Biodegradable analogues of DDT*

Bull. Org. mond. Sante 1971, 44, 363-374 Bull. Wld Hlth Org. Biodegradable Analogues of DDT * ROBERT L. METCALF,1 INDER P. KAPOOR 2 & ASHA S. HIRWE 2 Despite the immense utility of DDT for vector control its usefulness is prejudiced by its stability in the environment and by the low rate at which it can be degraded biologically. Metabolic studies in insects, in mice, and in a model ecosystem with several food chains have shown that DDT analogues with substituent groups readily attacked by multifunction oxidases undergo a substantial degree of biological degradation and do not appear to be stored readily in animal tissues or concentrated in food chains. Detailed metabolic pathways have been workedout and it is clear that comparative biochemistry can be used to developDDT analogues that are adequately persistent yet biodegradable. A number of new DDT analogues have been evaluated for insecticidal activity against flies and mosquitos and for their potential usefulness as safe, persistent, and biodegradable insecticides. After 20 years of intensive search for new insecti- cides for the control of insect vectors of human disease,3 it must be concluded that none of the newer materials equals or excels DDT from the point of view of the combined properties of dura- bility of residues on different surfaces, safety to humans and higher animals, and low-cost insecti- cidal effectiveness. However, there is growing con- cern about the continuing liberation of vast quanti- ties of DDT into the environment (annual world production is estimated to be about 200 000 tons). In the living world, the very qualities that make DDT such an effective residual insecticide-its stability, very low water solubility (0.002 ppm), and high lipid solubility (ca. 100 000 ppm)-result in its accumulation in the fatty tissues of animals and are responsible for its progressive accumulation and concentration in organisms of a food chain, a process that has been referred to as ecological magnification (Dustman & Stickel, 1969). The prob- lems of the biological accumulation of DDT are aggravated by its conversion to the even more stable * A contribution from the WHO International Insecticide Reference Centre, Departments of Entomology and Zoology, University of Illinois, Urbana-Champaign, Ill., USA. Supported in part by grants from the Rockefeller Foundation and from the World Health Organization. 1 Professor of Zoology and Entomology, University of Illinois, Urbana-Champaign, 1ll., USA. 2 Research Associate, Uniiversity of Illinois, Urbana- Champaign, Ill., USA. 3World Health Organization (1968) Evaluation of insecticides for vector control, part I, Geneva (unpublished document WHO/VBC/68.66). dehydrochlorination product DDE,f which is the principal environmental pollutant, and in many insect control programmes it must be replaced by a persistent, biodegradable substitute. The drug-metabolizing or multifunction oxi- dase enzymes (MFO) are known to play a domi- nant role in determining the absolute toxicity of insecticides to both insects and higher animals (Hodgson, 1968). The action of these enzymes is profoundly related to such factors as insecticide specificity and selectivity, to the genetic selection of insecticide-resistant races of insects, and to biodegradation in the environment. It is evident that DDT and its derivatives DDE f [1,1-dichloro-2,2- bis(p-chlorophenyl)ethylene] and TDE [1,1-dichloro- 2,2-bis(p-chlorophenyl)ethane] are highly resistant to detoxification by MFO enzymes, and this single factor accounts for their storage and accumulation in animal tissues, especially at the higher ends of food chains. On the other hand, DDT analogues such as methoxychlor are readily attacked by MFO enzymes, which bring about O-demethylation and rapid elimination of the compound as mono- and bis-phenols (Kapoor et al., 1970). Thus, methoxy- chlor is an example of a persistent but biodegradable insecticide that does not generally accumulate in animal tissues and that is a more prudent choice for a variety of uses where environmental pollution is an important factor. We believe that the concept of persistent but biodegradable insecticides is parti- t Names against which this symbol appears are identified by chemical name in the Glossary on pages 445-446. 2649 -363- 364 R. L. METCALF AND OTHERS Table 1 Ring-substituted 1,1,1 -trichloro-2,2-diphenylethanes GENERAL FORMULA H --_3C<; C Cl3 Substituents Melting Method point of NMR data b R I R 2 ( C) synthesis a CH30 CH30 88-9 A aH: 4.93(S); OCH3: 3.734(S) C2H50 C2H50 105 A aH: 4.9425(S); OCH2: 3.8-4.099(Q); CH3: 1.242-1.484(T) C3H70 C3H70 62 A aH: 4.933(S); OCH2: 3.775-3.984(T); CH2: 1.5-2.1 (M); CH3: 0.87-1.085(T) (CH3)2CHO (CH3)2CHO 52 D aH: 4.935(S); OCH: 3.82-4.1 (Q); CH3: 1.23-1.43(D) C4H9O C4H90 50 A aH: 4.94(S); OCH2: 3.82-4.01 (T); CH2-CH2: 1-2(M); CH3: 0.82-1.0(T) CH30 C2H50 88-9 B aH: 4.95(S); OCH2: 3.8-4.167(Q); CH3: 1.217-1.482(T); OCH3: 3.75(S) CH30 C3H70 65 B aH: 4.966; OCH2: 3.75-4(T); CH2: 1.6-1.95(Q); CH3: 0.867-1(T); OCH3: 3.75(5) CH30 C4H 90 42 B aH :4.958; OCH2: 3.85-4.05(T); CH2-CH2: 1.4-1.87 (M) ; CH 3: 0.835-1.067(T); OCH3: 3.85(S) CH30 C5H1 10 30 B aH: 5.059(S); OCH2:3.93-4.13(T); (CH2)3:1.4-1.89(M); CH3:0.933-1.65(M) CH30 C6H130 liquid B aH:4.967(S); OCH2: 3.8-4(T); (CH2)4: 1.67-1.835(M); CH3: 0.818-0.967(T); OCH3: 3.72(S) CH30 CBH170 43-5 B aH: 4.93(S); OCH2: 3.818-4.01 (T); (CH2)6: 1.1-1.735(M); CH3: 0.884-0.97(T); OCH3: 3.75(S) C2H50 C3H70 54-5 B aH: 4.936(S); (OCH2)2: 3.7675-4.167(M); CH2: 1.615-1.95(Q); CH3: 0.885-1.165(T); CH3: 1.258-1.488(T) C2H50 C4H9Q 53-4 B aH: 4.915(S); (OCH2)2: 3.8-4.15(M); CH2-CH2: 1 -1.835(M); CH3: 1.25-1.4675(T); CH3: 0.815-1.05(T) CH5S CH30 92-3 B aH: 4.966(S); OCH3: 3.7675(S); SCH3: 2.42(S) CH3S C2H50 103-4 B aH: 4.95(S); OCH2: 3.8-4.15(Q); CH3: 1.242-1.4675(T); SCH3: 2.416(S) CH3S C3H70 98 B aH: 4.95(S); OCH2: 3.785-4.0(T); CH2: 1.618-1.965 (M); CH 3 0.884-1.1 65(T); CH 3: 2.35(S) CH3 CH30 80 B aH: 4.966(S); CH3: 2.267(S); OCH3: 3.72(S) CH3 C2H50 96 B aH: 4.95(S); OCH2: 3.81-4.167(Q); CH3: 2.3(S); CH3: 1.25-1.5(T) CH3 C3H70 77 B aH: 4.95(S); OCH2: 3.75-3.985(T); CH2: 1.485-1.95(M); CH3: 0.885-1.133(T); CH3: 2.3(S) CH3 C4H 90 65-6 B aH: 4.97(S); OCH2: 3.8675-4.0(T); CH2-CH2: 1.33-1.8675(M); CH3: 0.834-1.01 (T); CH3: 2.31 87(S) CH3 C2H5 liquid C aH: 4.97(S); CH2: 2.384-2.785(Q); CH3: 1.066-1.33(T); CH3: 2.267(S) CH30 OCH20 87 C aH: 4.925(S); OCH2: 5.9(S); OCH3: 3.675(S) C2H50 OCH20 115 C aH: 4.916(S); OCH20: 5.9(S); OCH2: 3.835-4.167(Q); CH3: 3.7685(T) CH30 HC-CCH20 72 D aH: 4.966; CCH20: 4.659-4.69(D); CH: 2.46-2.54(T); OCH3: 3.7675(S) CH30 Cl 95 C aH: 5.04(S); OCH3: 3.86(S) CH30 F 77-8 B aH: 5.016(S); OCH3: 3.785(S) CH3 Br 104 C aH: 4.965(S); CH3: 2.317(S) a See text. b NMR data: (S) = singlet, (D) = doublet, (T) = triplet, (Q) = quartet, (M) = multiplet. BIODEGRADABLE ANALOGUES OF DDT 365 cularly important for achieving satisfactory residual insecticidal control of pests of both public-health and agricultural importance without causing long- term environmental pollution and the accumulation of pesticide residues throughout the biosphere. Therefore we are systematically exploring the attack of the MFO enzymes on a variety ofDDT analogues with the objective of characterizing mechanisms of biodegradation. We are seeking DDT-like com- pounds that, while effective and persistent insecticides in inanimate situations, when absorbed into living organisms will have weak points for attack by the MFO enzymes, promoting rapid detoxification and elimination as water-soluble metabolites. Such DDT analogues should have many advantages as safe, relatively stable, and potentially inexpensive residual insecticides. MATERIALS AND METHODS The compounds evaluated were all highly puri- fied chemicals synthesized in our laboratory by variations of the Baeyer condensation between chloral and appropriate substituted benzenes. Four general methods of synthesis were used for the compounds listed in Table 1: (A) condensation of 2 moles of substituted benzene with 1 mole of chloral in excess (10 volumes) of concentrated H2SO4 or 0.6 M AlCl3 (anhydrous) to form the sym- metrical 1,1,1-trichloro-2,2-diphenylethane; (B) con- densation ofp-alkoxyphenyl trichloromethylcarbinol (Kapoor et al., 1970) or p-alkylphenyl trichloro- methylcarbinol (Reeve et al., 1966) with 1.0 M AIC13 (anhydrous) in ethanol-free chloroform; (C) con- densation of substituted benzene and the carbinol referred to above in 10 volumes of concentrated H2S04 to produce the asymmetrical 1,1,1-trichloro- 2,2-diphenylethanes; and (D) treatment of 1,1,1- trichloro- 2-(p-hydroxyphenyl)- 2 -(p-methoxyphenyl) ethane in acetone with the appropriate alkyl bromide in the presence of potassium carbonate. The compounds were purified by recrystalli- zation or distillation until they were at least 99% pure by thin-layer chromatography. The structures were confirmed by NMR spectrometry as shown in Table 1 (see Kapoor et al., 1970). The insecticidal activities were determined by the standard methods used in Stage I of the WHO Insecticide Evaluation Programme.' Female house- flies, under CO2 anaesthesia, were treated by the topical application of 1-,ulitre droplets of standard solutions of the insecticides in acetone. Three replicates of 20 flies 2-4 days old were treated on the pronotum at each dosage, and at least 5 dosages were used to establish each dosage-mortality curve. Mortalities were determined by holding the flies at 22°C with 40% sucrose solution as food. Topical applications were also made to laboratory- reared black blowflies, Phormia regina, in exactly the same manner. The results of these evaluations are reported in the tables as LD50 values in micro- grams of toxicant per gram of insect, using the average weight of the housefly as 20 mg and of the blowfly as 40 mg. To determine the amount of detoxification of the insecticides in the insect body, the synergist pipe- ronyl butoxide was applied topically to the ventral portion of the abdomen of the flies 1 hour before treatment with the insecticide, applied to the prono- tum in a dosage of 1 ,ulitre of 5% w/v solution in acetone (50 Htg per insect). This dosage produced no observable mortality and did not materially affect the longevity of the flies. The toxicity of the DDT analogues for larvae and adults of the mosquitos Culex pipiens fatigans and Anopheles albimanus was evaluated by the WHO method 1 (see also Metcalf & Fukuto, 1968). Studies of the toxicity of some of the DDT ana- logues to female Swiss mice, 6-8 weeks old, are also reported. The compounds were dissolved in olive oil at 5-10% w/v and the requisite dosage was administered orally by a micrometer-driven Hamilton syringe. The mice were observed for symptoms of intoxication and for mortality over a 1-week period. EXPERIMENTAL RESULTS DDT analogues as substrates for multifuntction oxidases The MFO enzymes utilize molecular oxygen and a unique cytochrome, P-450, to form a " hydroxyl-- ating " free radical (-OH or *OOH) which is respon- sible for a large variety of reactions with xenobiotic compounds. There are at least 9 distinct types of biochemical metabolizing reaction and at least 4 of these can be demonstrated with insecticidally active DDT analogues, as follows: (1) hydroxylation of aromatic rings, which should take place most readily in unsubstituted analogues such as 1,1,1,-trichloro-2,2-bis(phenyl)ethaneor 1,1,1- I See footnote 3 page 363. R. L. METCALF AND OTHERS H C le| OH_ R3 H RO C\OR3/HR R3 H R / \ C / \ CH3 R3 H C e OH Cf R3 H RO / / OH - I - C R3 H R/ COOH - - Cf K3 H H 0 RS X3 C e S SR RS Ci SR - I-- -C C R3 R3 t^2 10223 trichloro-2-(p-chlorophenyl)-2-phenylethane, form- ing monophenolic and diphenolic compounds; (2) side-chain oxidation, which is demonstrable in 1,11,1-trichloro-2,2-bis(p-methylphenyl)ethane by oxidation to mono- and di-benzoic acids (Kapoor et al, unpublished data, 1971); (3) O-dealkylation, shown by the conversion of methoxychlor, 1, 1, 1 - trichloro - 2, 2 - bis(p-methoxy- phenyl)ethane, to mono- and bis-phenols (Kapoor et al., 1970); and (4) sulfide oxidation, shown by the oxidation of I, I, I -trichloro-2,2 - bis(p-methylthiophenyl)ethane to sulfoxide and sulfone derivatives (Kapoor et al., 1970). Typical reactions ofDDT analogues as substrates for these enzymes are shown in the accompanying figure. Synergistic ratio as a measure of biodegradability Much evidence indicates that methylenedioxy- benzene derivatives (1,3-benzodioxoles) such as piperonyl butoxide and sesamex t are effective and specific inhibitors of MFO enzymes. Inhibition of these drug-metabolizing enzymes is responsible for the pronounced synergistic effects of the methyl- enedioxyphenyl compounds when used together with pyrethroids or carbamates; they almost totally prevent detoxification of the insecticide and permit it to reach the target site of action. This subject has been reviewed by Metcalf (1967) and Casida (1970). The data in Tables 2 and 3 show topical LD50 values for 3-4-day-old susceptible (SNAmIM) and DDT- and dieldrin-resistant (Rsp) female houseflies as determined (a) with the compounds alone and (b) 1 hour after pretreatment with 50 [g of piperonyl 366 BIODEGRADABLE ANALOGUES OF DDT 'v-, 02 coE D-. c0E2~ff M _ Cl: C' 0 10 _- C')0 In 0 6 cc( % 00 0 5% - - 0 0 0 0 0 0 0 0 0 0 o0( C A A A o A CD ^ o o o o AA A 5 0 0C W - cn A A O co W _ --AA 0 0 A A A P. t- o _- o- 5% I.: -; N*W N o v _- C4 C4 _: LO N-l Lo = ts "LN I-) o t N IC 0 0- 5 '- 0 0 0 0 .-Dq W 1O 10 I _-5 W10 N N A A A - A 4 Co:cc 0 N 0 0) i-(0 '- 1001010 N 10 N N A A A ) 5% _ C N w 0 NO 0 IqLo A (0 U1 - m 0t o % AA N o 5 C' M ..0 0 00 0 0 - 0101 0 10 00 0o A C 105 00 00 0) N i 4 oCi4 6 oim w ~~~~~~~~A1N( A00A C') A to 1o La )L0 o01 0 r_N r_ A CD 0 0 04 o 1 r% 1- 0 t N 0 a0 N _ U1 0 0 N A A ANc 00 0 0c~~~~~~~ooA6~~0C U ~~~O° _ o o oh O Q C _C0 0 I -II e~~~~~ ~~ ~ 0 0I~C 000 00 -zM - - =- - > -5 _ x *0CL - 0. CE 00 0 .0C: v 0 CD 000 .2 - CD 00 _ 00CE 0 *0 - . U,_ 0 1 C O _ ._ Co2 367 -Q Q go x 0 a 0 '0 0 U9a 0 0 -J 0 01 10 -j -a ,U 0 0 C 0 C14 (D0) Wo I- 00 oU)0) 0 'a C ._ m'a0 M 0) E E (A I.- 0 0 0 LU -J I.z CD e ,, X-u Q i P co 0 X 0 C 6 25 l- 11 ~l C* "ON = :LE -0,., 0m to *I -- C4 -0= T EM,Q 368 R. L. METCALF AND OTHERS N_C,)OLACO) O O O ° ° tC) °o° J')U - cn C'i66orooooooo Io,o It 5. 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II Ez~~~~~~~ co > U CD C) a o m m o LO) _ _ _ _ _ _ LO L__o,_O __CD U) LO I ___o AOucnS-D- CND_1 C DNgv LO L)O o LO LO LO) sNS_ o~~~~~~~~~~~~~N 0 (D M N N M*Cs-MsNQU)U o~~~ ~ ~ ~ ~ ~ ~~~~~~~~CY)*; 0 I K~~~~~~~~~~~~ 0) Q L L O O Q Q Q O O O Q O O O O O Q Q O Q I I I uz ~~ ~~~~~0 0 Ln r, O O°, ° ° ° ,, 0 Lf tn LI) CM3_ I I I I'I INI-IT I)IO0 C I I I I IN C I _ I _ ~ ~~~~~~~~ u u u 0 u u uD 0 u u uJ u u u u u 0 o *0 00 R R x x x x x x x x ° X X X X X X X X X X X X X X X X X X X X X X X X X BIODEGRADABLE ANALOGUES OF DDT butoxide. The synergistic ratio (SR) gives a quantita- tive measure of the effect of the MFO enzymes in the detoxification of the DDT analogues. It is apparent from Tables 2 and 3 that there is a remarkable range in SR values for the different compounds against the SNAIDM fly-from 40 for 1,1,1-trichloro-2-(p-chloro- phenyl)-2-phenylethane (XXXIV) to 1.0 for 1,1,1- trichloro-2-(p-butoxyphenyl) - 2 - (p - methoxyphe- nyl)ethane (XXI). Thus, the former is rapidly detoxified in vivo while the latter seems not to be affected. Such data suggest that the intrinsic toxicity of the DDT analogue is best measured by the synergized LD50, as has been demonstrated for car- bamates (Metcalf & Fukuto, 1965), and that the SR value affords a quantitative measure of the detoxifica- tion of the compound by susceptible and resistant insects. The data in Table 2 were obtained with symmetri- cal DDT analogues. DDT (I) is detoxified only slowly by MFO action in S flies and somewhat more rapidly in R flies. The rates of detoxification of the simple p-substituents can be ranked as follows: Cl <H <CH3<CH30<CH3S, and this technique demonstrates the reactions described for MFO enzymes as shown in the figure. The rates of detoxifi- cation of the p,p'-dialkoxy analogues can be ranked as follows: C2H50 <C3H70 <CH30 <iso-C3H70. The asymmetrical compounds of Table 2 exhibit greater overall biodegradability. Compounds with high SR values included CH3-H (XXXV) and CH3-Br (XII); those with low SR values were C2H50-C3H70 (XXV), CH30-C3H70 (XX), CH3- C2H5 (XXX), and C.H50-OCH.O (XXXII). " Ethoxychlor ", 1, 1,1 -trichloro-2,2-bis(p-ethoxy- phenyl)ethane (IV), was outstandingly active against both S and R flies and was in fact more toxic than DDT both alone and synergized. It is evident from the magnitude of the LD50 values that the SNAIDM strain, which has been reared for more than 25 years on bran and alfalfa inadvertently contaminated with trace amounts of DDT, has become less susceptible to DDT. The LD 50value originally obtained in 1948 for this strain at 15.5°C was 0.04 ,ug per female. " Ethoxychlor ", with an LD50 of 0.058 jug per female at 15.5°C (Metcalf & Fukuto, 1968), was originally slightly less toxic than DDT but is now more toxic. We have recently obtained S strains from three other laboratories and have found their susceptibility to DDT to be about equal to or less than that of the SNAIDMI strain. With respect to DDT resistance as indicated by the response of the RSp housefly (Tables 2 and 3), the alkyl- and alkoxy-substituted analogues are substantially more effective than DDT. Compounds such as " ethoxychlor " and the CH30-C2H,O (XIX), CH30-OCH20 (XXXI), C2H50-C3H70 (XXV), and C2H50-C4H,O (XXVI) analogues have resistance ratios of <2.0, while the CH30-C3H70 (XX) and CH30-C4H90 (XXI) analogues are of nearly equal toxicity to the S and R strains. In the DDT molecule, the inductive and mesomeric effects of the ring substituents control the rate of dehydro- chlorination in response to DDT-ase (Metcalf & Fukuto, 1968), and this rate is minimal when the substituents are electron-donating-e.g., alkyl or alkoxy groups. Therefore, where DDT-resistance is controlled by DDT-ase, as in the Rsp housefly, such analogues should be substantially more effective than DDT. Autosynergism o.fDDTanaloguies The term autosynergism has been applied to phenyl methylcarbamate insecticides containing 3,4-methylenedioxyphenyl or 2- or 3-propynyloxy- phenyl moieties, which have been shown to inhibit the multifunction oxidases. These carbamates are much more toxic to the housefly than their relative anticholinesterase activity would indicate, suggesting that they also function as synergists by self-inhi- bition of the detoxifying enzymes (Metcalf, 1968). The DDT-type compound provides the structural framework for substitution with similar groups on one or both phenyl rings and these compounds have been examined for autosynergism. The data of Table 4 contrast the toxicity and the syner- gism with piperonyl butoxide of several pairs of compounds. The higher toxicity and lower SR values for the methylenedioxyphenyl compounds (XXXI, XXXII), especially at the LD90 level, suggest that autosynergism occurs. There is also evidence of a smaller degree of autosynergism with the propynyl- oxyphenyl compound (VIII). Further analysis of this problem is complicated by the lack of a quantita- tive measure of the interaction of DDT-type com- pounds with the receptor site (equivalent to the 150 values for carbamates with cholinesterase). However, the synergized LD,0 values for the fly may provide an approximate indication of the efficiency of receptor interaction. The data of Table 2 indicate that methoxychlor (III), whose synergized LD50 for Musca is 3.5 tkg/g, is a much more efficient inter- actant than 1,1,1-trichloro-2,2-bis(p-propynyloxy- phenyl)ethane (VIII), whose synergized LD50 is 28.0 ,ug/g. A more definitive examination of this 369 R. L. METCALF AND OTHERS Table 4 Autosynergism of DDT analogues GENERAL FORMULA H RI / \ C / R2 - cc'-13 Musca domestica SNAIDAI RSp SR slope LD50a SR slopeP (Mg/g) P 12.8 4.9 10.3 4.3 3.9 2.4 4.0 2.5 4.3 8.1 48 39 105 29 26 10.4 3.8 14.0 2.3 3.3 2.6 2.4 1.6 3.9 2.5 Phormia regina LD5oa SR slope(,Lg/g) 10 65 9.5 10 11.5 2.2 2.7 1.1 1.0 1.1 3.5 2.4 3.0 4.2 3.8 a Topical application. phenomenon as applied to DDT-type compounds does not seem feasible at present. Intrinsic toxicity of DDT analogues to Phormia and Musca Lack of specific knowledge about the DDT recep- tor site makes it difficult to relate precisely the mole- cular structures of DDT analogues to their intrin- sic toxicity. The most promising theory of the mode of action of DDT appears to be that of Mullins (1955), which relates the size and shape ofDDT ana- logues to their ability to enter into pores or inter- spaces between the macromolecules of the nerve axonic membrane. The toxic action of DDT, it has been suggested, results from its penetration into the interspaces with a specific orientation so as to produce ion leaks and consequent depolarization of the axon. This theory has been further refined by Holan (1969) to suggest that the active analogues of DDT function as molecular wedges within the Na+ cavities of the nerve membrane and cause ion leaks by keeping open the expanded sodium springs (Weiss, 1969). The present evaluation (Tables 2 and 3) provides some interesting leads to further refinement of these theories. It is suggested that the LD-,o for Phormia and the synergized LD50 for Musca provide a rough measure of reactivity at the DDT receptor site. Phormia differs substantially from Musca in its response to xenobiotics-for example, its topi- cal LD50 of carbaryl is 29 ,ug/g and the SR with piperonyl butoxide is 6.7, whereas the LD50 for Musca is 900 ,g/g with an SR of 72 (Brattsten & Metcalf, 1970). Tissues from Phormia are only about 0.26 times as active per mg of protein in hydroxylating naphthalene and only about 0.07 times as active in epoxidizing aldrin to dieldrin (Ter- riere, 1968). D. Nye in our laboratory has found Phormia to have very low levels of cytochrome P-450 compared with Musca. Therefore, the com- parisons, in Tables 2 and 3, between topical LD50 values and SR values for Phormia and Musca demonstrate the role of MFO detoxification in the intrinsic activities of the various DDT analogues. It is apparent that in Phormia the MFO enzymes play a very limited role in detoxification, with SR values of 1-3, while in Musca they are substantially higher, 2.5-40. Methoxychlor is more toxic to Phormia than DDT, unsynergized, and this- together with its higher synergized LD50 for Musca- suggests that methoxychlor has a greater affinity for the DDT receptor than does DDT. Almost Substituents R I R 2 CH30 CH30 CH30 C2HsO C2H50 LD50a (/Ag/g) 45 22 34 16 13.5 CH30 OCH20 HC-CCH20 CH30 OCH20 370 BIODEGRADABLE ANALOGUES OF DDT 371 every DDT analogue was more effective on a body- weight basis against Phormia than against Musca, and these data suggest that the apparent toxicity of these insecticidal compounds is determined by the levels of activity of the MFO enzymes in the test organisms. The data of Table 2 suggest that for the symmetri- cal analogues of DDT, the order of toxicity of p,p'-substituents is C2H50> CH3O> C1> CH3> CH3S> C3H70. This is contrary to the general impression that the Cl substituent (DDT) is the most active. For the asymmetrical analogues of Table 3, the order of intrinsic toxicity is CH3- C2H50 (XVI) > Br-CH3 (XII) > CH3-C3H70 (XVII) > CH3S-C3H70 (XXIX) = CH3S-C2H50 (XXVIII) = CH3S-CH30 (XXVII) > CH3-C2H5 (XXX) > C2H50-C3H70 (XXV) > CH30-HC- CCH20 (XXXIII) > C2H50-OCH,O (XXXII). The CH3-C2H50 (XVI) analogue was the most active compound studied, being slightly more active than " ethoxychlor " (C2H50-C2H50). The high intrinsic activity of these asymmetrical compounds together with the very adequate acti- vity of CH30-C4H90 and the considerable activity of CH30-C6H130 suggest a possible lack of symme- try of the receptor site. This is further emphasized by the high activity of synergized monosubstituted compounds. It has been assumed in almost every investigation that p,p'-disubstitution is essential for high toxicity and maximum receptor interaction. Yet the monochloro-DDT (XXXIV) is synergized 40 times and its synergized toxicity for Musca is greater than that of DDT. However, it is not highly toxic to Phormia. Similar results were obtained with the monomethyl-DDT (XXXV). Even more interesting from the standpoint of DDT mode of action is the toxicity of o,p'-DDT, 1,1,1-trichloro- 2-(p-chlorophenyl)-2-(o-chlorophenyl)ethane, whose LD50 is 190 ,ug/g alone and 15 ,ug/g synergized (SR = 12.6). This compound can be hydroxylated in the para-position by MFO and its alleged in- activity is clearly due to detoxification and not to steric factors, as is generally believed. These responses of the fly clearly suggest a need for sub- stantial revision of current ideas about the interaction of DDT and hypothetical receptors. This will be attempted in a subsequent paper. Selective toxicity to insects and mammals DDT obviously is of very low toxicity to mammals, as demonstrated by its enormous use without sick- ness or fatality. However, this seems to be largely a function of its low dermal toxicity, since recorded values for the oral LD50 for the mouse range from 150 ,tg/g to 400 ,ug/g; the oral LD50 for the male rat is 1 13 ,ug/g and for the female rat it is 118 ,ug/g (Hayes, 1963). Oral doses of at least 285 mg/kg have been taken by man without fatal result (Hayes, 1963). Methoxychlor, with an oral LD50 for the rat exceeding 6 000 mg/kg, is much less toxic, pro- bably because it undergoes rapid biodegradation (Kapoor et al., 1970). The difference in toxicity suggests that bio- degradable analogues of DDT may be substantially less toxic to mammals than to insects because of the generally higher titres of MFO enzymes in the mammalian liver. The data of Table 5 summarize the available information, including data obtained in our laboratory, on some of the compounds in Tables 2 and 3. Compounds with a single methoxy, methyl, or methylthio group were of low toxicity. It appears that these groups should be incorporated into appropriate DDT molecules where a high safety factor for mammals is required. " Ethoxy- chlor " is nearly as toxic as DDT to the mouse and its ethoxy-propoxy analogue is considerably more toxic. A comparison with methoxychlor illustrates the rather specific nature of O-dealkylation. Biodegradation ofDDT anialoguies within an ecosystem The principal problem involved in the wide- spread use of DDT today is its lack of biodegrad- ability and consequent " ecological magnification " in food chain organisms. Therefore any compounds proposed as substitutes should be free from this defect. Our laboratory has developed a model ecosystem with a seven-element food chain, which can be used with radiolabelled compounds to evaluate biodegradability. This technique has been applied to DDT, methoxychlor, " methiochlor " (Kapoor et al., 1970), " methylchlor", 1,1,1-tri- chloro-2,2-bis(p methylphenyl)ethane, and " ethoxy- chlor" (unpublished data). An account of these studies is beyond the scope of the present paper;' however, the data obtained demonstrate the utility of this approach in evaluating new candidate insecticides. As shown in Table 6, substitution in the p,p'-positions with groups that can be attacked by the multifunction oxidases effectively decreases the concentration of the DDT-type analogue in food- chain organisms. 1 A full account of these studies will be published else- where. R. L. METCALF AND OTHERS Table 5 Toxicity of some DDT analogues for the mouse Table 6 Accumulation of DDT analogues in an ecosystem GENER, R'I/ Substituents R R Cl Cl CH3 CH3 CH30 CH30 C2H50 C2H50 C3H70 C3H70 C4H9O C4H90 CH3S CH3S CH30 CH3 CH30 CH3S CH30 C2H5O CH30 -OCH20- CH30 C4H90 C2H50 CH3S C2H50 C3H70 C2H50 -OCH20- CH3 C2H50 CH3 C3H70 AL FORMULA H - \ R2 C Cl3 Oral LD5o (mg/kg) for the mouse Our laboratory Literature 200 200 (t 400 b 750 c, c 300-325 3 350 b 1 000 "IC 1 850 b 250 a 200 ' 1 QQU,c 1 000 >1 000 1 000 300-500 -1 000 500 -1 000 75-100 -1 000 1 000 500 " Van Oettingen & Sharpless (1 946) t' Dornenjoz (1946). c No effcct. Analoguies as DDT substitutes Because of increasing public concern about environmental pollution by DDT and legal restric- tions upon its use, the question of its replacement by persistent biodegradable substitutes has become urgent. Methoxychlor, which has been used to a substantial extent for many years, is a suitable DDT replacement for many uses (Kapoor et al., 1970). " Methylchlor" (II) was discussed as an in- secticide in the original DDT report (Muller, 1946). Concentration (ppm) in: Compound DDT DDE t TDE methoxychlor " ethoxy- chlor' " " methio- chlor ''" " methyl- chlor " "( Water 0.00022 0.0053 0.0004 0.00011 0.0006 0.0018 0.0001 Physa (snail) 7.6 103.5 3.3 13.2 58.5 0.539 72.16 Gambusia (fish) = 18.6 145.0 33.4 0.17 0.922 0 0.084 Con- centration ratio, fish/water 85 000 27 000 83 000 1 500 1 500 0 840 " These compounds are identified in the text. It should be re-examined because of its very low toxicity for mamumals, potential low cost, and biodegradability. These compounds appears to be particularly effective against adult mosquitos. "Ethoxychlor " (IV) was first reported as an insecti- cide by Prill et al. (1945) and subsequently by Ste- phenson & Waters (1946). " Ethoxychlor " was found to be an excellent insecticide but has re- ceived little further investigation. It appears to be slightly less acuLtely toxic to the mouse than DDT and is biodegradable to a substantial extent. " Propoxychlor " (V) and " butoxychlor " (VI) were also studied by the above investigators and found to be less effective. " Isopropoxychlor " (VII) was prepared as a guLm by Stephensen & Waters (1948) but was neither purified nor evaluated. Prill et al. (1946) investigated a number of asymme- trical analogues of DDT including CH30-C2H50 (XIX), CH3 O-OCH,O (XXXI), and C1-CH3 (XI). Schneller & Smith (1948) prepared the CH30- C2H50 (XIX), Cl-Br, and Cl-I analogues of DDT. However, they were unsuccessful in several attemiipts to prepare the Cl-CH30 analogue 1,1,I-trichloro-2-(p - chlorophenyl) - 2 - (p-methoxy- phenyl)ethane (XIII). The corresponding F-CH3O analogue XIV) was patented by Balaban & Sutcliffe (1948), together with the CH3-F and F-Cl ana- logues. We can find no record of the preparation or insecticidal evaluation of the other disubstituted 372 t BIODEGRADABLE ANALOGUES OF DDT 373 DDT analogues of Table 3. The C1-CH30 analogue (XIII) was prepared for the first time after numerous failures but was not outstandingly active. The most promising compounds for further evalu- ation appear to be the CH3-CH30 (XV), CH3-C2H50 (XVI), CH3-C3H70 (XVII), CH30-SCH3 (XXVII), C2H50-CH3S (XXVIII), C2H50-OCH20 (XXXII), and CH3-C2H5 (XXX) analogues, all of which are highly insecticidal, have low toxicity for mammals, and should readily undergo biodegradation. ACKNOWLEDGEMENTS It is a pleasure to acknowledge the skilful assistance of Mrs Ruth Millholin, Mrs Penny Hansen, and Mrs Arlene Pyle in carrying out the bioassay evaluations. REFERENCES Balaban, E. & Sutcliffe, F. K. (1948) British Patent No. 597 091, 19 January Brattsten, L. & Metcalf, R. L. (1970) J. econ. Ent., 63, 101 Casida, J. (1970) J. agric. Food Chem., 18, 753 Domenjoz, R. (1946) Helv. chini. Acta, 29, 1317 Dustman, E. H. & Stickel, L. F. (1969) Ann. N. Y. Acad. Sci., 160, 162 Hayes, W. J. (1963) Clinical handbook on economic poison?s, Atlanta, Ga., US Department of Health, Education, and Welfare, Communicable Disease Center Hodgson, E., ed. (1968) Enzymatic oxidation of toxicants, Raleigh, North Carolina State University Press Holan, G. (1969) Nature (Lond.), 221, 1025 Kapoor, 1. P., Metcalf, R. L., Nystrom, R. F. & Sangha, G. K. (1970) J. agric. Food Chiem., 18, 1145 Metcalf, R. L. (1967) Ann. Rev. Ent., 12, 229 Metcalf, R. L. (1968) The role of oxidative reactions in the mode ofaction of insecticides. In: Hodgson, E., ed., Enzymatic oxidation of toxicants, Raleigh, North Carolina State University Press, p. 151 Metcalf, R. L. & Fukuto, T. R. (1965) J. agric. Food Chem., 13, 220 Metcalf, R. L. & Fukuto, T. R. (1968) Bull. Wld Hlth Org., 38, 633 Muller, P. (1946) Helv. chim. Acta, 29, 1560 Mullins, L. J. (1955) Science, 122, 118 Prill, E. A., Hartzel, A. & Arthur, J. M. (1945) Science, 101, 464 Prill, E. A., Synerholm, M. E. & Hartzell, A. (1946) Contrib. Boyce Thompson Inst., 14, 341 Reeve, W., Mutchler, J. P. & Liotta, C. I. (1966) Canad. J. chem., 44, 575 Schneller, G. H. & Smith, G. B. L. (1948) J. Amer. chem. Soc., 70, 4057 Stephenson, 0. & Waters, W. A. (1946) J. chem. Soc., 339 Street, J. (1969) Ann. N. Y. Acad. Sci., 160, 274 Terriere, L. C. (1968) The oxidation of pesticides, the comparative approach. In: Hodgson, E., ed., Enzymatic oxidation of toxicants, Raleigh, North Carolina State University Press, p. 175 Van Oettingen, W. F. & Sharpless, N. (1946) J. Pharmacol. exp. Ther., 88, 400 Weiss, D. E. (1969) Aust. J. biol. Sci., 22, 1355 DISCUSSION ALDRIDGE: Do you think there are potentialities for the development of molluscidides, using as a starting point those compounds that you have shown to accumulate in snails ? METCALF: It is possible that some of these DDT-like com- pounds with appropriate solubility in water might be molluscicidal, and we are investigating this possibility. WEIDEN: Is the apparent lack of microsomal activity in the snail used in your micro-ecosystem typical of other molluscs ? METCALF: The snail used is the genus Physa, one of the largest of aquatic snails. We do not know much about other species or genera, but the one described in the paper has a very low level of microsomal enzyme activity. R. L. METCALF AND OTHERS WEIDEN: Were the sulfoxides and the sulfones of the alkylthio DDT analogues active? METCALF: The bis-sulfoxide and the bis-sulfone de- rivatives of 1, 1, 1 -trichloro-2, 2-bis(p-methylthiophenyl) ethane were completely nontoxic to Musca domestica when applied topically at 500 ,uglg. They were not sy- nergized by pretreatment with 50 jug of piperonyl but- oxide. KENAGA: Why has methoxychlor not met with greater commercial success for insect control? Does Dr Gysin think that it may be more widely used in the future as a replacement for DDT? GYsIN: Methoxychlor was considerably less active for fly control in Europe. Owing to this factor and to its greater production costs, it never found wider use in Europe. It may have to be reconsidered for use in malaria control, but not for crop protection. 374