homogenization. These

two treatments Were compared to a stock solution of 0.25 ~g per ~l

(without extraction) (Table 1).

In similar experiments to analyze metabolism of permethrin by

TLC, third-ins tar larvae were dosed with 40:60 (CiT), cis-, or trans­ permethrin. Treated larvae were held in the temperature cabinets for

24 and 48 hrs.

The extraction procedure of Bigley and Plapp (1978) was fol­ lowed. This procedure used TLC as a detection method for CiT meta­ bolites. Twenty-five larvae were rinsed 3 times each in 2 ml acetone

(after 24 or 48 hr) and this was termed "surface fraction". The larvae were homogenized with 2 ml methanol, the homogenate centri­ fuged at 4060 rpm (2000 g) for 20 min at 4°C, and the supernatant removed. Supernatants were pooled for each group and termed the "in­

ternal fraction"; they were placed in the freezer overnight to allow further precipitation of proteins. The methanol extract was centri­ fuged for 10 min at 2000 g to eliminate the proteins. Supernatants were pooled, equal volumes of water and chloroform added, and the whole mixture shaken. The chloroform layer (CL) was transferred to another separatory funnel, an equal volume of de-ionized water added and shaken for 2 min. The methanol-water layer (M-WL) of the homo­ genate was then extracted three times. CL was concentrated under a nitrogen stream to 10 ml. M-WL was concentrated with a rotatory eva­ porator at room temperature. The extracts were kept in a refrigerator for later TLC analysis on silica gel 60 F-254 chromatoplates (EM

Laboratories) at a gel thickness of 0.25 mm. 43 The following TLC solvent systems were employed:

BE Benzene-ethyl acetate (6:1),

BEM ~ Benzene-ethylacetate-methanol (15:5:1),

BFE ~ Two developments with benzene saturated with formic

~cid - ether (10:3),

CE Carbon tetrachloride-ether (3:1)

Standards were spotted on the chromatoplates and developed in one of the solvent systems as recommended by Shono et al. (1978). The chromatoplates were air-dried after being developed, sprayed uniform- ly with phosphomolybdic acid (10% in ethanol; Sigma Chemical Company;

Bigley and Plapp, 1978) or visualized with UV-light. The sprayed plates were air-dried, then heated at 120°c for 2 min until optimum color formation was obtained. Rf values were determined for the stan- dards (Tab Ie 2).

Metabolism of Permethrin Studied in vitro

Comparison of C. carnea and H. vires ens -

The method of Jao and Casida (1974) was followed with slight modification. Third-instar GLW larvae (120-150 larvae per each of three replicates) and fourth-ins tar TBW larvae (6 to 8 larvae per each of three replicates obtained from Department of Entomology,

Cotton Insect Laboratory) were weighed and homogenized. The homo- genization mixture was comprised of 0.25 M sucrose and 0.15 M Na 2 HP0 -KH2P0 buffer (PH 7.5). Homogenization was carried out in an ice 4 4 44 bath. The homogenate was centrifuged for 20 min at 800 g (2500 rpm) at o 4 C to eliminate nuclei and debris. After centrifugation, the super- natant was diluted with cold homogenization mixture to obtain 15 mg tissue per ml of buffer.

The effect of esterases on metabolizing permethrin isomers was studied after Shono and Cas ida (1978). Twenty ~g of permethrin (CiT)

(1 ~g per ~l acetone) were added to the bottom of a large test tube, and the acetone evaporated before introducing the enzyme preparation.

Two ml of larval homogenate (30 mg tissue) in the buffered sucrose solution were added, mixed thoroughly, and incubated for either 30,

60, 90 or 120 min at 30 0 C with continuous shaking. At the end of the incubation the contents were transferred to a separatory funnel and

5 ml of hexane added with shaking to stop the enzymatic activity. The test tube was washed twice with 5 ml hexane to recover all contents.

The esterases plus oxidases activities were studied by adding

NADPH (dihydronicotinamide adenine dinucleotide phosphate) at 2.2 ~M per test tube just prior to the introduction of the substrate. The R contents were mixed thoroughly, the tubes covered with parafilm , and a stream of compressed air allowed to blow gently on the surface of the reaction mixture. Incubation was performed as previously described.

For determination of NADPH-dependent mixed-function oxidases

(MFO) activity, DPVP (dimethyl-2,2-dichlorovinyl phosphate; Shell -3 Chemical Company, Agric. Chem. Div., New York) at 10 M was added in acetone, and the acetone evaporated before introducing the enzyme 45 preparation (Tsukamoto and Casida, 1967). Each test tube received 2 ml larval tissue, mixed thoroughly with DDPV, incubated at 300 C for 20 min to completely inhibit the esterases. NADPH (2.2 ~M per tube) was added just prior to the introduction of substrate. Twenty ~l of sub- strate injected into the reaction mixture and mixed thoroughly by shak- ing. All test tubes were covered with Parafilm and a stream of compressed air allowed to blow across the surfaces. Other conditions were as above.

All experiments had three replicated controls; subdivided tissue of controls was denatured by overheating. This control was compared with completely inhibited homogenate. Complete inhibition was achieved by adding DDVP and eliminating NADPH. For the esterases, 3.5 ml of buffer were added instead of the 2.2 liM (in 3.5 ml) NADPH. Two ml of tissue were extracted as previously described and analyzed by

GLC.

Metabolism by ~. carnea

In similar experiments to study the metabolism of permethrin

(CiT), cis- and trans-isomers by GLW homogenates, the following was performed: Third-instar GLW larvae (120 to 150 larvae per each of three replicates) were weighed and homogenized. The previously men- tioned homogenization mixture was used. The homogenate was centrifuged o for 20 min at 800 g at 4 C. The supernatant was diluted with cold homogenization mixture to obtain 15 mg tissue per mI.

The effect of total activity (esterases plus oxidases), ester- ases, and oxidases was studied as previously mentioned. The incubation 46 o - period was 120 min at 30 C with continuous shaking. At the end of the

incubation the contents were transferred to a separatory funnel and 5 ml of chloroform added with shaking to stop the enzymatic activity.

Each replicate ran parallel with a control with overheated larval homogenate. The test tube was washed twice with 5 ml of chloroform

to recover all contents, equal volumes of water and chloroform added, and the whole mixture shaken for 2 min. The chloroform layer (CL) was collected and the water layer discarded. CL was concentrated under a nitrogen stream and brought up to 10 ml. The extracts were kept in a refrigerator for later TLC analysis. TLC was performed as mentioned before. RESULTS

Met~bolism of Permethrin by Larvae of C. carne a Studied in vivo

Recovery Efficiency

The efficiency of recovery (ER) was determined to predict if there was a difference in permethrin isomers recovered in the first few hours from dosing compared to the end of the experiment at 50 hr. Re- sults of the extraction efficiency are presented in Table 1. The ER changed with the time after dosing. During the first four hr. after dosing, 98.2 + 0.3 of cis and 96.0 + 0.7 of trans were recovered. Be- tween five and 35 hr. recovery declined to 93.1 + 1.3 and 90.0 + 2.1% respectively for cis and trans. The trend continued for cis as its ER declined at the period from 40 to 50 hr. whereas the trans isomer showed increased availability for extraction (87.8 ± 0.3 and 92.9 ±

0.1%, cis and trans, respectively). Almost complete recovery of permeth- rin was obtained in the recovery standard (2486 ~g of the standard analytical stock solution). The ER study was used in calculating the total permethrin recovered from each treatment. Using the correct % ER for each treatment predicted the correct isomer concentration and, con- sequently, the correct % permethrin metabolized.

GLW larvae were able to metabolize almost 79.8 + 1.3% of cis and 71.2 + 2.0% of trans during the first two hr following topical application (Figure 3). Ninety-six percent or more of cis and 95% or 47 48

Table 1. Extraction efficiency of permethrin isomers in the presence of Chrysopa carnea tissue.

% Extraction Efficienc;l1/!:!./ Treatment Time (Hr) cid/ Trans~/

2-4 98.2 + 0.3 96.0 + 0.7

5-35 93.1+1.3 90.0 + 2.1

40-50 87.8 + 0.3 92.9 + 0.1 - 1/ x + SE

J:../ Based on 1000 ~g (40% of 2500 ~g dosed to 25 larvae) 100 ~g per larva. l/ Based on 1500 ~g (60% of 2500 ~g dosed to 25 larvae) 100 ~g per larva.

!:!./ Figures obtained by comparing: a) unextracted stock-solutions, b) extracted stock-solution, c) control insects spiked with 100 ~g per larva. 49 less of trans was metabolized-by 50 hrs. The two isomers were equally metabolized after 15 hrs.

The "surface fraction", obtained by rinsing the larvae with acetone followed by the extraction procedure, was taken at 24 and 48 hr1 (Table 3). In each case for CiT, cis and trans, less than 1% of the compound was found on the insect surface.

Table 4 reports the "cup fraction" or the residue in the plas­ tic cup as a result of larval contact with the cup's surface and as a result of excretion. Less than 1% of any of the compounds was left in the cup. The remainder constituted the "surface fraction" and the

"internal fraction."

The detectable peaks in the chromatograms were: 1) the solvent,

2) tissue artifact, 3) cis, and 4) trans. No metabolite peaks were detected when Ecn/GLC was used (Figure 4).

TLC of Standards

Rf values for 11 standards developed with four different sol­ vent systems, one-dimensional TLC, are reported in Table 5. Cis and trans gave almost identical Rf values for 3 out of the four solvent systems; with BE, cis gave a higher R value (0.90) than trans (0.77). f The BFE system resulted in identical Rf values for m-PBalc (V) and 4'-HO, t-perm (IX) (0.82). But these two potential metabolites had different R values in BEM (0.37 0.02 and 0.71 0.05), BE (0.14 f ± ± ± 0.00 and 0.59 ± 0.05), and CE (0.96 ± 0.01 and 0.47 + 0.02) (m-PBalc and 4'-HO, t-perm, respectively). so

100

Q LIJ N :J o CD ~ 60 LIJ

Z== ~o ::z:a: t- 40 LIJ • cis :::E A trans ffi 30 a. -ae 20

10

OI'---2.1.--3'&'--4.1.--~!5-/1 10 I~ io i5 ~O 3'5 ~ 45 50 TREATMENT TIME (HRS)

Figure 3. Metabolism of permethrin by larvae of Chrysopa carnea studied in vivo. 51

Table 2. Metabolism of permethrin (CiT) by larvae of Chrysopa carnea following topical application (25 larvae per replicate, three replicates)l.!'!:..1

Treatment Micrograms Recovered Mean Metabolized Time per Larva (percent)ll (Hrs) cis trans cis trans

2 20.2 + 1.3 28.8 + 2.0 79.8 + 1.3 71.2 + 2.0

3 13.7 + 0.9 19.5 + 0.8 86.3 + 0.9 80.6 + 0.8

4 10.5 + 1.0 16.2 + 1.2 89.5 + 1.0 83.8 + 1.3

5 10.1 :t 0.6 15.2 + 0.8 89.9 + 0.6 84.9 + 0.8 10 10.8 :t 0.5 17.3 + 0.4 98.2 + 0.5 82.7 + 0.4

15 8.6 + 0.6 8.7 + 0.3 99.2 + 0.6 99.2 + 0.3

20 8.6 + 0.8 15.1 + 0.3 99.2 + 0.8 84.9 + 0.3

25 1.4 + 1.2 4.3 + 0.7 98.6 + 1.2 95.7 + 0.7

30 2.2 + 1.6 5.4 + 0.7 97.1+1.6 94.6 + 0.7

35 2.2 + 0.2 4.1 + 0.5 97.8 + 0.2 95.9 + 0.5

40 3.6 + 0.4 5.4 + 0.6 96.4 + 0.4 94.6 + 0.6

45 4.3 + 0.7 4.3 + 0.7 95.7 + 0.7 95.7 + 0.7

50 3.6 :t 0.8 5.4 + 0.9 96.4 + 0.8 94.6 + 0.9

11 Each insect dosed topically with 100 ~g permethrin (CiT) and held at 25 :t 2oC, 65 :t 5% RH, and 16:8 light-dark cycle prior to homogeniza­ tion.

'!:..I The procedure of Shono et ale (1978) modified by Shour and Crowder (1980) to permit use of GLC/ECD.

11 Significant difference cis and trans, values followed t-test at 0.05. Table 3. Surface-fraction of permethrin (C/T) after dosing larvae of Chrysopa carnea with 100 ~g per larva.

Micrograms Recovered Eer Larval/ % Permethrin Recovered per Larva Treatment Time CIT CiT (Hrs) cis ---Trans cis ---Trans cis ---Trans cis Trans 3 4 24 0.4 + 0.2 0.2 + 0.0 0.3 + 0.1 0.5 + 0.1 0.1 0.05 3 X 10- 5 X 1,0- 3 4 48 0.4 + 0.2 0.2 + 0.0 0.2 + 0.1 0.4 + 0.2 0.07 0.03 2 X 10- 3 X 10-

1/ ~ + SEe

V1 N Table 4. Cup-fraction of permethrin (CiT) after dosing larvae of Chrysopa carnea with 100 ~g per larva.ll

Hicrograms Recovered Eer Cup % Recovered Eer CUE Treatment Time CiT CiT c(Hrs) cis Trans cis ---Trans cis Trans cis Trans

24 0.5 + 0.0 0.4 + 0.1 0.5 + 0.1 0.5 + 0.1 1.25 l.0 0.50 0.50

48 0.5 + 0.2 0.4 + 0.1 0.5 + 0.1 0.5 + 0.1 l.25 l.0 0.50 0.50

II x + SE

1Il W 54

A 8 LIJ UJ Z 0 Q. UJ LIJa: a: cLIJ c: 0 U LIJa:

1\I.a. T/ME C 0 , A ri

~ v.,a. ., \ c E F

I.a.

Figure 4. Typical chromatograms obtained from GLC analysis of extracts from permethrin metabolism studied in vivo (c=cis; t=trans; t.a.=tissue artifact) .-- A) control insects, 5-10 hrs; B) control insects, 25-50 hrs; C) permethrin blank; D) fortified control; E) analytical standard; F) dosed insect after 25 hrs. 'Table 5. Rf values for some standard compounds and pe~ethrin metabolites using four different sol- vent systems and thin-layer chromatograph. (x ± SE of trials).

R Values f Roman Mobile Phase Solvent S~stem Compound FMC Number Numeral BFE BEM BE CE

m-PBalc 30952 V 0.82 + 0.00 0.37 + 0.02 0.14 + 0.00 0.96 + 0.01 m-PBacid 30953 VI 0.65 + 0.00 0.67 + 0.02 0.35 ± 0.02 0.36 + 0.03 2'-HO-PBacid 53813 VII 0.62 + 0.00 0.13 + 0.00 0.88 0.00 - 0.89 + 0.03 + 2'-HO-PBa1c 53814 VIII 0.43 + 0.02 0.50 + 0.04 0.14 + 0.00 0.14 ± 0.01 4'-HO-PBacid 53808 III 0.49 + 0.04 0.15 + 0.01 0.94 ± 0.02 0.85 + 0.00 4'-HO-PBalc 53809 IV 0.47 + 0.00 0.41 + 0.04 0.11 + 0.02 0.09 + 0.01 4'-Ho, t-perm 53824 IX 0.82 + 0.02 0.71 + 0.05 0.59 + 0.05 0.47 ± 0.02 2'-HO-perm 53825 X 0.85 + 0.00 0.82 + 0.01 0.69 + 0.03 0.68 + 0.02 cis/trans-perm 30062 0.96 + 0.03 0.91 + 0.01 0.90 + 0.04 0.91 + 0.00 cis-perm 35171 I 0.92 + 0.04 0.91 + 0.04 0.90 + 0.04 0.91 ± 0.00 trans-perm 30980 II 0.91 + 0.04 0.89 + 0.04 0.77 + 0.00 0.91 + 0.03

17 BFE = 2 developments with benzene saturated with formic acid - ether (10:3). 1/ BEM = Benzene-ethyl acetate-methanol (15:5:1). l/ BE Benzene - ethylacete (6:1). 4/ CE ~ Carbon tetrachloride-ether (3:1).

111 111 56

A similar situation was true between m-PBalc (V) and 2'-HO-

PBalc (VIII) for BE (Rf a 0.14 ~ 0.00). The 2'-HO-PBacid (VII), cis, and trans compounds resulted in R values almost identical with BEM f

(0.89 ~ 0.04 and 0.90 ~ 0.04), and CE (0.88 and 0.91). In the other two solvents systems, BFE and BEM, there was a definite difference

(0.62 ~ 0.00 and 0.91 ~ 0.04, for BFE; 0.13 + 0.00 and 0.91 + 0.04 for BEM).

Metabolism of Permethrin by Chrysopa carnea Studied in vivo

CiT

Chloroform Extract. Table 6 lists the metabolites of CiT in the CL at 24 and 48 hr after dosing. BEM detected 9 spots at 24 hr after treatment; two were identified as cis and trans. There were three unknowns with R£ values of 0.97, 0.56 and 0.06. The remaining

4 were tentatively identified as 4 alcohol metabolites: 2'-HO-perm

(X), 4'-HO-PBalc (IV), m-PBalc (V), and 2'-HO-pBacid (VII).

BE showed 7 spots; three of them were unknowns with R values f of 0.28, 0.24, and 0.03. Three spots were tentatively identified as

4'-HO-PBacid (III), 2'-HO-PBacid (VII), and 2'-HO-perm (X). The seventh spot with an R value of 0.14 could be either m-PBalc (V) or f 2'-HO-PBalc (VIII) since both had the same R value in BE. It was f more likely to be V which was detectable with BEM since VIII did not appear in the latter system.

Therefore, all compounds were detected in the 24 hr treatment except VIII (2'-HO-PBalc) and IX (4'-HO, t-perm). Some of the Table 6. Metabolites of permethrin in the chloroform extract of Chrysopa carnea larvae within 24 and 48 hours of dosing in vivo.

24 Hrs. 48 Hrs. R f Rf Solvent 2/ Roman Roman System Valuesl/ Compound- Numeral Values Compound Numeral

BFE 0.97 + 0.01 cis/Trans-perm 0.98 + 0.00 Cis/Trans-perm 0.82 +" 0.03 m-PBale V 0.91 +" 0.01 cis-and/or trans I or II 0.74 +" 0.03 unk. 0.53 + 0.01 4'-HO-PBair-d---- III 0.52 + 0.06 4'-HO-PBaird III 0.39 +" 0.01 unk. 0.30 +" 0.02 unk. 0.33 + 0.00 unk. 0.18 + 0.00 unk. BEt-! 0.97 + 0.01 unk. 0.93 + 0.01 cis and/or trans I or II 0.96 + 0.00 unk. 0.89 +" 0.01 trans-perm II 0.86 +" 0.01 trans-perm II 0.81 + 0.02 2'-HO-perm X 0.67 + 0.03 m-PBacid VI 0.56 + 0.02 unk. 0.51 + 0.01 2'-HO-PBacid VIII 0.43+0.01 4'-HO-PBalc IV 0.40 +" 0.01 4'-HO-PBalc IV 0.36 + 0.05 m-PBalc V 0.26 + 0.02 unk. 0.12 + 0.01 2'-HO-PBacid VII 0.15 + 0.00 4'-HO-PBacid III 0.06 + 0.01 unk.

BE 0.96 + 0.01 4'-HO-PBacid III 0.97 + 0.01 4'-HO-PBacid III 0.86+0.01 2'-HO-PBacid VII 0.80 + 0.01 unk. 0.72+0.02 2'-HO-perm X 0.41 +" 0.02 unk. 0.28 + 0.01 unk. o . 30 + (). f.H unk. 0.24 + 0.03 unk. 0.28 + 0.00 unk. 0.14 + 0.01 m-PBalc or 2'-HO- pBalc V or VIII 0.26 + 0.01 unk. 0.03 + 0.01 unk. 0.09 + 0.01 4'-HO-PBalc VIII

I..n ...... Table 6, Continued.

24 Hrs. 48 Hrs. R R Solvent f Roman f Roman System Values Compound Numeral Values Compound Numeral

CE 0.91 + 0.02·!.! cis- and/or trans I and/or II 0.96 ± 0.01 m-PBalc V 0.57 + 0.02 unk.l/ -- 0.88 + 0.01 2'-HO-PBacid VII 0.36 "+ 0.05 m-PBacid VI 0.48 + 0.01 4'-HO, t-perm IX 0.09 + 0.01 4'-HO-PBalc IV 0.39 + 0.00 m-PBacid VI 0.34 "+ 0.01 unk. 0.24 "+ 0.02 unk. 0.07 "+ 0.01 unk. 0.03 + 0.00 unk.

1/ x + SE Ii unk. = unknown.

1I1 00 59 compounds did not appear in all solvent systems. BEM and BE proved to be the most dependable of the four systems.

For the 48 hr treatment, all systems were dependable; any system showed at least seven spots (Table 6). Four unknowns occurred with BFE (Rf m 0.39, 0.33, 0.18, and 0.06), a similar number with BE and CE, whereas only two unknowns were found in the BEM system. BEM manifested only two unknowns. In summary, all compounds were detected including VIII "and IX except X (2'-HO-perm).

The two time periods shared seven compounds, two of them being the two isomers of CiT. The other five were compounds III through

VII. The 48 hr treatment was distinguished by having VIII and IX, whereas the 24 hr treatment exclusively had X.

Methanol and Water Extract. Once again, the BE-system showed better separation of metabolites in M-WL at both 24 and 48 hr. Six spots were detected in both treatments with solvent system.

Table 7 reports both the presence of £is and trans in the first three solvent systems, while CE only showed trans. Besides these isomers, the two time periods shared only one metabolite: 2'-

HO-PBacid (VII). Two other metabolites occurred at 24 hr: III and

V. The presence of VI and X was seen at 48 hr.

In summary, the following compounds were found in CL and M-WL: cis; trans; 4'-HO-PBacid; m-PBalc; 2'-HO-PBacid; m-PBacid; and 4'-HO, t-perm. The following unknowns were noted: BFE - one at 24 hr and two at 48 hr; BEM - two unknowns at 24 hr but none at 48 hr; BE - three at 24 and two at 48 hr; CE - none for either treatment. Table 7. Metabolites of permethrin 1n methanol and water extract of Chrysopa carnea larvae within 24 and 48 hours of dosing, in vivo.

24 Hrs. 48 Hrs. R Solvent RfY Roman f Roman System Values Compound~/ Numeral Values Compound Numeral

BFE 0.99 + 0.01 cis/Trans-perm 0.94 + 0.01 cis and/or trans I and/or II 0.94 + 0.01 cis and/or trans I and/or II 0.70 + 0.01 unk. 0.84 + 0.01 m-PBalc -- V 0.04 + 0.01 unk. 0.23 + 0.00 unk.

BEM 0.99 + 0.00 unk. 0.91 + 0.02 cis and/or trans I and/or II 0.92 + 0.00 cis and/or trans I and/or II 0.85 + 0.01 unk.

BE 0.96 + 0.00 4'-HO-PBacid III 0.90 + 0.00 cis-perm I 0.87 + 0.00 2'-HO-PBacid VII 0.89 + 0.00 2'-HO-PBacid VII 0.78 + 0.01 trans-perm II 0.74 +" 0.01 unk. 0.44 + 0.00 unk. 0.63 + 0.00 4'-HO, t-perm X 0.33 + 0.01 unk. 0.37 +" 0.02 m-PBacid VI 0.25 + 0.01 unk. 0.27 +" 0.01 unk.

CE 0.93 + 0.01 trans-perm II 0.89 + 0.00 trans-perm II 0.88 + 0.01 2'-HO-PBacid VII

1/ x + SE 2/ unk. = unknown.

0"1 o 61

Cis-Permethrin

Chloroform Extract. The four solvent systems were able to separate a sizable number of metabolites in CL (Table 8). BE was still the leading solvent system on the basis of exhibiting the greatest num­ ber of spots.

Both hydrolysis and oxidation products of cis, in addition to traces of cis, were detected. At 24 hr, 2'-HO-perm; 4'-HO, t-perm; m-pBacid; and 4'-HO-PBalc (IV, VI, IX and X) were present. The num­ bers of unknowns were: BFE (3), BEM (4), BE (4), and CE (3).

The 48 hr treatment showed more oxidation and hydroxylation products than the 24 hr treatment. At 48 hr, 2'-HO-perm; 2'-HO-PBacid;

2'-HO-PBalc; and m-PBacid were observed. The two treatments shared only two metabolites: m-PBacid (VI) and 2'-HO-perm (X). Compounds IV and

IX disappeared after 24 hr; VII and VIII were present at the latter time period. At 24 hr the numbers of unknowns were: BFE (1), BEM

(5), BE (4), and CE (3), whereas the 48 hr numbers of unknowns were:

BFE (3), and four for each of the other three systems.

Methanol and Water Extract. Not as many compounds in M-WL were found by the BFE and BEM TLC system as in the BE and CE systems, especially for the 24 hr treatment, only one each for BFE and BEM

(Table 9). Compounds III, V and VI in addition to cis were produced by 24 hr. At 48 hr compound X also occurred (Table 10). At 24 hr the number of unknowns were: BFE (0), BEM (1), BE (1), and CE (2), where­ as the 48 hr number of unknowns were: BFE (2), BEM (1), BE (3), and

CE (2). Table 8. Metabolism of cis-permethrin in chloroform extract of Chrysopa carnea larva within 24 and 48 hours of dosing, in vivo.

24 Hr 48 Hr R R Solvent f Roman f Roman System Values Compound Numeral Values Compound Numeral

BFE 0.96 + 0.01 cis/trans-perm 0.96 + 0.01 cis/trans perm 0.84 + 0.01 2'-HO-perm X 0.85 + 0.01 2'-HO-perm X 0.75 + 0.03 unk. 0.76 + 0.00 unk. 0.67 + 0.01 m-PBacid VI 0.71 + 0.01 unk. 0.28 + 0.02 unk. 0.67 + 0.00 m-PBacid VI 0.05 + 0.00 unk. 0.60 +" 0.01 2'-HO-PBacid VII 0.23 +" 0.02 unk.

BEM 0.98 + 0.00 unk. 0.97 + 0.01 unk. 0.89 + 0.02 trans-perm II 0.95 + 0.00 cis-perm I 0.80 + 0.00 unk. 0.87 + 0.01 unk. 0.72+0.01 4'-HO, t-perm IX 0.77+0.01 unk. 0.55 + 0.01 unk. 0.54 + 0.02 2'-HO-PBalc VIII 0.28+0.03 unk. 0.22 + 0.00 unk. 0.09 + 0.00 unk.

BE 0.98 + 0.01 unk. 0.97 + 0.00 unk. 0.83 + 0.00 unk. 0.79 + 0.01 unk. 0.33 + 0.00 m-PBacid VI 0.46 +" 0.05 unk. 0.28 + 0.00 unk. 0.26 + 0.01 unk. 0.23 + 0.01 unk. 0.14 + 0.00 2'-HO-PBalc/or m-PBalc VIII or V 0.12 + 0.03 4'-HO-PBalc IV

0\ N Table 8, Continued.

24 Hr 48 Hr R R Solvent f Roman f Roman System Values Compound Numeral Values Compound Numeral

CE 0.98 + 0.01 unk. 0.94 + 0.01 unk. 0.91 + 0.01 cis-perm I 0.28 + 0.01 unk. 0.32 + 0.00 unk. 0.24 + 0.02 unk. 0.26 + 0.00 unk. 0.03 + 0.00 unk.

1./ ~ :t- SE li unk. = unknown.

0\ W Table 9. Hetabolites of cis-permethrin in methanol and)Water extracts of Chrysopa carnea larvae within 24 and 48 hours, in vivo.

24 Hr 48 Hr R Solvent Rf Roman f Roman System Value Compound Numeral Value Compound Numeral

BFE 0.99 + 0.01 cis/trans-perm 0.94 + 0.03 cis-perm I 0.55 +" 0.00 unk. 0.51 +" 0.00 4'-HO-PBacid III 0.34 +" 0.00 unk.

BEH 0.97 + 0.01 unk. 0.99 + 0.01 unk. 0.81 + 0.01 2'-HO-perm X

BE 0.96 + 0.00 4'-HO-PBacid III 0.96 + 0.00 4'-HO-PBacid III 0.74 "+ 0.00 unk. 0.83 "+ 0.00 unk. 0.70 + 0.01 2'-HO-perm X 0.35 "+ 0.00 m-PBacid VI 0.29 +" 0.00 unk. 0.18 + 0.00 unk. 0.16 "+ 0.00 m-PBalc V

CE 0.97 + 0.00 m-PBalc V 0.89 + 0.02 m-PBacid VI 0.86 +" 0.01 4'-HO-PBacid III 0.29 +" 0.02 unk. 0.36 "+ 0.00 m-PBacid VI 0.04 "+ 0.00 unk. 0.31 + 0.00 unk. 0.05 +" 0.00 unk.

1/ ~ + SE 2/ unk. = unknown.

0\ +0- 65 In summary, the following compounds were found in CL and M-WL:

cis (I): 4'-HO-PBacid (III), 4'-HO-PBalc (IV), m-PBa1c (V); m-PBacid

(VI); 2'-HO-PBacid (VII); 2'-HO-PBa1c (VIII); 4'-HO, t-perm (IX); and

2'-HO-perm (X).

-----Trans-Permethrin Chloroform Extract. Table 10 lists the CL compounds detected by 24 and 48 hr. At 24 hr, trans and three unknowns were present in

BFE; compounds II, VI, VIII and IX and two unknowns were detected with

BEM; compound IV and three unknowns were exhibited by BE: VI, IX and five unknowns were detected with CEo

At 48 hr there were II, VI, IX and four unknowns with BFE. In addition to the metabolites VI and X, three unknowns were detected by using BEM. BE detected II and two unknowns; whereas CE resulted in II,

VI, IX, and four unknowns.

The two time intervals shared only three compounds: II, VI, and IX. Compounds IV and VIII were exclusively detected at 24 hr, whereas X (2'-HO-perm) was exclusively detected at 48 hr.

Methanol and Water Extract. Trans (II) was common in BFE,

BEM and BE at 24 hr; and BFE, BEM and CE at 48 hr for M-WL (Table

11). Trans (II), VI, VII, IX, and X were recorded at 24 hr. By 48 hr, II, III, V, VIII, and IX were noted. The only shared compound between the two treatments was II. At 24 hr the numbers of unknowns were: none for BFE and BEM, BE (1), and CE (2): whereas the 48 hr numbers of unknowns were: one each for BFE and BEM, BE (4), and CE (2). Table 10. Metabolites of trans-permethrin in chloroform extracts of Chrysopa carnea larvae within 24 and 48 hours of dosing, in vivo.

24 Hr 48 Hr R );} R Solvent f Roman f Roman 2/ System Value Compound- Numeral Value Compound Numeral

BFE 0.94 + 0.01 trans-perm II 0.93 + 0.01 trans-perm II 0.77 + 0.01 unk. 0.80 + 0.01 4'-HO, t-perm IX 0.69 +" 0.00 unk. 0.67 + 0.01 m-PBacid VI 0.25 + 0.00 unk. 0.28 + 0.03 unk. 0.12 + 0.00 unk. 0.08 + 0.00 unk. 0.04 +" 0.00 unk.

BEM 0.96 + 0.01 unk. 0.96 + 0.01 unk. 0.85 + 0.01 trans-perm II 0.83 + 0.02 2'-HO-perm X 0.76 + 0.01 4'-HO, t-perm IX 0.65 + 0.01 m-PBacid VI 0.66 + 0.00 m-PBacid VI 0.59 + 0.01 unk. 0.54 + 0.01 2'-HO-PBacl VIII 0.26 + 0.00 unk. 0.26 + 0.00 unk.

BE 0.83 + 0.01 unk. 0.79 + 0.02 trans-perm II 0.27 + 0.00 unk. 0.26 + 0.00 unk. 0.22 + 0.01 unk. 0.18 + 0.02 unk. 0.10 + 0.01 4'-HO-PBalc IV

CE 0.99 + 0.01 unk. 0.94 + 0.01 trans-perm II 0.91 + 0.01 trans-perm II 0.46 + 0.00 4'-HO, t-perm IX 0.62 + 0.00 unk. 0.37 + 0.00 m-PBacid VI 0.47 + 0.01 4'-HO, t-perm IX 0.23 + 0.01 unk. 0.34 + 0.01 m-PBacid VI 0.11 + 0.00 unk.

0\ 0\ Table 10, Continued.

24 Hr 48 Hr R R Solvent f Roman f Roman System Value Compound~/ Numeral Value Compound Numeral

CE 0.12 + 0.01 unk. 0.07 + 0.00 unk. 0.07 + 0.00 unk. 0.04 + 0.00 unk. 0.04 +" 0.00 unk.

1/ x + SE '!:../ unk. unknown

(j\ --.J Table 11. Metabolites of trans-permethrin in methanol and water extract of Chrysopa carnea larvae within 24 and 48 hours of dosing, in vivo.

24 Hr 48 Hr R Solvent Rfll Roman f Roman 2/ System Value Compound- Numeral Value Compound Numeral

BFE 0.96 + 0.01 trans-perm II 0.91 + 0.01 trans-perm II 0.64 + 0.00 m-PBacid VI 0.84 + 0.00 2'-HO-perm X 0.49 +" 0.01 4'-HO-PBacid III 0.04 + 0.00 unk.

BEM 0.90 + 0.01 trans-perm II 0.97 + 0.00 unk. 0.81 + 0.00 2'-HO-perm X 0.88 "+ 0.01 trans-perm II 0.71 + 0.00 4'-HO, t-perm IX 0.77 +" 0.00 4'-HO, t-perm IX 0.48 + 0.00 2'-HO-PBalc VIII 0.36 +" 0.01 m-PBalc V

BE 0.78 + 0.00 trans-perm II 0.84 + 0.01 unk. 0.23 + 0.00 unk. 0.74 + 0.01 unk. 0.56 +" 0.00 unk. 0.29 + 0.00 unk.

CE 0.86 + 0.01 2'-HO-PBacid VII 0.94 + 0.00 trans-perm II 0.43 + 0.00 unk. 0.84 + 0.01 4'-HO-PBacid III 0.32 +" 0.00 unk. 0.74 + 0.00 unk. 0.31 + 0.00 unk.

)j x = SE l/ unk. = unknown.

0\ (Xl . 69 Metabolism of Permethrin Studied in vitro: Comparison of Chrysopa carnea and Heliothis virescens

A study was conducted to examine the effects of the enzyme systems, esterase(s) and oxidases, when they were acting simultan- eously and individually, in the tolerant GLW and a susceptible species"TBW. Figure 8 shows typical chromatograms obtained from GLC analysis.

Esterases plus Oxidases

GLW esterases plus oxidases were able to metabolize cis and trans at higher rates than could the TBW (Figure 5). GLW metabolized

39% of cis during the first 30 min of incubation, whereas TBW meta- bolized only 19.1% within the same incubation time. GLW metabolized cis and trans at higher rates at the other time periods except at 90 min with trans where both insects metabolized the compound at almost similar rates (42.6% for GLW and 41.3% for TBW). By 120 min, the greatest difference was recorded: GLW metabolized 71.1% of cis and

42.0% of trans, while TBW metabolized only 24.9% cis and 26.2% trans.

GLW was able to metabolize cis at higher rates than could TBW at all times. For trans, GLW metabolized the isomer at higher rates at all incubation times except the 90 min as mentioned before.

GLW metabolized cis at a higher rate than trans for the first and last time periods. Trans was metabolized at a higher rate between

60 and 90 min. The greatest difference was at 120 min: 71.3% for cis and 42.0% for trans.

TBW metabolized cis at higher rate for the 30 min and 60 min intervals (19.1 to 14.7, and 23.5 to 18.6%, respectively). At the 70

70

C ILl N o~ ~ 4~ I- ILl ~ 40 z :I:a: 3~ t- ILl a::~ 30 w a. 25 'J!.

• GLW-cis o GLW-tr(Jns .. TBW-cis 6 TBW-IrOn$

30 60 90 120 INCUBATION TIME (MIN)

Figure 5. Metabolism of cis- and trans- by Chrysopa carnea and Heliothis virescens: effect of esterases plus oxidases studied in vitro. 71

later time, the trend was reversed: 30.4 to 41.3, and 24.9 to 25.2%,

cis to trans, 90 and 120 min, respectively.

Oxidases Alone

When the oxidases of GLW and TBW were studied alone, GLW was able to metabolize cis and trans at a dramatically higher rate at the

time points (Figure 6). GLW metabolized 24.9% of cis and 21.8% of

trans within 30 min, compared to 35.5 and 34.3% for TBW. Eighty-nine percent and 88.2% of cis and trans were metabolized within the first one hr for GLW, and 21.6 and 25.3% for TBW. By the last two intervals

(90 and 120 min), GLW and TBW metabolized cis and trans as follows:

90 min - 84.1, 83.8; and 16.4 and 18.8%; 120 min - 86.4, 85.8 and

31.8 and 38.4%.. There did not appear to be any difference in the rates between isomers for both GLW and TBW.

Esterases Alone

GLW metabolized both isomers at 1.5 to 2-fold higher than the

TBW when the esterases were studied alone (Figure 7). GLW was able to metabolize 32.1% of cis and 28.0% of trans within 30 min, whereas TBW metabolized 9.9 and 12.1%, respectively. After one hr, 46.5 and 49.5% for cis and trans were observed with GLW, while TBW metabolized about

21.6 and 21.1% of cis and trans. Forty-two and 35% cis and trans were recorded for GLW at 90 min, while TBW metabolized 28.4 and 19.4%, respectively. The dramatic difference between the two species in metabolizing cis and trans occurred at 120 min. GLW metabolized 56.4%, cis and TBW metabolized only 29.9%. For trans, 39.4% was metabolized by GLW and 27.6% by TBW .. 72

100

90

80

o 70 lU N ::J • GLW-cis o GLW-frons CD 60 o ~ ... TBW-cis LIJ :;: 6 TBW-frons Z !50 ii: ::z:: I­ LIJ :E 40 Il: lU CL oe 30

20

10

30 60 90 120 INCUBATION TIME (MIN)

Figure 6. M~tabolism of cis- by Chrysopa carnea and Heliothis virescens: effect of oxidases alone studied in vitro. 73

• GLW-cis o GLW-Irons • TBW-cis ~ TBW-Irons 60 Q UJ N ::io eo CD ~ l&J :E 40 z a:: ::z:: ~ 30 :E a:: UJ c- o 20 ~

10

30 60 90 120 INCUBATION TIME (MIN)

Figure 7. Metabolism of cis- and trans- by Chrysopa carnea and Heliothis VIrescens:--effect of esterases alone, studied in vitro. 74

A 8 1.0. c r.· 0 ,. I .•. on '"Z o "­on a::'" 0:: 01 '"0:1 o ...UI 0:

I .•.

TIME

II E ~~ F G f.O. II!o • I \ I ! I.a. 1 1 I I I I.a. I I •. 'a. I

Figure 8. Typical chromatograms obtained from GLC analysis of permethrin metabolism by Chrysopa carnea and Heliothis virescens, studied in vitro (c=cis; t=trans; t.a.= tissue artifact; s=solvent).--A) analytical standard at 2 n GLW; B) esterases plus oxidases of GLW, 90 min; C) esterases plus oxidases of TBW, 90 min; D) oxidases of GLW, 90 min; E) oxidases of TBW, 90 min; F).esterases of GLW, 90 min; G) esterases of TBW, 90 min. Table 12. Metabolism of permethrin studied in vitro; comparison of Chrysopa carnea and Heliothis virescens (30 mg tissue per test tub~Temperature = 30oC, pH = 7.5 permethrin concen- tration = 20 g per test tube (x = SE).

C. carnea H. virescens Incubation Permethrin Recovered % Permethrin Permethrin Recovered % Permethrin Time Enzyme ( g) Metabolized ( g) Metabolized (min) System(s) cis Trans cis ---Trans cis Trans cis ---Trans 3D Esterases + 4.9 + 0.1 8.1 + 0.1 39.0 33.5 6.5 + 0.1 10.3 + 0.2 19.1 14.7 oxidases Oxidases 6.0 + 0.2 9.4 + 0.2 24.9 21.8 5.2 + 0.9 7.9 + 0.8 35.5 34.3 Esterases 5.4 + 0.1 8.6 + 0.2 32.1 28.0 7.2 + 0.0 10.6 + 0.5 9.9 12.1

60 Esterases + 5.6 + 0.1 8.0 + 0.1 29.8 39.0 6.1 + 0.1 9.8 + 0.1 23.5 18.6 oxidases Oxidases 0.8 + 0.1 1.4 + 0.1 89.5 88.2 5.5 + 0.7 9.0 + 0.8 30.8 25.3 Esterases 4.3 + 0.3 6.1 + 0.5 46.5 49.6 6.3 + 0.7 9.5 + 0.1 21.6 25.3

90 Esterases + 4.9 + 0.1 6.9 + 0.7 38.9 42.6 5.6 + 0.2 7.0 + 0.7 30.4 41.3 oxidases Oxidases 1.3 + 0.1 1.9 + 0.2 84.1 83.8 6.7 + 0.2 9.8 + 0.3 16.4 18.8 Esterases 4.6 "+ 0.3 7.7 + 0.5 42.1 35.7 6.3 + 0.2 9.7 + 0.2 28.4 19.4

120 Esterases + 2.3 + 0.3 7.0 + 0.5 71.1 42.0 6.0 + 0.2 8.9 + 0.7 24.9 26.2 oxidases Oxidases 1.1 + 0.1 1.7+0.2 86.4 85.8 5.5 + 0.6 7.4 + 0.5 31.8 38.4 Esterases 3.5 +" 0.2 7.3 + 0.2 56.4 39.4 5.7 "+ 0.1 8.7 "+ 0.1 29.9 27.6

-...J 1Il . o 76

In summary, GLW metabolized the cis-isomer of permethrin at a higher rate than the trans-isomer during 30, 90 and 120 min. The trans- isomer was only metabolized in a higher rate at 60 min. TBW metabolized cis at higher rates at all time periods except at the first 30 min in which the difference was not dramatic (9.9% cis vs. 12.1 trans). In addition, GLW metabolized the CiT isomers more rapidly than TBW.

Metabolism of Permethrin by Chrysopa carnea Studied in vitro

The purpose of this study was to compare the formation of sev- eral metabolites by esterases and oxidases of larval homogenates of

GLW separately and acting in combination. The parent and both isomers of permethrin were studied.

Metabolites of Permethrin (CiT)

Esterases plus Oxidases. Nine spots were detected in the BFE chromatograms from CiT when both esterases and oxidases were active

(Table 13). Two pots represented the two isomers, cis and trans; there were four unknowns (Rf = 0.76 ~ 0.01, 0.69 ~ 0.01, 0.10 + 0.00 and

0.04 ± 0.00) and three identifiable metabolites: 2'-HO-PBacid (VII)

4'-HO-PBacid (III), and 2'-HO-PBalc (VIII). BEM exhibited six spots, three of them being unknowns (Rf ~ 0.97 ± 0.00, 0.64 + 0.00 and 0.11 ± 0.00). Compounds IV and VIII, in addition to the parent compound were identified. VI, VII, and three unknowns (R 0.50 + 0.01, 0.40 f = ± 0.01, and 0.26 ~ 0.02) were demonstrated in BE. CE resulted in IV,

VII, VIII and three unknowns (Rf ~ 0.74 ± 0.02, 0.55 ± 0.02, and 0.06 ± 0.00). 77

Table 13. Metabolites of permethrin (C/T) by Chrysopa carnea homogen­ ates studied in vitro: esterases plus oxidases.

R Value Solvent f Roman System (x ± SE) Compound Numeral

BFE 0.98 + 0.00 cis/trans-perm 0.93 "+ 0.01 cis and/or trans I and/or II 0.76 + 0.01 unk.l.! 0.69 +" 0.01 unk. 0.61 + 0.01 2'-HO-PBacid VII 0.52 +" 0.01 4'-HO-PBacid III 0.42 + 0.01 2'-HO-PBalc VIII 0.10 +" 0.00 unk. 0.04 + 0.00 unk.

BEM 0.97 + 0.00 unk. 0.93 "+ 0.00 cis and/or --trans I and/or II 0.64 "+ 0.00 unk. 0.52 + 0.01 2'-HO-PBalc VIII 0.45 +" 0.00 4'-HO-PBalc IV 0.11 + 0.00 unk.

0.89 + 0.02 2'-HO-PBacid VII 0.50 +" 0.01 unk. 0.40 + 0.01 unk. 0.33 "+ 0.01 m-PBacid VI 0.26 "+ 0.02 unk.

CE 0.89 + 0.01 2'-HO-PBacid VII 0.74 + 0.02 unk. 0.55 +" 0.02 unk. 0.15 +" 0.00 2'-HO-PBalc VIII 0.10 "+ 0.00 4'-HO-PBalc IV 0.06 + 0.00 unk.

1./ unk. =: unknown. 78

Therefore, in the presence of both enzyme systems, CiT was meta­

bolized to the following compounds: III, IV, VI, VII and VIII. Com­

pounds V, IX, and X (m-PBalc; 4'-HO, t-perm; and 2'-Ho-perm,

respectively) were not found.

Esterases Alone. BFE exhibited nine spots when only esterases were active on CiT, similar to that found in the presence of both

enzyme systems (Table 14), but five of these were unknowns (Rf = 0.78

± 0.00, 0.72 ± 0.01, 0.22 ± 0.00, 0.19 ± 0.00, and 0.12 ± 0.01) (Table 14). Compound V (m-PBacid) was detected only in BFE. Compound IX

(4'-HO, t-perm) was exclusively exhibited by BEM. Overall, the com­

pounds identified with' esterases were: I, II, IV, V, VI, VII, VIII

and IX. Compounds III (4'-HO-PBacid) and X (2'-HO-perm) were not found.

Oxidases Alone~ Only eight spots were observed from oxidases

activity on CiT with BFE, two of them being the parent isomers, I and

II (Table 15). Compound III, VI, VIII and four unknowns (Rf = 0.74 ± 0.01, 0.58 ± 0.01, 0.12 ± 0.00, and 0.04 ± 0.00) were also found. BEM detected seven compounds: I, II, IV, V, VI, VIII and one unknown with

an Rf of 0.87 + 0.01. Three unknowns (Rf = 0.80 ± 0.00, 0.27 ± 0.00, and 0.17 ± 0.01) and III, VI, VIII and X were observed with BE, whereas CE exhibited the two isomers, IV and four unknowns (Rf = 0.75 ± 0.01, 0.53 ± 0.01, 0.16 ± 0.00 and 0.12 ± 0.00). Therefore, in the presence of oxidases alone, the following com­

pounds were detected: I through VI, VIII and X. Compounds VII (2'­

HO-PBacid) and IX (4'-HO, t-perm) were absent.

BFE exhibited more spots when either esterases plus oxidases

or esterases alone wer~ active, than when oxidases alone were 79

Table 14. Metabolites of permethrin (C/T) by Chrysopa carnea homogen- ates studied ---in vitro: Esterases alone.

R Values Solvent f Roman System (x .:t SE) Compound Numeral

BFE 0.96 + 0.01 cis and/or trans (C/T) I and II 0.89 + 0.00 cis and/or trans I and/or II 0.83 + 0.01 m-PBalc V 0.78 + 0.00 unk.l:./ 0.72 + 0.01 unk. 0.64 +" 0.00 m-PBacid VI 0.22 +" 0.00 unk. 0.19 + 0.00 unk. 0.12 +" 0.01 unk.

BEM 0.96 + 0.01 cis unk. or tra.ns I or II 0.91 + 0.01 cis and/or trans I and/or II 0.71+0.01 4'-HO, t-perm IX 0.60 + 0.01 unk. 0.46 +" 0.00 2'-HO-PBalc VIII 0.43 +' 0.00 4'-HO-PBalc IV

BE 0.89 + 0.01 2'-HO-PBacid VII 0.50 + 0.01 unk. 0.33 +" 0.01 m-PBacid VI 0.28 + 0.00 unk. 0.24 +" 0.00 unk.

CE 0.90 + 0.00 cis and/or trans I and/or II 0.83 +" 0.01 unk. -- 0.27 + 0.01 unk. 0.17 +" 0.01 unk. 0.13 + 0.00 2'-Ho-PBalc VIII 0.11 + 0.00 unk.

)) unk. = unknown. 80

Table 15. Metabolites of permethrin (C/T) by Chr~soEa carnea homogen- ates studied in vitro: oxidases alone.

R Values f Roman Solvent 1/ System (x ± SE) Compound- Numeral

BFE 0.95 + 0.01 cis- and/or trans I and/or II 0.74 + 0.01 unk. 0.67 + 0.01 m-PBacid VI 0.58 + 0.01 unk. 0.53 + 0.01 4'-HO-PBacid III 0.43 + 0.01 2'-HO-PBa1c VIII 0.12 + 0.00 unk. 0.04 +" 0.00 unk.

BEM 0.96 + 0.00 unk. 0.93 + 0.01 cis-and/or trans- I and/or II 0.87 + 0.01 unk. -- 0.65 + 0.01 m-PBacid VI 0.52 + 0.00 2'-HO-PBalc VIII 0.44 + 0.01 4'-HO-PBalc IV 0.39 + 0.01 m-PBalc V

BE 0.93 + 0.01 4'-HO-PBacid III 0.80 + 0.00 unk. 0.72 + 0.00 2'-HO-perm X 0.35 + 0.02 m-PBacid VI 0.27 + 0.00 unk. 0.17 + 0.01 unk. 0.14 + 0.00 2'-HO-PBalc VIII

CE 0.89 + 0.01 cis-and/or trans I and/or II 0.75 + 0.01 unk. 0.53 + 0.01 unk. 0.16 + 0.00 unk. 0.12 + 0.00 unk. 0.08 + 0.00 4'-HO-PBalc IV

1:/ unk. CI unknown. 81

metabolizing CiT. On the other hand, BEM and BE showed more spots for

the oxidases than either of the two enzyme treatments. Six spots were

found in CE for each of the experiments.

Summary of CiT

Oxidases were responsible for the production of compounds III

(4'-HO-PBacid) and X (2'-HO-perm). The esterase system was responsible

for VII (2'-HO-PBacid) and IX (4'-HO, t-perm). In the presence of both

~nzyme systems, the homogenate restored the ability to produce III and

VII, but lost the ability to produce IX and X.

Metabolites of cis

Esterases plus Oxidases. When cis was incubated with active

esterases and oxidases, eight, seven, six, and eight spots were ob­

served with BFE, BEM, BE and CE, respectively (Table 16). The iden­

tifiable spots were: I, IV, V, VI, VII, VIII and X. The unknowns

were: BFE (4), BEM (3), BE (3) and CE (7).

Esterases Alone. For esterase activity with £is, five spots

were detected with BFE and BEM, three for BE, and five for CE (Table

17). BE was only able to exhibit I and two unknowns (Rf ~ 0.84 +

0.00 and 0.25 0.02). Three unknows were found with BFE (R 0.97 ± f =

~ 0.00, 0.09 0.01 and 0.04 ~ 0.00), and two for each of BEM (R ± f 0.32 + 0.00 and 0.04 ± 0.00) and CE (Rf = 0.24 + 0.00 and 0.20 +

0.00).

Overall, the identifiable compounds were: I, III, IV, V, VI,

and VIII. Compound III was not found when both enzyme systems were 82

Table 16. Metabolites of cis-permethrin from Chrysopa carnea homogen­ ates studied in vitro: esterases plus oxidases.

R Values Solvent f Roman System (i ±. SE) Compounds Numerals

BFE 0.96 + 0.01 ciS-term I 0.69 + 0.02 unk._1 0.60 + 0.00 2'-HO-PBacid VI 0.53 +" 0.01 4'-HO-PBacid VIII 0.47 + 0.01 4'-HO-PBalc IV 0.39 +" 0.00 unk. 0.12 + 0.00 unk. 0.05 + 0.00 unk.

BEM 0.93 + 0.01 cis-perm I 0.83 +" 0.01 2'-HO-perm X 0.59 + 0.02 unk. 0.46 +" 0.01 2'-HO-PBalc VIII 0.39 + 0.00 4'-HO-PBalc IV 0,34 +" 0.01 unk. 0.29 + 0.00 unk.

BE 0.90 + 0.00 cis-perm I 0.81 + 0.01 unk. 0.64 +" 0.00 unk. 0.19 + 0.00 unk. 0.15 +" 0.00 m-PBalc V 0.12 + 0.00 4'-HO-PBalc IV

CE 0.94 + 0.01 unk. 0.88 +" 0.01 2'-HO-PBalc VIII 0.80 +" 0.01 unk. 0.74 + 0.02 unk. 0.25 +" 0.01 unk. 0.19 + 0.01 unk. 0.07 +" 0.01 unk. 0.03 + 0.00 unk. 83

'Table 17. Metabolites of cis-permethrin by Chrysopa carnea homogen­ ates studied in vitro: esterases alone.

R Values Solvent f Roman System (i .:t XE) Compound Numerals

1/ BFE 0.97 + 0.00 unk.- 0.66 + 0.00 m-PBacid VI 0.49 + 0.00 4'-HO-PBacid III 0.09 +" 0.01 unk. 0.04 +" 0.00 unk.

BEM 0.91 + 0.00 cis-perm I 0.45 "+ 0.00 4'-HO-PBalc IV 0.38 +" 0.00 m-PBalc V 0.32 "+ 0.00 unk. 0.04 + 0.00 unk.

BE 0.88 + 0.00 cis-perm I 0.134 +" 0.00 unk. 0.25 + 0.02 unk.

CE 0.89 + 0.00 cis-perm 0.86 + 0.02 4'-HO-PBacid III 0.24 "+ 0.00 unk. 0.20 +" 0.00 unk. 0.15 + 0.00 2'-HO-PBalc VIII

1:/ unk. '" unknown. 84 active. Compound X, detectable when both enzyme systems were active, was not observed when oxidases were not active.

Oxidases Alone. From Table 18, the oxidases produced six metabolites from cis with BFE, five with BEM, five for BE, and four with CEo BFE revealed compound I, III, VI and three unknowns (Rf =

0.97 ± 0.00, 0.50 ± 0.00 and 0.12 ± 0.01). Compounds I, VI, VIII and two unknowns (Rf a 0.84 ± 0.01 and 0.57 ± 0.00) were exhibited by BEM. In addition to III and X, three unknowns were discerned by BE (Rf = 0.57 ± 0.00, 0.52 ± 0.00 and 0.47 ± 0.00); whereas CE demonstrated

III, II and two unknowns (Rf = 0.32 + 0.00 and 0.11 ± 0.00).

Summary of cis

For cis with both esterases and oxidases active, compound III

(4'-HO-PBacid) and VIII (2'-HO-PBalc) were found. Compound X (2'-HO­ perm) was only discerned when oxidases were active. Also, compound

III (4'-HO-PBacid) was only found when either oxidases alone or ester­ ases alone were active, but not when both were simultaneously active.

Hetabolites of Trans

Esterases plus Oxidases. Of the seven spots demonstrated with

BFE from esterases plus oxidases acting on trans, only compound VIII

(2'-HO-PBalc) was identified (Table 19). The other six were unknowns

(Rf = 0.97 ± 0.01, 0.60 ± 0.02, 0.38 ± 0.00, 0.20 ± 0.00, 0.08 ± 0.00 and 0.03 ± 0.00). In addition to IV, VIII, and X, four unknowns were discerned by BEM (Rf = 0.95 ± 0.01, 0.29 ± 0.01, 0.11 ± 0.01, and

0.03 ± 0.00). BE differentiated VII plus four unknowns (Rf = 0.81 + 0.01,0.26 ± 0.01, 0.20-± 0.00, and 0.03 + 0.00), whereas CE separated 85

Table 18. Metabolites of cis-permethrin by Chr;:lsoEa carnea homogen- ates studied ---in vitro: oxidases alone.

R Values Solvent f Roman System (x ± SE) Compound Numeral

BFE 0.97 + 0.00 unk)./ 0.95 + 0.00 cis-perm I 0.61 +" 0.01 2""CHO-PBacid VI 0.52 + 0.00 4'-HO-PBacid III 0.50 +" 0.00 unk. 0.12 ± 0.0; unk. BEM 0.91 + 0.01 cis-perm I 0.84 + 0.01 unk. 0.69 +" 0.01 m-PBacid VI 0.57 + 0.00 unk. 0.54 +" 0.00 2'-HO-PBa1c VIII

BE 0.96 + 0.00 4'-HO-PBacid III 0.67 +" 0.01 2'-HO-perm X 0.57 + 0.00 unk. 0.52 +" 0.00 unk. 0.47 + 0.00 unk.

CE 0.85 + 0.00 4'-HO-PBacid III 0.38 +" 0.00 m-PBacid VI 0.32 + 0.00 unk. 0.11 +" 0.00 unk.

1/ unk. a unknown. 86

Table 19. Metabolites of trans-permethrin by Chrysopa carnea homgen­ ates studied in vitro: esterases plus oxidases.

R Values Solvent f Roman System (x ± SE) Compound Numeral

BFE 0.97 + 0.01 unk.1:/ 0.60 + 0.02 unk. 0.46 +" 0.01 2'-HO-PBalc VIII 0.38 + 0.00 unk. 0.20 +" 0.00 unk. 0.08 +" 0.00 unk. 0.03 + 0.00 unk.

BEM 0.95 + 0.01 unk. 0.82 +" 0.02 2'-HO-perm X 0.49 + 0.01 2 1-HO-PBalc VIII 0.41 +" 0.01 4'-HO-PBalc IV 0.29 + 0.01 unk. 0.11 +" 0.01 unk. 0.03 + 0.00 unk.

BE 0.88 + 0.01 2'-HO-PBacid VII 0.81 + 0.01 unk. 0.26 +" 0.01 unk. 0.20 + 0.00 unk. 0.03 +" 0.00 unk.

CE 0.90 + 0.00 trans-perm II 0.81 +" 0.01 unk. 0.22 + 0.00 unk. 0.16 +" 0.01 2'-HO-PBalc VIII 0.13 + 0.00 unk.

1:/ unk. "" unknown. 87

II, VIII and three unknowns (Rf = 0.81 ± 0.01, 0.22 ± 0.00 and 0.13 +

0.00). There£ore~ when the esterases and oxidases were acting together, the identified compounds were: II, IV, VII, VIII and X.

Esterases Alone. BFE resulted in the following compounds from esterase enzymes on trans: II, III, VIII and four unknowns (Rf = 0.98 ± 0.00, 0.63 ± 0.00, 0.39 ± 0.01, and 0.03 ± 0.01) (Table 20). Com- pound VIII was shared with the esterases plus oxidases. The rest of the spots revealed different R£ values except for one unknown (R ~ f 0.03 ± 0.00) under both tests. BEM separated seven compounds: III, III, IV, VIII and three unknowns (Rf = 0.96 + 0.01, 0.31 ± 0.01, and 0.03 ± 0.00). Trans and four unknowns (R = 0.90 + 0.00, 0.84 + f - 0.01, 0.27 ± 0.00, and 0.21 + 0.00) were differentiated by BE. Six spots resulted from CE = II, IV, VIII and three unknowns (Rf = 0.79 + 0.02, 0.22 ± 0.00, and 0.11 ± 0.00).

Compounds o£ trans produced by esterases were: II, III, IV and

VII. The common metabolites between the esterases plus oxidases tests and the esterase alone were: IV (4'-HO-PBalc) and VIII (2'-HO-PBalc).

Oxidases Alone. Four compounds were separated by BFE: III,

VIII, and two unknowns (Rf = 1.00 ± 0.00 and 0.97 ± 0.00) when oxidases were acting alone on trans (Table 21). BEM differentiated six com- pounds II, VIII, IX, and three unknowns (Rf = 0.84 ± 0.01, 0.64 ± 0.01, and 0.55 ± 0.55). Compounds VII, IX, and two unknowns (R = 0.52 + f 0.00 and 0.44 ± 0.00) were detected by BE. In addition to IV, V, and

VI, one unknown (R£ a 0.43 ± 0.00) was separated by CEo The overall compounds produced by the action of oxidases were II through IX, inclu- sive. 88

Table 20. Metabolit"es of trans-permethrin by Chrysopa carnea homogen­ ates studied in vitro: esterases alone.

R Values Solvent f Roman System (~ :t SE) CompoundY Numeral

BFE 0.98 + 0.00 unk. 0.94 +" 0.00 trans-perm II 0.63 +" 0.00 unk. 0.51 + 0.01 4'-HO-PBacid III 0.43 +" 0.01 2'-HO-PBalc VIII 0.39 + 0.01 unk. 0.03 +" 0.01 unk.

BEM 0.96 + 0.01 unk. 0.90 + 0.00 trans-perm II 0.51 +" 0.01 2'-HO-BPalc Viii 0.43 +" 0.00 4'-HO-PBalc IV 0.31 + 0.01 unk. 0.16 +" 0.00 4'-HO-PBacid III 0.03 + 0.00 unk.

BE 0.90 + 0.00 unk. 0.84 +" 0.01 unk. 0.78 + 0.00 trans-perm II 0.27 +" 0.00 unk. 0.21 +" 0.00 unk.

CE 0.89 + 0.00 trans-perm II 0.79 + 0.02 unk. 0.22 +" 0.00 unk. 0.15 + 0.01 2'-HO-PBalc VIII 0.11 +" 0.00 unk. 0.09 + 0.00 4'-HO-PBalc IV

1./ unk .... unknown. 89

'Table 21. Metabolites of trans-permethrin by Chrysopa carnea homogen­ ates studied in vitro: oxidases alone.

R Values Solvent f Roman System (i ± SE) Compound Numeral

BFE 1.00 + 0.00 unk.1/ 0.97 + 0.00 unk. 0.48 +' 0.02 4'-HO-PBacid III 0.42 +' 0.02 3'-HO-PBalc VIII

BEM 0.92 + 0.01 trans-perm II 0.84 +" 0.01 unk. 0.72 +' 0.00 4'-HO, t-perm IX 0.64 +" 0.01 unk. 0.55 +' 0.00 unk. 0.51 + 0.00 2'-HO-PBalc VIII

BE 0.86 + 0.04 2'-HO-PBacid VII 0.58 + 0.01 4'-HO, t-perm IX 0.52 +' 0.01 unk. 0.44 + 0.00 unk.

CE 0.95 + 0.00 m-PBalc V 0.43 +' 0.00 unk. 0.39 + 0.00 m-PBacid VI 0.08 +" 0.00 4'-HO-PBalc IV

1/ unk. Q unknown. 90

Esterases plus o~idases shared the following metabolites with the oxidases alone: IV, VII, and VIII. Meanwhile the esterases shared the following with o~idases: III, IV, and VIII. Compound X was only produced when both enzymes were active.

Summary of Trans

From the above, oxidases metabolized trans to seven identi- fiable metabolites, whereas esterases metabolized it to only three identifiable metabolites. The two enzymes acting separately did not share any of the unknowns.

Comparison of the Two EnZyme Systems in vitro

Cis and trans shared the following metabolites when both es- terases and oxidases were active: IV, VII, VIII, and X. The follow- ing compounds were common when only esterases were active: III, IV, and VIII. When oxidases acted alone, III, VI, and VIII were identical between cis and trans. From this comparison, VIII (2'-HO-PBalc) was the common metabolite from both enzyme systems and with both isomers.

Comparison of the in vivo and in vitro Metabolism of Permethrin by Chrysopa carnea TLC

Permethrin (CiT) For CiT, the 24 and 48 hr in vivo and in vitro experiments, showed only on common compound III (4'-HO-PBacid) in the BFE solvent system on TLC. In vitro shared metabolite VIII (2'-HO-PBacid) with the 24 hr experiment, and VII (2'-HO-PBalc) with the 24 hr experiment 91 using BE. They all exhibited metabolite IV (4'-HO-PBalc) with CEo

Meantime, metabolite VI (m-PBacid) was discerned at both the 24 and 48 in vivo experiments. This latter compound was only detected in vitro with BE.

Overall, compounds detected in the 24 hr in vivo experiment were I through VII and X; those of 48 hr were I through IX, inclusive.

The in vitro products were I through IV, VI, VII and VIII. Thus, V, II, and X were not present in vitro, whereas V was detected at both the 24 and 48 hr in vivo studies. Compound IX was absent at 24 hr and X was missing at 48 hr.

Cis

The 24 hr in vivo products for cis were I, IV, V, VI and IX.

Those of the 48 hrs were I, III, V, VI, VII and VIII. The in vitro experiment produced I, IV, V, VI, VII and X. Compounds shared among the two treatments and both times were I, V, and VI. In addition to the above mentioned compounds, the in vitro study shared VII with the

48 hr and IV with the 24 hr. Compound VIII was produced exclusively at 48 hr, IX at 24 hr, and X only in the in vitro experiment.

Trans Products of trans at 24 hr were II, IV, VI, VIII and IX. The

48 hr test produced II, IV, V, VI, IX and X. The in vitro study re- sulted in II, IV, VII, VIII and X. The common products were II

(tralls-permethrin) and IV (4'-HO-PBalc). Compounds II, IV, and VIII were common for in vitro and the 24 hr, whereas II, IV and X were 92

'shared among in vitro-and 48 hr. Compound VII (2'-HO-PBacid) was de­

tected only in the in vitro study.

The shared unknown among all studies, in vivo and in vitro,

with CiT, cis and trans was a compound exhibiting an R value ranging f

between 0.03 and 0.04 when CE was used. A compound with an Rf value ranging between 0.75 and 0.78 was detected in all experiments in BFE.

BE revealed the presence of a compound with an Rf value between 0.25

and 0.28 for all experiments. For the rest of the unknowns, some of

them shared the same range of Rf values, especially between the 24 and

48 hr, and the others did not. DISCUSSION

Methodology

Gas-Liquid Chromatography/ Electron-Capture Detector

The modification of Shono's et ale (1978) procedure by Shour and Crowder (1980)_~llowed the use of GLC/ECD instead of TLC. Tissue art~facts were determined by extracting larval tissue, followed by the same experimental conditions and extraction procedures. No meta- bolites arising from CiT in GLW were detected (Figure 4); only cis and trans were recorded. Metabolites might be expected because the acidic moiety of CiT contains chlorine (Shour, 1979). However, for the detection of such metabolites by ECD, they should first be methy- lated. Instead, TLC was used for metabolite identification rather than the methylation process.

Efficiency of Recovery (ER)

When ER was determined for spiked tissue of control larvae over time, a declining trend was obtained. The best recovery occurred for insects dosed two- to four-hr, and the lowest for 40- to 50-hr, after dosing (Table 1). There was a difference of more than 10% in the ER of cis when extracted at two- to four-hr than when extracted at 40- to 50-hr. Binding of CiT to the tissues might explain the presence of an unextractable fraction (Shour, 1979). Based on tissue extracts (Figure 4A and 4B), two small tissue artifact peaks were

93 94

'observed at five- to-ten-hr after dosing. At 25- to 50-hr, two dif-

ferent peaks were found; one had the same retention time but different

height; the second was exclusive for 25- to 50-hr, with a longer re-

tention time and higher peak height than any of the other artifacts.

The physiological changes which might have occurred since the insects

were dosed (beginning of the third-ins tar) until the end of the ex-

periment (two days) could be responsible for the different binding

capacities with CiT and its isomers. These experiments confirm the

work ofShour (1979) which showed that the majority of the compound did

not bind to the GLW tissues. Our techniques recovered a higher per-

centage (> 89%) than did Shour « 80%).

Conjugated Compounds

An experiment was conducted to study the possibility of con-

jugated metabolites occurring in the in vitro studies. After two hr

of incubation, the mixture was extracted with chloroform and methanol-

water as previously mentioned. With TLC, the only detected spots

belonged to the parent compound; thus, evidence of conjugated com-

pounds was lacking for this reason, only CL was used for the in

vitro studies.

Metabolism of Permethrin by Chrysopa carnea Studied in vivo

GLC Surface Fraction

The surface fraction secured at 24 and 48 hrs showed that less

than 0.1% of CiT or either of its two isomers was present as unmeta­

bilized compound (Table 3). Bigley and Plapp (1978) using l4C_acid 95 and alcohol-labelled cis and trans with the bollworm. recovered 7.3% of trans-alcohol, and 15.5% of cis-alcohol from the surface fraction.

Therefore, with the same procedure, there was a dramatic difference be­ tween bollworm and GLW: between 73 to lSS-fold for trans, and 123- to

ISS-fold for cis. Even though the authors were comparing the bollworm with TBW, they did not publish data for the surface fraction from TBW.

The bollworm was known to be susceptible to CIT, yet the surface frac­ tion results indicated the slow penetration of CIT through the cuticle contrary to what was found in the present work with the tolerant GLW.

The surface fraction studies were by no means indicative of cuticular penetration as a tolerance mechanism. Even though less than

0.1% of the parent was obtained on the surface, these compounds could still be bound within cuticular layers. Thus, cuticular penetration as a mechanism of tolerance remains unresolved.

Cup-Fraction

When CIT was used, about 1.3% of cis and 1.0% of trans were detected in the cup fraction even after 48 hr from dosing (Table 4).

Less than 1.0% was found when the pure isomers were used. These residues resulted from the insect's contact with the cup's surface and excretion. Bigley and Plapp (1978) showed that TBW excreted

15.9% of cis-acid, 27.6% of trans-acid, 22.2% of cis-alcohol, and

32.3% of trans-alcohol, while bollworm, the more susceptible species, excreted 18.3, 19.6, 19.4 and 22.0%, respectively. These are at least 159 to 323-fold higher than what was found for GLW in the cup fraction. 96

Bond (1978) observed copious amounts of what he called urine being excreted from GLW larvae, but only when the insects had finished

their meals and were inactive. Shour (1979) referred to a brown liquid

excreted irregularly by the control larvae in the cups; this excretion became regular when the insects were dosed. Our results showed regu1-

arity in both cases. Anyhow, this brown liquid, which was part of the

cup fraction, did not exhibit significant amounts of any of the parent

compounds and appears not to be an explanation for CUI tolerance.

Killington (1937) and Imms (1977) reported that chrysopid

larvae as well as Apocrita (Hymenoptera), Glossina and other viviparous ~- ~L r.--', ," ~~ "e Diptera are unique among insects because their midgut was separated

from their hindgut by an embryonic plug, thus preventing bulk move-

ment of ingesta through the tract. Solid waste materials

were eliminated following the imaginal molt. This solid waste was

homogenized with the larvae in the present work. Therefore, unless

the solid waste is removed from the larvae before homogenization and

extracted separately, the contribution of excretion to GLW tolerance

will remain unanswered.

Internal Fraction

GLW larvae metabolized both isomers of CiT to a very high per-

centage (Table 2). Almost 80% of cis and 71% of trans were degraded within the first two hr after dosing. The cis isomer, which proved

to be more toxic to all previously tested insects (Bigley and P1app,

1978; Shono et al., 1978; Jao and Casida, 1974; Ishaaya and Casida, 97

,1980; Abd-Aal and Soderlund, 1980), was metabolized at a significantly'

higher percentage than trans in GLW.

In Shono's et al. (1978) study of CiT metabolism in adult

American cockroach, adult housefly, and cabbage looper larvae, cis

was recovered in slightly larger amounts than trans from the cockroach,

meaning that trans was metabolized at a higher rate than cis. In

houseflies and their excreta 48 hr after topical application, approx­

imately two-fold greater cis than trans was found. Cabbage looper

larvae metabolized trans much more rapidly than cis; unmetabolized CiT

in the larvae after 48 hr contained approximately eight-fold more cis

than trans. These authors concluded that cis is generally more insec­

ticidal than trans. This may be partially attributable to the greater

resistance of cis than trans to metabolism, particularly in cabbage

looper and housefly.

GLW was more susceptible to trans than to cis (Shour and

Crowder,1980). Shour (1979) reported that cis was metabolized more

than trans but failed to study the cup fraction lending doubt to his

conclusion. The results reported herein confirm Shour's conclusion -

GLW are able to metabolize both isomers at high rates but cis is

metabolized more rapidly than trans; this could be a primary factor

concerning the tolerance mechanism in GLW. The higher toxicity of

trans compared to cis in GLW is therefore ascribed to ~ higher

stability against degradation in this insect which is different from

other insects susceptible to CiT. 98

Comparison of Permethrin Metabolism by Chrysopa carnea and Heliothis virescens Studied in vitro

In vivo studies showed that GLW degraded CiT to a very high percentage, and the question was raised whether this rate had a bearing on GLW tolerance. A study was therefore conducted to compare CiT metabolism of the tolerant GLW with a susceptible insect, TBW.

After two hr of incubation, TBW homogenates degraded cis and trans almost equally, 24.9% and 26.2%, respectively, when both enzyme systems were active. Data on the in vivo metabolism of CiT by TBW were reported by Bigley and Plapp (1978). They showed that trans prepara- tions (acid- and alcohol-labelled) were metabolized more readily than the cis preparation. From the internal fraction they recovered 22.9% and 45.6% of cis-acid and alcohol-labelled for fifth instar larvae; meanwhile, these larvae excreted 15.9 and 22.2%, in that order. For trans, 6.0% and 13.2% acid- and alcohol-labelled were recovered from the internal fraction, but 27.6% and 32.3% were excreted. This means fifth ins tar larvae of TBW were able to metabolize 61.2% of cis-acid and 32.2% of cis-alcoho1-1abelled. On the other hand, 66.4% trans- acid and 54.5% trans-alcohol-1abelled were metabolized. It should be pointed out in comparing these values to those reported herein, fourth ins tars were used here instead of the fifth ins tar larvae. The reason for using fourth instars was the desire for susceptibility to compare the GLW and TBW larvae. The latter tend to be more tolerant in the advanced instars (P1app, 1973).

GLW degraded 71% cis and 42% trans when both esterases and oxidases were active. This repr.esents a 1.7-fold faster cis degradation 99 than tran~, whereas TBW metabolized trans in a slightly higher rate than cis. GLW metabolized cis at 2.85-fold faster than TBW; again for trans, it was 1.6-fold in favor of GLW. Comparing Shono's et al.

(1978) work with cockroach, housefly, and cabbage looper larvae, the cockroach metabolized cis at almost the same percentage as GLW, but the housefly degraded it at higher percentage than both; the cabbage looper was the lowest. For trans, all three insect species metabolized it at a higher percentage than GLW. All three species metabolized trans more intensively than cis, the reverse occurred with GLW. The

TBW strain used in the present work behaved similarly to the afore­ mentioned three species, but degraded CiT at significantly lower rates than cockroach, housefly, cabbage looper, and GLW.

Oxidases of GLW degraded cis 2.72-fold faster than TBW, and trans 2.23-fold faster. Similarly, esterases of GLW degraded cis

1.89-fold and trans 1.43-fold faster than TBW. Soderlund and Cas ida

(1977) showed that trans was more vulnerable to hydrolysis than oxi­ dation, and cis degradation was oxidative with a very slow hydrolytic rate when mouse liver microsomal enzymes were incubated with CiT.

The results of the present work for TBW showed that cis was equally degraded by means of hydrolytic and oxidative mechanisms, yet trans was affected more by oxidation. Either system, when acting alone, was able to degrade CiT isomers at higher percentages than when both systems were simultaneously active. This finding agreed with Shono et al. (1978) who reported that when one pathway was blocked, the other increased its degrading activity. Bigley and Plapp (1978) working 100 with TBW and bollworm declared that CiT metabolism was both hydrolytic

and oxidative. They emphasized the importance of hydrolysis in cis and oxidation in trans metabolism. The present work agreed with their finding about trans, but for cis, both hydrolysis and oxidation were equally important for TBW.

With GLW, oxidative mechanisms were more important in detox­

ifying CiT isomers than hydrolysis. Both isomers were equally meta­ bolized when oxidases were active alone, but cis was more vulnerable

to hydrolysis than trans. The reverse was found with mouse liver microsomal enzymes by Soderlund and Casida (1977). Ishaaya and Casida

(1981) declared that GLW larval enzymes had unusually high activity and unique specificity for hydrolyzing cis two- to three-fold faster

than trans. They reported that pyrethroid esterase(s) was the major factor contributing to the natural pyrethroid tolerance of GLW larvae.

However they used a California strain of GLW. Shour (1979) reported

that the California strain responded differently from Arizona GLW to­ ward the five pyrethroids tested. The latter author studied the ef­ fects of these pyrethroids on paralysis, failure to pupate, and mortality. No difference was observed between the two strains con­ cerning failure of pupation; however, Arizona insects showed prog­ ressive effects over 24, 48, and 72 hr whereas effects increased up to

48 hr with California GLW. The California insects were more suscept­

ible to paralysis when treated with pydrin or CiT than Arizona GLW.

The hypothesis of the present work was whether GLW tolerance was due to a high metabolic rate of CiT. Previous studies with r~sis­

tant TBW showed that resistance was due primarily to increased 101

- detoxication mechanisms involving a1i-esterases, MFO, and conjugating enzymes (Whitten and Bull, 1970; Bull and Whitten, 1972; Williamson and

Schecter, 1970; Plapp, 1973). Bigley and P1app (1978) reported that metabolism was more rapid in TBW (resistant) than the bollworm (sus- ceptible), a factor probably responsible for the two-fold greater tolerance of TBW to CiT.

GLW degraded cis and trans faster than the susceptible TBW.

Therefore, tolerance of GLW larvae was strongly related to their fast metabolism of CiT isomers. When oxidases alone were active, GLW meta- bo1ized cis and trans faster than TBW. Also esterases of GLW hydro- lyzed cis and trans faster than TBW. Thus, CiT detoxication was attributed to both hydrolytic and oxidative mechanisms. The results herein agreed with Shono et al. (1978) that when one pathway was blocked, the other increased its degrading activity. This was obvious when esterases were inhibited and oxidases were acting alone; oxi- dases clearly dominated detoxication.

Holden (1979) working with CIT on P. americana obtain similar results. He stated that cis, although more resistant to metabolic attack, was slowly hydrolyzed and oxidative degradation was probably

the major component. The present work showed that: 1) both isomers were equally metabolized when oxidases were active alone, 2) cis was more vulnerable to hydrolysis than trans, 3) the presence of both enzyme systems was important for detoxication, and 4) cis was more readily degraded than trans in the presence of both active sys-

terns for GLW. Therefore, metabolism of CiT by GLW was primarily oxidative with hydrolysis as a secondary mechanism for both isomers. 102

Trans appears to be more toxic than cis because of trans slower rate of metabolism by GLW larvae.

In summary, the hypothesis that GLW tolerance toward CiT is related to a high detoxication rate proved to be true. Both oxidative and hydrolytic mechanisms are involved in CiT metabolism. Oxidation was the primary mechanism while hydrolysis is secondary in importance.

Toxicity of trans to GLW is probably related, at least in part, to its slower rate of detoxication.

Metabolism of Permethrin by Chrysopa carnea Studied in vivo

Permethrin (CiT)

The following non-conjugates were formed from CiT by GLW at 24 hr: 4'-HO-PBacid (III); 4'-HO-PBalc (IV); m-PBalc (V); m-PBacid (VI);

2'-HO-PBacid (VII); and 2'-HO-perm (X). In the work of Bigley and P1app

(1978), TBW exhibited III, IV, VI and VIII when trans was employed; and

III, IV, VII, and VIII when cis was used. The bollworm metabolized trans to III through VIII, inclusive, which was two more metabolites than TBW (V and VI). Cis metabolites were identical for both species.

Compound VII, observed in GLW, TBW and bollworm, was not found in the following species: adult American cockroach, adult housefly, cabbage looper, and the rat (Shono et al., 1978; Gaughan et al., 1977).

Actually all three insects studied were not able to produce compounds hydroxylated at the 2'-position. The rat was able to form only one compound (2'-HO, c-perm) with cis, but not trans. The 2'-HO, c-perm was then hydrolyzed to 2'-HO-PBacid-sulfate. 103

Compounds VIII and IX did not occur in GLW. But compound VII, which is the acid form of VIII, was present. This could be explained by the existence of a highly active oxidative system, i.e., MFO system.

Perhaps all of the alcohol had been converted to an acid (VIII to VII) by 24 hr. Compared to the rat, the alcohol (VIII) was present but the acid (VII) was found in a conjugated form with sulfate (Gaughan et al.,

1977). Compound X (2'-HO~erm) was not present in any of the afore­ mentioned species, but both TBW and bollworm formed a compound which was hydroxylated in the 2'_ and 4'-positions at the same time (2',

4'-HO-perm).

The presence of the alcoholic moiety metabolites would result from hydrolysis, and the existence of X points to the hydroxylation at the phenoxybenzyl (PE) group before hydrolysis occurs. Both the 2'_ and 4'-positions of the PB group were vulnerable to hydroxylation.

The two other positions, 5'_ and 6'_, were not identified because of the unavailability of these standards. However Shono et al. (1978) reported that the 6-position was found only in house flies and the 2'_ position in rats for CiT. The S'-position was described only for de­ camethrin. Therefore, GLW is comparable to the TBW and bollworm in most cases, and nearer to the rat than cockroach, housefly, and cabbage looper. Thus, GLW produced an oxidase metabolite (X), and esterase metabolite (V), and secondary metabolites (III through VIII inclusive).

2'-HO-perm (X) was the only standard not detected at 48 hr.

This could mean that after being hydroxylated, X was then hydrolyzed 104 with esterases. As was previously mentioned, not one of the other

species wa~ able to produce this compound. TBW and bollworm formed

2', 4'-perm (not available as a standard). Compounds VIII (2'-HO-

PBa1c) and IX (4'-HO, t-perm) were also found at 48 hr but absent from

the 24 hr examination. The presence of VIII might mean that the hydro-

lytic process was still occurring at 48 hr. This compound was also de-

tected in the rat from cis, TBW with trans, and bollworm with both

isomers.

4'-HO, t-perm (IX), which is a product of trans-permethrin

hydroxylation, was found in GLW, similar to the adult American cock-

roach and the rat. This finding could again be explained by the ac-

tivity of the MFO system. GLW could have been hydroxy1ating the

remainder of the parent as a first step followed by hydrolysis. This

last step has been shown in both the rat and cockroach (Shono et a1.,

1978; Gaughan et al., 1977; Holden, 1979).

Even after 48 hr, the two isomers of CiT (I and II) were still

detected as non-conjugates. But traces of I and II were also detected

in M-WL at both 24 and 48 hr. This means that GLW was not able to

completely detoxify CiT. This unmetabo1ized portion of CiT was the

same portion that was recorded in the in vivo studies employing GLC/ECD.

In the American cockroach, 22.5 and 24.7% of trans and cis were not metabolized even after 24 hr; at that time the cockroaches were para-

1yzed. After 48 hr, the housefly exhibited 1.9 and 8.8% unmetabo1ized

trans and cis in the insect and 5.1 and 6.6% in excreta. In the

cabbage looper, unmetabo1ized trans and cis were 5.6 and 47.1% in

insects, and 2.1 and 0.8% in excreta (Shono et a1., 1978). 105

Other metabolites found in M-WL of GLW were: 4'-HO-PBacid (III), m-PBalc (V), and 2'-HO-PBacid (VII) at 24 hr; m-PBacid (VI), VII, and X at 48 hr. In the cockroach, housefly, and cabbage looper, III and IV were conjugated with glucosides. Compound VI was conjugated with glutamic acid in the cockroach and housefly, and glutamine in cockroach.

The type of conjugate for GLW still awaits to be revealed.

Compound VII was exhibited only by GLW and rat. The rat con­

jugated VII immediately with sulfates. 2'-HO-perm (X) was vulnerable

to conjugation with glucosides in housefly and cabbage looper. It was hydrolyzed by both the cockroach and rat.

In general, the absence of 2'-HO-PBalc (VIII) and 4'-HO-PBalc

(IV) at 48 hr could be explained only by high MFO activity of GLW.

That means a further oxidation of the alcohol to the acid form: VIII

to VII, and IV to III. These oxidation processes would have been very rapid because the alcoholic compounds were detected in all the previous animals.

Cis

GLW were able to metabolize cis to all available standard com­ pounds. Compound IV disappeared after 24 hr and was probably oxidized to V. Compound IX also disappeared and could have been hydrolyzed or conjugated as mentioned earlier. Compounds VII and VIII were formed instead of IV and IX; that means the 2 '-position was favored over the

4 '-position when cis was employed.

It appeared that the 2'-position was the favored site of hydro­ xylation with cis. The only 4'-position compound discerned was 4'-HO, 106

t-perm (IX). It should not have been detected, probably indicating cis was not 100% pure. None of Shono's et al. (1978) subject insects were

able to produce the 2'-position hydroxylation; nevertheless, TBW, boll­ worm and rat were hydroxylating cis at the 2'-position. One metabolite of trans (VIII) and 2',4'-HO-perm wereproduced by TBW and bollworm. The rat did not produce any 2 '-position metabolite from trans. Therefore,

GLW was behaving to some extent like the Heliothis complex.

Trans

Compo~~d II, IV, VI, VII through X were produced by 24 hr from

trans. In addition to what was found at 24 hr, the 48 hr examination exhibited III and V instead of IV. Compound III could have resulted

from the oxidation of IV. Compound V could have been a product of further unmetabolized trans hydrolysis as shown by Shono et al. (1978) and Gaughan et al. (1977).

2'-HO-PBacid (VII) and 2'-HO-perm were detected only in M-WL and could have been proceeding toward conjugation. As previously dis­ cussed, VII was found only in rats, but X was not formed by any of these aforementioned species.

Trans was vulnerable to hydroxylation in GLW at both the 2'­ and 4'-positions, unlike cis which was favored at the 2'-position.

Cis is the more toxic isomer to all insects except GLW. All other in­ sects were able to hydroxylate cis at the 4'-position most of the time.

Thus, hydroxylation at the 4'-position does not appear to explain the tolerance of GLW. The latter insect as well as the resistant TBW were able to hydroxylate cis at the 2'-position. The results of the in vivo 107 metabolism of CiT by GLW showed that cis was metabolized more readily than trans. Therefore, the 2'-position hydroxylation of the PB is suggested to be one of the reasons for GLW tolerance to cis-permethrin as such, and CiT in general.

Metabolism of Permethrin by Chrysopa carnea Studied in vitro: TLC

When esterases plus oxidases were active, GLW metabolized CiT to all available standard metabolites except m-PBalc (V), 4'-HO, t- perm (IX); and 2'-HO-perm (X). Compound V was not produced by TBW but was found in the bollworm (Bigley and Plapp, 1978). It was found in the rat (Gaughan et al., 1977), cockroach, housefly and cabbage looper (Shono et al., 1978). In the latter insects, V was either con- verted to VI then conjugated with an amino acid, or immediately conjugated with glucosides. This compound was produced by either CiT isomer. The 2'-HO compounds were not produced by any of the following insects: cockroach, housefly, and cabbage looper (Shono et al.,

1978), whereas GLW produced two metabolites hydroxylated at the 2'- position. These metabolites were VII and VIII. Rat (Gaughan et al.,

1977), TBW and bollworm (Bigley and Plapp, 1978) produced the 2'- position metabolites. The latter two species were able to hydroxy- late CiT at two positions at the same time leading to 2', 4'-HO-perm.

When esterases of GLW were active alone, CiT resulted in all metabolites including V and IX. The missing metabolites were 4'-

HO-PBacid (III) and 2'-HO-perm (X). Compound IX, which is a hydroxy- lation product of permethrin was not produced by TBW, bollworm 108

(Bigley and P1app, 1978), rat (Gaughan et al., 1977), housefly, nor

cabbage looper (Shono et al., 1978). The only insect reported able to

produce it was~. americana, perhaps produced from the cis isomer be-

cause Holden (1979) reported that cis was more resistant to hydrolysis;

and the oxidative attack was the major component in ~. americana. It

is suggested that GLW first hydroxylated the cis isomer minimizing its

toxicity, followed by hydroxylation, whereas trans was open for hydro-

lysis and hydroxylation was not required.

The action of oxidases on CiT secured the following metabol-

ites: III through VI, VIII and X; 2'-HO-PBacid (VII) and 4'-HO, t-

perm (IX) were absent. Compound X which was not produced by either

este,rases plus oxidases, or esterases alone, was formed when oxidases were active alone.

Elliott et ale (1976) stated that CiT was hydroxy1ated to some extent before hydrolysis. Compound III was also produced by cockroach, housefly (Shono et a1., 1978) and rat (Gaughan et a1.,

1977) but not by cabbage looper. Compound IV was produced by all four species and GLW; the same was true for V and VI. Compound VIII which was found in GLW was also formed by TBW and bollworm (Bigley and P1app, 1978). TBW produced III, IV, and V, but not VI which was found with bollworm. Neither degraded CiT to IX or X. -3 Surprisingly, despite the use of 10 M DDVP, hydrolysis products of CiT were found; the same finding was reported by Shono et al. (1979) when TEPP was applied. The range used by other in- vestigators was at least 100-fold less than here. This could explain 109 the finding of Abd-Aal-and Soderlund (1980), and Ishaaya and Casida (1980) who stressed the presence of a number of esterases acting on

CiT. These esterases had different sensitivities to inhibitors.

To summarize, hydroxylation at the 2'-position seems to be important for GLW tolerance to CiT. Hydroxylation could be the first step in detoxifying CiT and its isomers, followed by hydrolysis. Com­ pound III and X were exclusively produced when oxidases were active; esterases were responsible for VII and IX. With both enzyme systems active, the homogenate restored its ability to produce III and VII, but not IX and X which ~ould be explained by the rapid hydrolysis after hydroxylation.

Cis was metabolized to IV through VIII and X when both systems were active. Compound VII (2'-HO-PBacid) was not formed by any of the" previously mentioned insect species or rat except the bollworm. In the rat, VIII went through conjugation with the sulfate as 2'-HO­

PBacid-sulfate; this means it was conjugated as fast as it was produced.

All species exhibited IV, V, VI, VIII, but not X which occurred in GLW.

All species including GLW did not degrade cis to IX. Esterases alone behaved the same as esterases plus oxidases with one exception, IX was not formed. This means that GLW behaved like all other species except for the occurrence of 2'-position compounds. Oxidases degraded cis to

III, VI, VIII and X. Compounds IV, V, VII and IX were not discerned.

This means all alcohol metabolites (IV and V) were oxidized to acids

(III and VI) as soon as they were produced because of the availability of NADPH. None of the 2'-HO compounds was formed. 110

When trans was used as a substrate, esterases plus oxidases de­ graded it to IV, VII, VIII and X. 4'-HO-PBacid (III), m-PBalc (V), m-PBacid (VI), and 4'-HO, t-perm (IX) which were formed when the rat was the subject (Gaughan et al., 1977) were missing from GLW. The cab­ bage looper, similar to GLW, did not form III or X (Shono et al., 1978).

But the cabbage looper as well as f. americana, the housefly and the rat produced V and VI (Shona et al., 1978; Gaughan et al., 1977). Com­ pound IX (4'-HO, t-perm) could undergo hydrolysis followed by gluco­ sidation or glucuronidation, or immediate glucosidation or glucuronida­ tion without being hydrolyzed. The former route was found in the cockroach, housefly, and cabbage looper; the latter pathway occurred in housefly and cabbage looper (Shono et al., 1978). In the rat X first went through hydrolysis (Gaughan et al., 1977). Esterases,-when acting on trruls, produced III, IV, and VII; the oxidases produced III through

IX, inclusive. This clearly showed the effectiveness of oxidases in degrading CiT especially trans. Therefore, oxidases used several hydroxylation routes to degrade trans whereas esterases went through one hydrolytic route. Compound III was produced by the esterases of the following species: rats (Gaughan et al., 1977), housefly, cockroach, cabbage looper (Shono et al., 1978), TBW and bollworm (Bigley and Plapp,

1978). All the species except the rat were able to produce IV whereas

VII was produced only by the bollworm.

As stated earlier Shour and Crowder (1980) stated that trans was more toxic to GLW than cis. This could be further explained by the limited routes by which esterases acting alone can degrade this 111 isomer. Only three metabolites were produced with esterases while the oxidases produced seven metabolites. SUMMARY

Larvae of GLW are known to be tolerant to pyrethroids including

CiT. Tolerance could be due to one or more of the following mechanisms:

1) high esterase activity, 2) high MFO activity (MFO), 3) cuticular penetration, 4) excretion, and 5) low nerve target sensitivity. The first two mechanisms were studied to determine if they were related to this tolerance.

GLW larvae treated with CiT were able to degrade 80% of cis and

71% of trans within two hr from dosing; approximately 95% of both cis and trans were degraded by 50 hr. GLW were able to degrade cis-faster than the trans-isomer.

Metabolism of CiT by GLW and a susceptible insect, TBW, was com- pared in vitro. GLW dE\graded cis faster than trans while TBW larvae metabolized trans at a slightly higher rate than cis. Cis and trans were metabolized about 3- and 2-fold faster by GLW than by TBW when both esterases and oxidases were active. Oxidases of GLW acting alone again metabolized cis and trans about 3- and 2-fold faster than TBW oxidases; esterases of GLW also hydrolyzed CiT faster than TBW ester- ases. Thus, tolerance of GLW to CiT was attributed to an efficient detoxication mechanism.

When studied separately, both isomers were equally metabolized by GLW when oxidases were active alone. Cis was more vulnerable to hydrolysis than trans. The presence of both enzyme systems was impor- tant for detoxication and cis was more readily degraded than trans.

112 113

I Therefore, metabolism of" CiT by GLW was primarily oxidative with hydro­

lysis as a secondary mechanism for both isomers. Trans appeared to be

more toxic to GLW because of its slower rate of detoxication.

The fate 'of CiT, cis and trans-isomers of permethrin was

studied at 24 and 48 hr after topical application to GLW larvae. CiT

metabolism resulted in the identification of: cis- and trans- permeth­

rin; 4'-HO-PBacid; m-PBalc; 2'-HO-PBacid; m-PBacid; and 4'-HO, t-perm.

A number of unknowns was found depending on the solvent system used

for TLC. The unknowns might have been metabolites of the acidic

moiety andlor conjugated compounds. Cis metabolites found were: 4'-HO­

PBacid; 4'-HO-PBalc; m-PBalc; m-PBacid; 2'-HO-PBacid; 2'-HO-PBalc; 4'­

HO, t-perm; and 2'-HO-perm. Therefore, based upon the number of

identified metabolites and unknowns, cis was metabolized more inten­

sively than trans by GLW.

The 2'-position could be one of the reasons for GLW to toler­

ance because the American cockroach, housefly, cabbage looper, and

rats were reported unable to hydroxylate either isomer at the 2'­

position, whereas the resistant TBW could. Furthermore all the species,

tolerant and susceptible, were able to hydroxylate CiT at the 4'­

position.

The fate of CiT, cis- and trans-isomers of permethrin was

studied in vitro with larval homogenates of GLW. The effects of

esterases plus oxidases, esterases alone, and oxidases alone were in­

vestigated. For CiT, esterases plus oxidases resulted in the identi­

fication of: 4'-HO-PBacid, 4'-HO-PBalc, m·"PBacid, 2'·-HO-PTIacid, and

2' -HO-PBalc. When esterases were active alone, the following ~vas 114

,found: 4'-HO-PBalc~ m-PBalc, m-PBacid, 2'-HO-PBalc, 2 '-HO-PBacid , and

4'-HO, t-perm. With oxidases, the following was observed: m-PBacid,

m-PBalc, 4'-HO-PBalc, 4 '-HO-PBacid , 2'-HO-PBalc, and 2'-HO-perm. Thus,

oxidases were responsible for the production of 4'-HO-PBacid and 2'-HO­

perm, while esterases were responsible for 2'-HO-PBacid and 4'-HO, t­

perm. In the presence of both enzyme systems, the homogenates ability

was restored to produce 4'-HO-PBacid and 2'-HO-PBacid. But it failed

to secure 4'-HO, t-perm and 2'-HO-perm which could be explained by the

immediate hydrolysis of these two compounds as soon as they were formed

and/or immediate conjugation.

The following was observed from cis when esterases plus oxi­

dases were active: 4'-HO-PBalc, m-PBalc, m-PBacid, 2'-HO-PBacid, 2'­

HO-PBalc, and 2'-HO-perm. When estereases were active alone, cis

produced the following compounds which were identified: 4 '-HO-PBacid ,

4'-HO-PBalc, m-PBacid, m-PBalc, and 2'-HO-PBalc. From oxidases: 4'­

HO-PBacid, m-PBacid, 2'-HO-PBalc, and 2'-HO-perm were recorded.

Identified metabolites of trans when esterases plus oxidases

were active were: 4 '-HO-PBacid , 4'-HO-PBalc, 2'-HO-PBacid, and 2'-HO­

perm. Esterases acting alone produced: 4 '-HO-PBacid , 4'-HO-PBalc,

and 2'-HO-PBacid. Based on the number of chromatographic spots, oxi­

dases were able to produce more compounds than either esterases plus

oxidases or esterases alone. The following were recorded with oxi­

dases alone: 4'-HO-PBacid, 4'-HO-PBalc, m-PBalc, m-PBacid, 2'-HO­

PBacid, 2'-HO-PBalc, and 4'-HO, t-perm.

These studies indicated that GLW was able to degrade CiT in­

tensively. Both hydrolytic and oxidative mechanisms were playing 115 major roles. Hydroxylation at the 2'-position could be the reason for

GLW tolerance toward CiT. APPENDIX A

TYPICAL THIN-LAYER CHROMATOGRAMS SHOWING METABOLITES OF PERMETHRIN

116 117

Figure A.l. Typical thin-layer chromatogram showing metabolites of cis-permethrin using CE solvent system: esterases alone studied in vitro. 118

Figure A.2. Typical thin-layer chromatogram showing metabolites of cis-permethrin using CE solvent system: oxidases alone studied in vitro. 119

Figure A.3. Typical thin-layer chromatograms showing meta­ bolites of trans-permethrin using CE solvent system: esterases alone studied in vitro. 120

Figure A.4. Typical thin-layer chromatograms showing meta­ bolites of trans-permethrin using BEM solvent system: oxidases alone studied in vitro. 121

Figure A.5. Typical thin-layer chromatograms showing metabolites of cis-permethrin using BFE solvent system studied in vivo. 122

Figure A.6. Typical thin-layer chromatogram showing meta­ bolites of trans-permethrin using BFE solvent system studied in vivo. 123

Figure A.7. Typical thin-layer chromatogram showing meta­ bolites of trans-permethrin using BEM solvent system studied in vivo. LITERATURE CITED

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