Degradation of Phenothrin in Stored Wheat Grains Kenji NAMBU

J. Pesticide Sci. 6, 183-191 (1981)

Degradation of Phenothrin in Stored Wheat Grains

Kenji NAMBU, Yoshiyuki TAKIMOTO and Junshi MIYAMOTO Research Department, Pesticides Division, Sumitomo Chemical Co., Ltd., Takarazuha, Hyogo 665, Japan (Received October 11, 1980)

14C-Labeled (+)-traps and (+)-cis isomers of phenothrin [3-phenoxybenzyl, (+)-cis traps-chrysanthemate] were each applied at the concentration of 4 ppm to wheat grains with 11% moisture content, and stored at 15 or 30C in the dark. Both traps and cis were decomposed slowly, and 79 and 87% of the applied radiocarbon remained intact in after 12-monthstorage at 30C, respectively. Both isomers majorly metabolized via hydrolysis of ester linkage, oxidation of the benzyl alcohol to the benzoic acid and of the benzoic acid. The joint application with piperonyl butoxide and fenitrothion inhed the degradation of phenothrin isomers to some extent. The phenothrin isomers and their decomposition products were mainly located at seed coat during 12-month storage, and the residue levels of both isomers in flour and bran were 0.77 and 11.4 The phenothrin residues in flour somewhat decreased through the baking process, leaving of phenothrin isomers in bread. These findings were compared with those of 14C-m which was rapidly decomposed to desmethylmalathion, malathion-monoacids, -and 14CO2i with respective half-lives of 8 and 1.1 months. at 15 and 30C

INTRODUCTION MATERIALSAND METHODS Phenothrin [3-phenoxybenzyl (+)-cis, trans- 1. Chemicals chrysanthemate] is one of the new synthetic (+)-Traps and (+)-cis isomers of pheno- pyrethroids and currently used in household, thrin labeled with 14C at benzyl-methylene public health and livestock production because carbon in the alcohol moiety ethyl synthesized of its outstanding insecticidal activity and its by Hazue and Kamata8) of Sumitomo Chemical extremely low acute mammalian toxicity.1) Co. The specific activity of both preparations Recently this insecticide proved to have an was 7.3 mCi/mmole with radiochemical purity advantage over pyrethrins and bioresmethrin of more than 99%, as evidenced by thin-layer in cost/efficiency and reliable availability as chromatography (tlc). Malathion [O, O-di- a potential grain protectant.2,3) This com- methyl S-(1, 2-dicarboethoxy)ethyl phospho- pound combined with piperonyl butoxide and rodithioate] labeled with 14C at 1,2-ethyl fenitrothion was found to control a broad positions was purchased from Radiochemical spectrum of insect pests including or- Centre, Amersham, England and its specific ganophosphorus-resistant strains. 2,3) Although activity was 2.78 mCi/mmole with radio- metabolic fates of phenothrin in rats,4-6> chemical purity of 98%. plants7) and soil7) already established, its The following unlabeled authentic com- fate in stored grains has not been clarified. pounds synthesized in the authors' This report deals with the degradation and laboratory4,7,9): 3-phenoxybenzyl (+)-trans- distribution of (+)-lrans and (+)-cis isomers chrysanthemate [t-phenothrin], 3-phenoxy- of phenothrin in stored wheat grains and also benzyl (+)-cis-chrysanthemate [c-phenoth- their decomposition through processing and rin], 3-phenoxybenzyl alcohol [PBalc], 3- cooking. phenoxybenzoic acid [PBacid], methyl 3- phenoxybenzoate [PBacid-Me], 3-phenoxybenzyl (+)-2,2-dimethyl-3-Mans-formyl-cy- 184 日本農薬学会誌第6巻第2号昭和56年5月 clopropane-l-carboxylate [formyl-t-pheno- containing 30 ml of 6 N potassium hydroxide thrin], 3-phenoxybenzyl (+)-2, 2-dimethyl-3- solution was placed inside each vessel. cis-formyl-cyclopropane-l-carboxylate [for- The moisture content of the wheat grains myl-c-phenothrin], malathion, O-hydrogen O- was measured at intervals by keeping them methyl S-(1, 2-dicarboethoxy)ethyl phospho- at 120C for 4 hr.10) Immediately after ap- rodithioate [desmethylmalathion], O, O-di- plication of the 14C-preparations, the moisture methyl S-(1-carboxy-2-carboethoxy)ethyl content of the grains was 11.0%. Thereafter phosphorodithioate [malathion z-monoacid], O it gradually increased with time, amounting , O-dimethyl S-(1-carboethoxy-2-carboxy)- to 12.8 and 12.0% at 15 and 30C after 12 ethyl phosphorodithioate [malathion ss-mono- months, respectively. acid], O, O-dimethyl S-(1, 2-dicarboxy)ethyl phosphorodithioate [malathion diacid]. 3. Analysis of Phenothrin Isomers, Mala- Piperonyl butoxide [PBO] with the purity thion, Fenitrothion and Their Metabolites in of 98.4% was purchased from Takasago Wheat Grains Perfumery Co., Hiratsuka, Japan. Fenitro- At specified intervals a 10 g-aliquot of thion or Sumithion(R) [O, O-dimethyl O-(3- wheat grains was powdered with a mill (Kyo- methyl-4-nitrophenyl) phosphorothioate] of ritsu-Riko Co., Tokyo, Japan) for 2 min and technical grade with the purity of 96.8% was 30 ml methanol was added. The mixture was synthesized by Sumitomo Chemical Co. 14C- mechanically shaken and kept standing over- t-phenothrin, 14C-c-phenothrin or 14C-mala- night at 4C. The solvent phase was separated thion in emulsion was prepared as follows; by centrifugation and the residue was extracted the 14C-preparation, Sorpol 3005X (Toho further four times with 30 ml methanol. The Chemical Co., Tokyo, Japan) and xylene were combined methanol extracts were analyzed mixed at the rate of 1/1/9 (w/w), and diluted for 14Cand metabolites by liquid scintillation suitably with water. An emulsion of 14C- counting and tlc as reported previously.7) The phenothrin plus PBO or 14C-phenothrin plus tlc Rf values for phenothrin isomers, malathion PBO plus fenitrothion was also prepared from and their metabolites are listed in Table 1. each mixture of 14C-phenothrin-PBO-Sorpol To observe grossly the distribution of radio- 3005X-xylene (1/5/1/9) and 14C-phenoth- carbon, the grains were embedded in paraffin rin-PBO-f enitrothion-Sorpol 3005X-xylene and cut transversely and longitudinally, then (1/5/1/1/9). the sections were exposed to X-ray films for two weeks.11) 2. Treatment of Wheat Grains For determination of f enitrothion residues Wheat grains, variety Horoshiri, harvested in the grains, aliquots of methanol extracts in Hokkaido, Japan, in August, 1978, with obtained above were subjected to gas-liquid residues of less than the detectable amount chromatography in the following conditions: (2 ppb) of both malathion and fenitrothion apparatus; Shimadzu GC-5A equipped with were kept at 20+2C for 3 weeks over saturat- KBr single crystal FTD,12) column; 5% XE-60 ed magnesium nitrate solution to adjust the on Gas Chrom Q (100-120 mesh) packed into moisture content to 10.5%. The grains were q3 mmXl 1.0 m glass column, gas; carrier gas treated with each 14C-preparation in emulsion (He) 48 ml/min, hydrogen 27 ml/min, air 0.3 at the rate of 1 % relative to grain weight in l/mm, temperature; column 186C, injection October, 1978, then mixed well and air-dried. port 275C. The retention time of fenitrothion The application rates of phenothrin, pheno- was 3.6 min. thrin plus PBO, phenothrin plus PBO plus fenitrothion and malathion were 4, 4+20, 4. Decomposition of Phenothrin Isomers 4+20+4 and 15 ppm, respectively. Each through Processing and Cooking treated sample was placed in a stoppered glass The flour and bran were prepared as in the vessel (i.d. 14 cm, height 14 cm) and kept at following. Thirty-five grams of the grains 15+2 or 30+2C in the dark. To trap the were mixed with 2 ml of distilled water and volatile radioactive products, a 50-ml beaker stirred with a glass rod to peel off pericarps Journal of Pesticide Science 6 (2), May 1981 185

Table 1 Tic Rf values for phenothrin isomers, malathion and their metabolites.

a) A: n-hexane/toluene/acetic acid (3/15/2 , v/v), B: n-hexane/acetone (4/1), C: benzene saturated with formic acid/ether (10/3), D: n-hexane/ether (50/1), 7 developments, E: benzene saturated with formic acid/ethyl acetate/acetic acid (8/2/1), F: toluene/ethyl formate/formic acid (5/7/1), G: benzene/ether/acetic acid (8/2/1), H: benzene/ethyl acetate/acetic acid (50/50/3), I: toluene/ ethyl acetate/isopropanol/acetic acid (8/12/5/3).

from the grains. Germs were separated from duplicate experiments. the peeled grains by picking them up with a pincette. Then the grains were powdered RESULTS with a coffee mill (Nihon Philips Co., Tokyo, 1. Degradation of Phenothrin Isomers in Stored Japan) for 2 min and the powdered endosperm Wheat Grains (flour) was obtained by passing through a 100- As shown in Fig. 1, both isomers of pheno- mesh sieve to separate from aleurone layers. thrin were decomposed slowly in the grains The germs, pericarps and aleurone layers were even at higher temperature storage. After combined and the fraction was designated as 12-month storage at 30C, 79 and 87% of the bran. Each flour and bran fraction was applied radiocarbon were recovered as intact extracted with methanol and analyzed as t- and c-phenothrin, respectively, and t- described above. The bread was prepared as follows. Dry yeast (0.3 g), sugar (1.1 g), salt (0.27 g), skim milk (0.5 g), butter (1.0 g) and distilled water (12 ml, 40C) were added to the flour (20 g), then mixed well and subjected to fermentation (1st, 37C for 60 min; 2nd, 37C for 30 min) and baking (200C for 30 min) with an oven (Type TO-120, Hitachi, Tokyo, Japan). The bread was powdered with a mill and extracted with methanol using a Polytron(R) (Kinematica GmbH, Luzern, Steinhofhalde, Switzerland). The methanol extracts were concentrated and then were partitioned between acetonitrile and petroleum ether. The acetonitrile layer was analyzed as described above. Fig. 1 Residue amounts of phenothrin isomers, All experiments were duplicated. The malathion and fenitrothion in wheat results are shown as the average values of the grains stored at 15C and 30C. 186 日本農薬学会誌第6巻第2号昭和56年5月 Journal of Pesticide Science 6 (2), May 1981 187 188 日本農薬学会誌第6巻第2号昭和56年5月

t-Phenothrin c-Phenothrin Malathion

Upper: 1 month, middle: 6 months, lower: 12 months.

Fig. 2 Radioautograms of transverse and longitudinal sections of wheat grains treated with 14C-

phenothrin isomers and 14C-malathion and stored at 30C for 1, 6 and 12 months.

phenothrin was somewhat more degradable formyl-t- and formyl-c-phenothrin were pro- than c-phenothrin (Tables 2 and 3). At lower duced at the early stage of incubation, amount- temperature storage, decomposition of both ing maximally to 2% of the applied radio- isomers was negligible. The joint application carbon, but their contents decreased with time. of phenothrin isomers with PBO or PBO plus Unknown products which contained at least fenitrothion did not significantly affect the five spots on tic, and volatile radioactive prod- residual lives of both isomers for up to 6 ucts were detected in small amounts. months. But after 12 months, PBO and PBO plus fenitrothion gave the higher recovery of 2. Distribution of 14C in Wheat Grains and t-phenothrin by 4 and 10% of the applied Decomposition of Phenothrin Isomers radiocarbon at 30C, respectively. This through Processing and Cooking tendency was also observed in the case of As shown in Fig. 2, the distribution of radio- c-phenothrin. carbon in wheat grains did not change sub- The degradation products obtained from stantially during 12-month storage. The radio- phenothrin isomers were isolated and then carbon was mainly located at seed coat includ- identified by two-dimensional tic cochromato- ing the crease area, while relatively little graphy with authentic standards. PBacid-Me radiocarbon was found in germ and endosperm was further confirmed not to be an artifact by regions. Actually the contents of phenothrin extraction with acetone or benzene instead of isomers in flour obtained with our milling methanol. Among these products identified, procedure were 13 to 15 times lower than ester-hydrolyzed products such as PBalc, those in bran (Table 4). Furthermore the PBacid and PBacid-Me increased gradually, plienothrin residues in flour decreased through and the sum of these metabolites amounted the baking process to some extent. No trans/ to 6-14% after 12-month incubation at 30C. cis isomerization was observed during baking. These metabolites were produced in larger Most of the decomposition products in bread amonts from trans isomer than cis isomer and were ester-hydrolyzed products, and c-formyl- also at 30C than 15C. PBO and PBO plus phenothrin was detected in a trace amount fenitrothion decreased the production of these (Table 5). metabolites by 1-4 and 3-9% of the applied radiocarbon after 12 months, respectively. Other than the above decomposition products, Journal of Pesticide Science 6 (2), May 1981 189

Table 4 Residues of phenothrin isomers prior to and following processing and cooking.

a) μg phenothrin/g (wet weight). b) The wheat grains were treated with 14C-phenothrin isomers at 4 ppm alone and stored at 30C

in the dark. c) Wet weight ratio of milled fractions.

Table 5 Contents of phenothrin isomers and their decomposition products in flour and bread.

a) μg Phenothrin equivalent/g flour or bread (wet weight).

Table 6 Decomposition of malathion in wheat grains stored at 15C and 30C. 190 日本農薬学会誌第6巻第2号昭和56年5月

3. Degradation of Fenitrothion and Malathion more slowly than fenitrothion and malathion in Stored Wheat Grains in stored wheat grains. The degradation of As shown in Fig. 1, fenitrothion combined pyrethroids on stored wheat grains was studied with PBO and each phenothrin isomer was under similar conditions. The respective half- rapidly decomposed with respective half-lives lives of pyrethrin I and allethrin in the grains of about 12 and 1.5 months at 15 and 30C. with 15% moisture content at 30C were 3 and Malathion was decomposed a little faster than 4 weeks.13) The half-lives of bioresmethrin at fenitrothion. The half-lives of malathion were 30-35 and 20C were 8-10 and more than 26 about 8 and 1.1 months at 15 and 30C, weeks, respectively.14) From these results, the respectively. Malathion was decomposed degradation of phenothrin isomers in wheat majorly to desmethylmalathion, amounting to grains seems to be much slower than these 29% at its maximum, and to malathion chrysanthemic esters. monoacids and diacid, as shown in Table 6. Phenothrin isomers were mainly decom- No malaoxon residue was detected throughout posed via cleavage of the ester linkage to incubation at both temperatures. Volatile PBalc which was subsequently oxidized to radioactive products increased gradually with PBacid. PBacid was further converted to time, amounting to 9% of the applied radio- PBacid-Me. Both isomers in part underwent carbon after 12-month incubation at 30C. cleavage of the isobutenyl double bond to Most of these products were identified as yield formyl-t-and formyl-c-phenothrin, prob- 14CO2. The unextractable residues (Bound in ably due to air oxidation.7) No trans Jcis Tables) also increased with time, amounting to isomerization occurred during storage. Oxida- 20-50% of the applied radiocarbon after 12 tion products at methyl groups of the isobute- months, but further characterization of these nyl moiety or ring hydroxylation products at residues was not conducted. 2'- and 4'-positions of 3-phenoxybenzyl The distribution pattern of 14C-malathion moiety, which were found in metabolic studies residues is similar to that of 14C-phenothrin with rats,4-6) were not detected in the present residues, as shown in Fig. 2. experiment. The degradation pathways for DISCUSSION phenothrin isomers in stored wheat grains are proposed in Fig. 3. Rawlands13) showed that Phenothrin isomers were decomposed much pyrethrin I and allethrin were maj orly de-

Fig. 3 Proposed degradation pathways for phenothrin isomers in stored wheat grains. Journal of Pesticide Science 6 (2), May 1981 191 graded to the chrysanthemic acid and the 5) T. Suzuki, N. Ohno & J. Miyamoto: J. Pesticide corresponding alcohols and no oxidative prod- Sci. 1, 151 (1976) ucts were detected in stored wheat grains. 6) H. Kaneko, H. Ohkawa & J. Miyamoto: J. From these findings it seems that little or no Pesticide Sci. 6, 169 (1981) oxidative metabolism of chrysanthemic esters 7) K. Nambu, H. Ohkawa & J. Miyamoto: J. Pesticide Sci. 5, 177 (1980) occurs in the first steps, hydrolytic degradation 8) M. Hazue & T. Kamata: unpublished being the usual pathway in stored wheat grains. 9) T. Suzuki & J. Miyamoto: Pestic. Biochem. The preferential hydrolysis was observed in Physiol. 8, 186 (1978) the decomposition of malathion in the present 10) S. W. Pixton: J. Stored Prod. Res. 3, 35 (1967) study, where no malaoxon was detected. 11) Y. Takimoto, M. Ohshima & J. Miyamoto: J. The radioautograms of sections of wheat Pesticide Sci. 3, 277 (1978) grains (Fig. 2) show that little radiocarbon was 12) Y. Sato, J. Miyamoto & S. Suzuki: Botyu- located in endosperm, however the contents of Kagaku 33, 8 (1968) 13) D. G. Rawlands: "Residue Reviews," ed. by phenothrin isomers in flour were relatively high. The gap is presumed to be due to F. A. Gunther, Vol. XXXIV, Springer-Verlag, New York, p. 116, 1971 contamination of seed coat at the crease area 14) FAO/ WHO: "1975 Evaluation of Some Pesti- where the radiocarbon was localized into flour cide Residues in Food," p. 67, 1976. during our milling process. In a normal com- 15) J. Moss: Private communication (1979) mercial flour milling, only a very small portion 16) K. Kawamura, M. Takeda, M. Uchiyama, K. of the crease area will contaminate into flour. 15) Sakai & H. Ishikawa: Shokuhin-Eiseishi 21, In fact, the malathion contents in flour were 70 (1980) 10-14% of those in wheat grains when a 17) K. Nambu, Y. Takimoto & J. Miyamoto: un- milling machine was used, as reported by published Kawamura et al. 16) The malathion contents in flour obtained with our milling procedure were about 22% of those in wheat grains. 17) There- 要約 fore, in practice the contents of phenothrin フェノスリンの貯蔵中の小麦穀粒における分 isomers in flour appear to be lower than the figures that we found. 解 These laboratory findings suggest that 南部健二, 滝本善之, 宮本純之 14Cで標識したフェノスリンの(+)-トランスおよび phenothrin isomers applied to wheat grains seem to remain intact at seed coat and exhibit (+)-シス体を水分含量11%の小麦穀粒におのおの4 an excellent insecticidal activity against stored ppmの濃度で処理し, 15または30。Cの暗所に貯蔵し grain pests for a prolonged period. Most of the た. トランスおよびシス両異性体は徐々に分解し, 12カ intact residues on the grains are likely to be 月後には30Cにおいてそれぞれ添加量の79および removed during milling process and a low 87%が小麦穀粒に残存していた. 両異性体ともおもに level of phenothrin residues will appear in エステル結合の加水分解, ベンジルアルコールの安息香 flour or bread. 酸への酸化さらに安息香酸のメチル化を経て代謝された. ピペロニルブトキサイドおよびフェニトロチオンと REFERENCES の混合処理によりフェノスリンの分解はいくぶん抑えら 1) K. Fujimoto, N. Itaya, Y. Okuno, T. Kadota れた. フェノスリンとこれらの分解物は12カ月間おも & T. Yamaguchi: Agric. Biol. Chem.37, 2681 に種皮に存在し, 製粉により, 小麦粉およびふすま中の (1973) 2) J. H. Ardley & G. R. H. Halls: Chemical 両異性体の残留量はそれぞれ0.77および11.4ppmと Marketing and Economics Division of the なった. この小麦粉を製パンすると残留量は0. 57ppm AmericanChemical Society, Preprint, Honolulu, に減少した. 一方, 14c-マラチオンは小麦穀粒中15お Hawaii,pp. 38-54, 1979 よび30Cにおいてそれぞれ半減期8および1. 1カ月 3) J. H. Ardley, M. Bengston,J. M. Desmarchelier & P. Skidmore: J. StoredProd. Res. in press の速度で分解し, デスメチル体, モノカルボン酸体, ジ 4) J. Miyamoto, T. Suzuki & C. Nakae: Pestic. カルボン酸体および14CO2を生成した. Biochem. Physiol. 4, 438 (1974)