The Neurotoxicity of O-(2,5-Dichlorophenyl) O-Methyl Phenyl- phosphonothionate, an Impurity and Photoproduct of

Leptophos (Phosvel) Insecticide

JAMES R. SANUOKN, ROBERT 1,. I'V~ETC.~LI~~,AND LAHRY Ci. HAMEN

Illinois Natural History Surury, Illinois Agrimltrrral Exprinrent Station and thr University of Illinois, Cirbana, Illinois 61801

IXeceived November 24, 197.5; ac*cepted May 19, 1976

The neurotoxic efferts of leptophos and three degradation products of leptophos have beer1 evaluat,ed in whit,e leghorn pullets. The data derived demon&ate that, desbromo leptophos and leptophos oxon are approximately three and t,wo time?, respectively, more effective neurotoxic agents than leptophos in the white leghorn pullet.

The problems associat,cd wit,h application of lcptophos (Phosvel) [0-(4-bromo-2,.5- dichlorophenyl) 0-met,hyl phmylphospho- nothionate] in Egypt to cont!rol cotton p&s have been well documented and in- volved t,he poisoning of several hundred wat,er buffalo as well as some humans (1). The poisoned water buffalo had signs of ascending paralysis of, the hind legs which suggestSed that the cause of t,hr sickness was related t,o demyelination of t#hc long nerves of the legs. The first indication t,hat# lept#o- phos might have neurotoxic propert’irs was described in a short, publication by Abou- Donia et al. (2), who dcmonst,rat8cd conclu- sively that chickens fed single oral doses of leptophos at, 180 mg/kg had t#ypical signs of ataxia which are generally associated with nrurot,oxic esters of phosphoric acid (3).

Recent data indicate that. sheep and cattle fed leptophos-t,rcat,ed silage t,hat, was then exposed to sunlight came down with signs quicker than animals treated \vit,h non- sunlight-exposed lept*ophos-treakd silagr. This is somewhat perplexing as it might bc

cxpccted that, phot’olysis could yield chcmi- cals t,hat, were less biologically active t,han lcptSophos. However, photolysis of lcptophos yields substantial amount,s of desbromo leptophos, [O-(L’,5-dichlorophenyl) O- methyl phcnylphosphonotShionat’e] (4). In

t’his communication it, is demonskated t,hat, dcshromo lcptophos is at least tShrrc~ times more effechive t,han lcptophos, as report,cd by Abou-Donia et al., as a causat,ive agent, of nrurot80xicity in white leghorn pullets. In addition it is demonstrated that Icpto- phos 0x011 will also cause ncurotoxicity in lvhitc leghorn pullets.

AIETHOLW ASD ?,IATERIALS

The two samples of technical lcpt~ophos utilized in this study contained 2.67 and 1 .!Xojn desbromo lept,ophos, and were designated leptophos-S and lcpt,ophos-T, rcspect’ivcly. Confirmat’ion of kc: presence of dcsbromo leptophos in t#hrsc samples was carried out, by gas chromat80graphy \I-it.h a flame ionization dct,ector (Varian Acrograph 1400; X2, ‘15 ml/min; air, 120 ml,/min; HZ,

142

Copynght 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

THE NEUROTOXICITY OF DESBROMO LEPTOPHOS 143

30 ml/min ; column, 250-265°C ; detector, 260°C; injector, 265°C) using 6-ft glass columns packed with 3% OV-17 on acid- washed Supelcoport (SO/l20 mesh), or 2.r>% OV-210/1.25y0 OV-17 on acid-washed Supelcoport (100/120) mesh, and employ- ing an authentic sample of desbromo lrptophos as a reference st,andard. Con- densation of O-methyl phenylphosphono- t#hioic chloride and 2,5-dichlorophenol in benzene in the presence of triet#hylaminc gave desbromo lept’ophos (mp 44.5-46.O”C, ethanol). Mass spectromet8ry dat,a (M+ 332, 297, 282, 172) and nuclear magnetic resonance data (1.80-2.20 7, nl; 2.5-3.21 7, ?H; 6.10, 6.30 7, s) for desbromo lcpt,ophos were consistent with t,hc assigned structure. Two other potent#ial leptophos metabolites wcrc prepared for this study. Lcptophos oxon was prepared by the m-chloroper- benzoic acid oxidation of lcptophos-S ac- cording t.o the met,hod of Relict and Casida (5). Lrptophos oxon was purified by column chromatography on acidic alumina with hcxanc and chloroform, 1: 1 (v/v), as

clution solvent (nmr 1.90-2.40 7, w; 2.45-2.70 T, ~1; 5.95, 6.20 7, s) . The prepara- tion of S-methyl leptophos was carried out by reaction of iodomethane and the di- cyclohexylamine salt of 0- (4-bromo-2,5- dichlorophenyl) phenylphvsphonothioic acid in benzene. The dicyclohexylamine salt of 0- (4-bromo-2,5-dichlorophenyl) phenylphosphonot,hioic acid was prepared by demethylation of leptophos-S with benzcnet,hiol and dicyclohcxylamine in re- fluxing acetone:DMF, 9: 1 (mp 193”C, et’hanol ; FDMS Mf 576). Purification of lcptophos-S was carried out by chroma- tography on acidic alumina using chloro- form: hexane, 1: 1 (v/v), as the elut,ion solvent (nmr 1.35-2.70 T, ~1; 7.65, 8.90 T, s). All compounds were at least 99% pure under the previously mctnt.ionctl g;is chromatographic condit~ions.

Biological

TwAy-w&-old white leghorn pullets weighing l-l.3 kg were given a single oral

Chemical

on White Leghorn-P&As ~___

Dose (mg) Chicken weight (g)

Leptophos-S” 200 1213 J,eptophos-S 200 1150 Leptophos-S 200 1190 Leptophos-Tb 100 1042 Leptophos-T 100 1198 Leptophos-T 200 1415 Leptophos oxonc 100 1124 Leptophos oxon 100 1143 &methyl leptophos 100 146s &methyl leptophos 100 1201 Ijesbromo leptophos 100 1373 Desbromo leptophos 100 134.5 Ijesbromo leptophos 50 1302

Desbromo leptophos 30 1240 Desbromo leptophos 23 3213 Desbromo leptophos 2.5 132.5

a 2.67% desbromo leptophos. b 1.63y0 desbromo leptophos. c Prepared from leptophos-Y.

Dose (mg/kg) Neurotoxicit,y

TABLE 1

Treatment and Biological E.fects qf Leptophos and its Derivutiues

164.6 110

173.9 II0

16X. 1 I10

191.9 110

166.9 II0

141.3 110 89.0 110

x7.3 yes 68.1 110

83.3 no 72.8 yes 87.8 yes 38.4 yes 40.3 110

20.5 no 1X.9 n0

144 SANBORN, METCALF ANL, HANSEN

tlosr in gdat,in c:~~~sulw awording to the sc~ht:dulr in Table 1. They kver( maint aincd in individual la.ycr cages, receiving feed and Avater ad lihitu~l. Pullets \\-crc observed at, least four times daily for the one month duration of the cxporiment by a vctcri- narian at, intervals of no more than (3 hr. Signs of cholinrst~crase inhibition (rye in- scnsitivit,y to finger stimulus), as well as posterior paralysis, were noted. At nccropsy, the leg muscles of all pullets showing toxic signs had atrophied. Xo other gross lesions were apparent.

RESULTS

The data in Table 1 indicat’c that lepto- phos at the doses given to t#he chickens did not develop delayed neurotoxic signs in one month. This is not part,icularly surprising, as in the experiments reported by Abou- Donia (2) only one out of two chickens given 180 mg/kg in a single dose developed dclaycd neurot80xic signs. One plausible cxplanat,ion lies in t hc conccntrat,ion of dcsbromo lept.ophox in t,he sample ut,ilizcd by Abou-Donia (2) for his st,udics, which could have been much higher t,han t#hat utilized in this study. From t8he present work, the presence of a greater quant,ity of desbromo leptophos in t-he Egyptian st#udy would account, for the ncurotoxic signs occurring in chickens t’reatcd with 1X0 w/k.

The second pertinent point is the ob- scrvcd ncurotoxicit$y in one chicken treat,ed \vit,h 100 mg lcpt,ophos 0x011 after 14-16 days at a dose which is lowr t,han the amount of leptophos given to the chickens in the Abou-Donia st(udy. If ncurot,oxicity in chickens involves hhe inhibition of selected esterases (6) in the my&n sheath, then the neurotoxiciby of leptophos oxon at, a lower dose than leptophos is reasonable since t,he oxon, which requires no prior metabolic act,ivation, is a much bct,ter csterase inhibitor than lcpt80phos because of the greater elcctrophilicity of the phos- phorus in P=O as compared to t.hc P=S in Ieptophos (3).

Finally tllcb nrosi. itl~l)ort:lnt, litl(lilbg of this ~vorlt is the Iuuch cwhunr~c~l u~wrot~osic effect of dcsbromo lcpt,ophos, which pro- tluccd neurotoxic signs aft.w 14--N days in bot,h chickens treated w\-it,h 100 mg, and one out of t,\vo chickens trcakd with 50 mg. The ncurot,oxicity of t.his ester apparentI> has never been reported. The observation that dcsbromo lcptophos is one of t,hc phot,oproduct,s from lept,ophos provides an excellent explanatNion for t hc great cr ncuro- toxicity of silage treated wit8h leptophos and t,hen exposed to sunlight, as compared t,o silage t,rratcd Cth leptophos and not exposed to sun irradiation (4).

Recent, dat’a obtained in this laboratory on the fate of [14C]n~cthoxy-labelrd lepto- phos in the terrestrial-aquat,ic model eco- system devcloped by RIckcalf et al. (7) corroborat’e t hc previously obtained en-

vironmental residue data, as it n-as ncccs- sary to extend t,hr ccosyst~em experiment, an addit,ional 16 days bccausc t,he mosquit,o larvae died after their introduct,ion int,o t,he aqueous segment. of t.his ecosyst,em (S). The mostluito morta1it.y during the sddi- tional time of the experiment clearly point,s to the stabilit#y of lept,ophos, as most insecbicides examined in t,his ecosyst8cm are sufhciently degraded so that int,roduct,ion of mosquito larvae on the 27t,h day of the 33-da.y regimen did not, result in their mortaky. Examinat,ion of t,he residues of leptophos and its metabolitcs in the fish and snail at’ the end of the experiment demonstrated that lept.ophos was accumu- latcd over t.hc rvat.cr concentration in these fish and snail 1,443 and 4S,398 times, respectively. Purt,hcr, t’hc amount of lepto- phos in the extractable radioactivity ob- tained from t’he fish and snail was 55.0 and 97.3%, respectively. In summary, the extension of the model ecosystem experi- ment an addit’ional 16 days beyond the normal 33 days and the high percentage of leptophos in the fish and snail demonstrate conclusively the lengthy environmental st.abi1it.y of t.he lipid soluble insecticide leptophos.

THE NEUROTOXICITY OF DESRROMO LEPTOPHOS 14.5

Ikcause of the ncurotoxicity of leptophos (2) and a photoproduct of leptophos, 0- (2,5-dichlorophenyl) O-methyl phenyl- phosphonotNhionate, the use of leptophos on cotton and vegetables in the United States could lead to undesirable human hea1t.h problems. Since a degradation product, desbromo leptophos, is more neurotoxic to the chicken than leptophos, and because it is formed via sunlight irradiation, it can bc concluded that even careful purification of technical leptophos will not reduce the neurotoxic ha,zards of lcpt,ophos.

ACKNOWLEDGMENTS

The authors express their thanks t,o Dr. Ross P. Tarara for providing clinical assistance during the rcjurse of these experiments. This work was sup- ported in part by the Illinois Nat,ural History Survey, Illinois Agricultural Experiment Stat,ion, and the N(1rt.h C’ent.1~1 11egicmal I’rojec:t,, NC-M.

REFERENCES

1. K. P. Shea, Nerve damage-The return of the “Ginger Jake,” Environment 16, 6 (1974).

2. M. B. Abou-Donia, M. A. Othman, G. Tantawy, A. Z. Khalil, and M. F. Shawer, Neurotoxic effect of leptophos, Experientia 30, 63 (1974).

3. Xlorifusa Eto, Organophosphorus Pesticides, in “Organic and Biological Chemistry,” p. 219, CRC Press, Cleveland, Ohio (1974).

4. Velsicol Corporation, Technical Data Sheet (1971).

5. E. Bellet and J. E. Casida, Products of peracid oxidation of organothiophosphorus com- pounds, J. Agric. Food Chem. 22, 207 (1974).

6. RI. K. Johnson, Organophosphorus and other inhibitors of brain “neurotoxic esterase” and the development of delayed neurotoxicity in hens, Biochem. J. 120, 523 (1970).

7. 1~. L. Metcalf, G. K. Sangha, and I. P. Kapoor, Model ecosystem for the evaluation of pesti- cide biodegradabilit,y and ecological magnifi- cation, Environ. Sci. I’echnol. 5, 709 (1971).

8. I:. I,. hfetcaalf and J. lt. Sanborn, Pesticides and environmental quality in Illinois, Illinois Sal. His/. Strnw!/ Hull. 31(9), 3X1 (1975).