THE RADIOCHEMICAL ANALYSIS OF PHOSPHINE RESIDUES IN FUMIGATED TOBACCOS 1

By J. G. UNDERWOOD British-American Tobacco Co. Ltd.,

Group Research and Development Gentre, Tobacco Science, 1972, 16-39, p. 123-126, ISSN.0082-4523.pdf Regent's Park Road, Southampton, England

This paper describes a radiochemical investigation Experimental fumigation of tobacco with radioactive phosphine has demonstrated that the level of residual phosphorus compounds into the level and identity of the residual phosphorus after fumigation was less than 5 ppm in the tobacco. This value compounds in tobacco after fumigation with phosphine­ is dependent on tobacco type, being lower for Burley than for flue-cured tobacco at a given moisture _content, and is increa~ed P32, and the fate of these compounds on smoking. by a rise in the moisture content orf a given tobacco. On smoking cigarettes made from each of these fumigated tobaccos, ~he MATERIALS AND METHODS residual phosphorus was retained entirely in the _ash, none be-1'"!g transferred to main-stream smoke. There is no evidence from this Tobacco work of the presence, after phosphine fumigation, o,f any phos­ Flue-cured and Burley strips were conditioned to ap­ phorus compounds not normally present in tobacco or ash. It is seen that the phosphine gas may not be regarded as a "n~­ proximately 12% water content. Portions of these tobac­ residue" fumigant· however it is shown that the residues identi­ cos were sprayed with the calculated amount of water fied in tobacco ar~ non-toxic and are not transferred into smoke. and equilibrated to give approximately 20% water con­ tent. These values were confirmed prior to each exper~­ ment by Near Infra-Red Spectroscopic Assay, and were INTRODUCTION calculated from a wet base. Studies by Childs et aL (6-10) and by Edmond et al. Cigarettes and Smoking (13, 14) have shown that phosphine gas is an effective Strip tobacco was cut into 1 mm slivers with a scalpel fumigant for the control of the tobacc·o beetle, Lasio­ and made into cigarettes by introducing 1 g portions of dertna serricorne (F) under various storage conditions. this cut tobacco into a "Laredo"2 cigarette maker. The It has been reported that phosphine fumigation has tip of each cigarette was removed to give plain ciga­ no measurable effect on the taste or aroma of ciga­ rettes, 70 x 2-5 mm, which were conditioned over satur­ rettes (7), or on the nicotine, protein, sugar or moisture ated sodium bromide solution for 24 hours.. These were level of tobacco (21, 22). smoked to an 8 mm butt on a B-A.T. bellows type Work in this laboratory (1), using a colorimetric restricted smoking machine, using a 2 second puff dura­ method, has shown tha,t the residual phosphine ga,s in tion, 35 ml puff volume, frequency 1 puff/minute. tobacco after phusphine fumigation was less than 2 Radiochemical Reagents parts in 10G. However, recent communications (2, 5, Phosphine-P32 was supplied (by the Radiochemical 1 2, 20) have indicated a low level of "chemisorbed" (3) Centre, Amersham, England) in sealed ampoules. Each Phosphine, i.e. residual phosphorus c_ompounds, in cere­ ampoule, sufficient for one fumigation, contained 9.1 al products after fumigation, not removed by prolonged mg, 2.9 mCi of phosphine-P32, specific activity 10.8 a:ration or baking (20), but not retaining the insecti­ mCi/mM ou 23rd October, 1970. This phosphine-P32 cidal activity. Inefficient aeration can lead to low con­ was free from detectable impurities :including dipho,s­ cen~rations of phosphine remaining in grain for long phine (Radiochemical -Centre, batch analysis). Periods, retaining its toxicity to insects. It was also Reagents were B.D.H. "Analar" grade or o,f equiva­ ~oted that the characteristic odour of phosphine ( due to lent purity; solvents were redistilled befure use. \~rrities, e.g. diphosphine) is absorbed by wheat flour Liquid Scintillation Counting Packard Liquid Scintillation Counters. Model No . . Attempts to determine the "chemisorbed" phosphine 1n gr · . 3375. Channels (a) conventional liquid scintillation h arn indirectly by measuring loss from the gas counting: (i) 50-1000 (1.7%). (ii) 400-1000 (1.9%), P ase have produced conflicting results (2, 11). (iii) 300-700 (2.0%); (b) Cerenkov counting: (i) 0-oo

~. 2 ference J.:re!en_ted at the Twenty Fifth Tobacco Chemists' Research Con­ • Registered Trade Ma,rk of Brown and Williamson Tobacco Corp., Louis­ , oir1sv1l/e, Kentucky, 6th October, 1971. ville, Kentucky, U.S.A.

(Tobacco Science 123) (100%), (ii) 50-1000 ( 4.2%), (iii) 50-1000 (8.8%). 66% aqueous ethanol, to collect the, phosphine remaining Aliquots of radioactive solutions were counted in one in the gas phase (26). One experiment using flue-cured or more of the following ,scintillation solutions: tobacco indicated that this solution was a more efficient (a) 2,5-diphenyloxazole (PPO) 7.0 g absorbent for phosphine than permanganate solution dime thy1-2,2-pheny Jene-bis- ( 5-phenyloxazole) (see Table 1). (dimethyl-POPOP) 0.3 g After aeration, the following assays were performed: naphthalene (scintillation grade) 100 g (i) Radioactivity in the gas phrrne. The contents of the dioxan (A.R.) 1 litre mercuric chloride traps were treated with bromine this scintillator was used for aqueous solutions; count­ water to dis,solve the precipitate. Excess bromine was ing efficiencies were determined by external standard­ removed and aliquots of the resulting solution sub­ isation and by the channels ratio method. mitted for scintillation c6unting. (ii) Radioactivity ad­ (b) PPO 5.0 g sorbed onto the walls of the apparatus. The apparatus dimethyl-POPOP 0.3 g wa,s carefully washed with a known amount of ,a 2% so­ toluene (A.R.) 1 litre lution of 44 Decon"3 at 80° until complete removal of the for organic s1olutions; counting efficiencies were deter­ radioactivity was effected. Aliquots of this solution were mined by external standardisation and by the channels assayed. (iii) Radioactivity in the tobacco. (a) Digest. ratio method. Tobacco (1 g) was heated under reflux for 2 hourn with I (c) Cerenkov counting was effected in aqueous solution a solution of nitric acid (5 ml, concentrated) in per­ or in dilute aqueous acid, following the method due to chloric acid ( 5 ml, 72 % ) , the solution evaporated to 4 Haviland and Bieber (17). Counting efficiencies were ml .and made up to 50 ml for radio-assay. This solution t determined by the channels ratio method. Activities gave a measure of the "chemisorbed" phosphine. (b) Tobacco Science, 1972, 16-39, p. 123-126, ISSN.0082-4523.pdf were all calculated from the published value for t112, Cold water extract. Ground tobacco (1.0 g) was allowed 14.29 days for P32 (15). to stand in co.Id water (50 ml) overnight. The solution Fumigation of Tobacco with Radioactive Phosphine was filtered and assayed. ( c) Hot water extract. A f The apparatus was assembled as 'Shown in Figure 1. further portion of the tobacco (lg) was ground and ex­ Flask D contained tobacco (250 g). Taps 1, 2 and 3 tracted with water (50 ml) for 4 hours in a Soxhlet ap­ were opened and the apparatus was swept (from A) paratus, the extract being made up to a known volume with a slow stream of nitrogen for 1 minute to preclude for assay. ignition or explosion on release of the phosphine. Tap 1 Identification of the Phosphorus Residues in Phosphine was then closed and a partial vacuum (approximately Fumigated Tobacco 10 cm Hg) applied at B, tap 3 being then closed. On A phosphine-P32 fumigation of flue-cured tobacco opening tap 4, ammonia and carbon dioxide were re­ (12% moisture content) was carried out as previously, leased (as with "Phostoxin") and the apparatus except that "Phostoxin"' (1 tablet = 1 g phosphine) brought to atmospheric pressure. After closing tap 2 was placed in ves1sel F, instead of the ammonium car­ the phosphine-P32 (approximately 9 mg, 3 mCi) was bamate. This exposure of tobacco to a greater weight of released using a magnetic breaker from the break-seal phosphine should give detectable amounts of phos­ ampoule C. The system was allowed to stand for 96 phorus-containing residues. After thorough aeration, hours at ambient temperature to effect exposure of the the hot water extract of ,the tobacco (10 g) and its tobacco (E) to radioactive phosphine. The apparatus digest were subjected to paper chromatography, follow­ was swept for 96 hours with a stream of air from A, ing the method of Robinson and Bond ( 11). through flask D, into three Dreschel traps (at B). These The relative intensities were estimated from (i) the traps contained a 1.0% solution of mercuric chlnride in intensity of the developed spot and (ii) the readings on a Geiger-lVIuller type radioactivity monitor held over the developed area. The limits of detection were approx­ imately 1 x 10-6 g for the -spray reagent and approx­ timately 1 x 10-8 g for the radio-monitor, correspond­ ing to levels in the tobacco of about 0.01 % and 0.0001 % 0) phosphorus respectively. Chromatographs were developed using a molybdate spray due to Harrap(16). The Fede of the Residual Phosphine-P32 in Tobacco on Smoking A. Five cigarettes made from each of the fumigated tobaccos were smoked into a Cambridge filter, followed by a mercuric chloride Dreschel trap, then a cold trap (liquid oxygen). The Cambridge filter pad and the cold trap were extracted with methanol and the resultant

solutions ass1ayed. The mercuric chloride trap was treated as previously. B. Five cigarettes were smoked into a Cambridge filter followed by the cold trap then the mercuric chlor· ide trap, work-up being as previously. In both cases the butt and ash were assayed after

A INLET digestion with nitric/perchloric acid (as before). B OUTLtT The results of the radio-assays from these experi~ C PHOSPHINE: - P32. ments are given in T·able 2. Where the count,ing rate O to 2 e"LASK WITH 100mm FLANGE E TOBACCO (Z50g) r AMMONlUM CARBAMATC G TEFLON COATED MAGNET M WATtR 0 J MANOMtTEA 3 • Registered Trade Mark of Decon Laborator.ies Ltd. Figure 1. Apparatus for the experimental fumigation of tobacco 'With 4 • Registered Trade Mcwk of Deutsche Gesellschaft fUr Schlid.Iingsbr• radioactive phosphine. klimpfung (Degesch). Frankfurt am Main, West Germany.

(Tobacco Science 124) --> was l"''Sr,,_, than 1 •25 x background level of radioactivity is 2. P,H, + H,O + %0, 2 H,PO, given as zero. 3. 2 H,PO, + Oo ➔ 2 H,PO, 1. 2 H,PO, + o; ➔ 2 H,PO, 5. H,PO, H,0 ➔ pyrophospbates and RESULTS AND DISCUSSION - polyphosphates. The Le,ud of Residual Phosphorus Compounds in Tobac- The diphosphine formed in step 1 accounts for t~e odour of phosphine. The preferential absorption of th~s co The effect of phosphine-P32 on flue-cured tobacco at diphosphine in wheat fl.our (4) may also occur m 12%, 1:3S>~ and 19% moi,sture content and on iburley to­ alkaline tobacco accounting for the adverse effoct on the ba~co at 19% and 22% moi~•ture content was dete~mmed, taste of smoke from cigars made with phosphine fumi- · e · n the case of gram the level of chem1sorbed srnc 1 . gated tobaccos (18). . phosphine was shown to be ve·ry dependent on the mois- Phosphine generated from "Phostoxin" tabl~ts 1s p~o­ ture content(l2). . . tected from ignition and explosion by the ambient mois­ The tobacco, in strip form, was expo_se~ to radioactive ture (and by the carbon dioxide and ammonia generated h 0 Sphine in the presence of carbon d10x1de and ammo- p • .. • F' 1 d simultaneously). However, there is no protection from nia (usmg the apparatus shown m 1gure , un er air-o·xidation of moist phosphine to give involatile prod­ conditions closely simulating those recommended for ucts found as residues. Since this work has demon­ warehouse fumigation with "Phostoxin" (6, 7, 8~. The strated the presence only of phosphates, of the type level of phosphine-P32 employed in these experiments naturally present in tobacco, this residue would not (9.1 mg in 10 litres) corresI?on~s to a dose of 26 g per appear to constitute a health hazard. . 1 000 cu. ft. Warehouse •fum1gat10ins normally use 20-30 The Transfer of Residual Phosphorus Compounds into "Phostoxin" tablets (= 20-30 g phosphine) per 1,000 Smoke Tobacco Science, 1972, 16-39, p. 123-126, ISSN.0082-4523.pdf cu. ft. (G). After contact with the fumigant. for 96 The transfer into main-stream ,gmoke of these residu­ hours, the tobacco was swept with a stream .of ·a~r for a al phosphorus-containing compounds in to~acco a~ter further !=J6 hours. The non-absorbed phoS'phme, 1.e. that fumigation with phosphine-P32 was determmed. Ciga­ remaining in the gas phase during fumigation, was rettes made from each of the fumigated tobaccos were collected in mercuric chloride solution (12). The phot:i­ smoked and the main-stream smoke collected on a Ca1:1-­ phine adsorbed onto the walls of the apparatus was bridge filter and in a subsequent cold trap and mercuric removed for assay. That aeration was, complete after 96 chloride trap. The radioactivity present in thes•e traps hours was checked by additional aeration of three sam­ Was measured by liquid scintillation counting. The butt ples of tobacco for a further 96 hours. Portions of the and ash were assayed after acid digestion. fumigated .tobacco were (i) digested with an acid ox­ The results of these experiments a.re given in Table idant (ii) extracted with cold water and (iii) extracted 2, and it is seen that, in all cases, the radioactivity with 'hot water, to give solutions for radio-assay by present in the tobacco is entirely retained in the ash, liquid •scintillation counting. . . there -being no transfer of any "chemisorbed" phos­ The results of these experiments. are summarized 1n phorus-containing compound into main-stream ,smoke. Table 1. The chemical form of this residue in the ash was shown From this work it is seen that all phosphine gas was by paper chromatography (20) to be, Irul.inly phosphate. removed from the tobacco by aeration for 96 hours, but The limits of detection by paper chromatography are that there is a residue of chemisorbed phosphorus com­ approximately 1 x 10-6 g whereas by scintillation pounds in the tobacco of 0.45-3.22 ppm, the value being dependent on the type of tobacco and its moisture con­ tent. Most of these radioactive phosphorus compounds are removed by hot water extraction, but not with cold water. Table 1. The recovery of radioactive phosphine after fumigation of tobacco The values obtained in this work agree with those of Tobac:c:o Type Flue•c:ured Burley Kuhn (18) who obtained 2.6-2.9 ppm using phosphine­ Water, % 12.2 13.0 !9.0 18.5 21.8 P32 remaining in gas phase, % 56.4 48.711 66.8 62.4 - b P32. These results are similar to those obtained for P32 adsorbed onto apparatus, % 19.3 5.4 12.7 13.8 14.6 grain and cereal products of 1-20 ppm (12, 20), depen­ Chemisorbed P32 residue, digest of tobacco, % 5.8 8.1 8.9 1.24 0.81 dent inter alia on moisture content and physical form. P32 residue in tobacco after The Nabue of the Residual Compounds in Tobacco extra aeration, % 5.8 8.9 1.24 Recovery of radioactivity, % 81.5 62.2" 88.4 77A 15.711 The solubility of the residual phosphorus compounds Residue of P32 in tobacco expressed as ppm of PH3 2.04 2.61 3.22 0.45 1.B9 in water was demonstrated (see Table 1). To obtain P32 extracted from tobacco sufficient of these residual phosphorus compounds in by cold water, % 1.4 1.6 3.0 0.2 0.3 P32 extracted from tobacco tobacco to establish their chemical 1identity, flue-cured by hot water, % 5.6 7.0 8.2 1.2 0.8 tobacco was exposed to a high concentration of phos­ a low va/iw· due to 1tse of potassium permanganate collection traps. :Phine-P32 (of lower s·pecific activity than used :in previ­ b gas phase value not given, due to a leak in the collection appa.ratus. ous experiments). After thorough aeration, the tobacco Was extracted with hot water and thi:s. aqueous solution Table 2 The fate on smoking of residual P32 compounds from subjected to paper chromatography (20) .. · phosphine.P32 fumigated tobacco By this method the major constituent of the phos­ Tobac:c:o Type Flue-c:und Burley Phorus-containing residue in tobacco was shown to be Collection method a A B A Rodioac:tivity d/m % d/m % d/m % orthophosphate, plus a small amount of pyrophosphate Total activity in cigarette 2.3 X 106 IO0 2.3 x ]QG l00 3.2 X ]()5 IO0 Activity in mainstream and a trace o,f polyphosphate. In contrast to the corre­ Cambridge filter 0 0 sponding analysis of the residues in grain (20) there Activity in mainstream mercuric chloride trap 0 0 0 0 Was no trace of phosp:hite or hypopho,sphite. A chromat­ Activity in mainstream cold trap 0 0 0 0 0 0 ographic analysis of the oxidising acid digest of the Tota\ mainstream activity 0 0 0 0 0 0 tobacco revealed the presence of phosphate only. Activity butt ,2.7 X 10' 11.7 2,1 X 105 ,., 3.6 X 104 11.3 Activity in'" ash L6 x 106 69.6 2.0 X 1()8 87.0 2.2 X ]{)5 68.7 Hence the oxidative environment in the tobacco al­ Total recovery of activity l.9x IQd 81.3 2.2 x 11)11 96.1 2.6 X IQ5 80.0 lows the formation of the oxyacids of pho.sphorus, even­ 11 A; Ciga..rette - Cambridqe Filte,r - Mer-e~c Ch!oride - Cq/d Trap iual!y all being converted to phosphates: STMking Mach_ine. B: Ciqarette -:- Cambridge Filter - Cold Trap - Mercuric Chloride - Smoking Machine, · 2 PH, + ½O, -> P.,H, + H,O

(Tobacco Science 125) counting they are approximately 2 x 1010 g of pho,g­ l\f. A. Baylis for assistance with ~he experimental work. phorus, thus a main-stream transfer of approximately 0.01 % would be detected by this method. LITERATURE CITED The direct method of applying labelled phosphine to 1. Ayers, C. \V., Personal Communication, 1969. tobacco in a closed system dne,s not closely duplicate the 2. Berck, B., Sorption of Phosphine bY Cereal Products. J, Agr, Food Chem, 16: 419-425. 1968. slow-release, open-ended type of exposure found in prac­ 3. Berck, B. and Gunther, F. A., Rapid Determination of So:rp­ tice, but it is unlikely that this will -cause more than a tion Affinity of Phosphine by Fumigation within a Gas Chroma.to. graphic Column. J. Agr. F-ood Chem. 18: 148-153. 1970. S'light difference in level of residual phosphorus com­ 4. Bond, E. J. and Dumas·, T., Loss of VVarning Odour fro:tn Phosphine. J. Stored Prod. Res. 3: 389-392. 1967. pounds; certainly it will not affect their nature. 5. Bruce, R, B. et al., Phosphine Residues from Phostoxin Since diphosphine is an intermediate in the oxidative Treated Grain. J. Agr. Food Chem. 10: 18-21. 1962. 6. Childs, D. P. et al. Phosphine Fumigation of Flue-cured degradation of phosphine, the- urSe of purified pho,s,phine­ 'L'obacco VVarehouses for Control of the Cigarette Beetle. Tobacco Sci. 13: 64-69. 1969. P32, rather than the crude phosphine liberated from the 7. Childs, D. P. et al., Phosphine Fumigation of Flue-cu:red aluminium phosphide in "Phostoxin" tablets, will not Tobacco for Control of the Cigarette Beetle-Laboratory Tests Tobacco Sci. 13: 160-163. 1969. · result in large discrepancies in the residue levels be­ 8. Childs, D. P. and Overby, J. E. Phosphine Fumiga,tion of To]jacco in Louvered Warehouses. Tobacco Sci. 14: 49. 1970. tween laboratory and warehouse fumigations. 9. Childs, D. P. et al., Phosphine Fumigation of Tobacco in Freight Containers (Part I). Tobacco Sci. 15: 1-4. 1971. 10. Childs, D. P. et al., Pho,sphine Fumigation of Tobacco in SUMMARY Freight Containers (Part II). Tobacco Sci. 15: 5-6. 1971. 11. Dieterich, W. H. et al., Hydrogen Phosphide as a Fumigant Experimental fumigation of tobacco with radioactive for Foods, Feeds and Processed Food Produc.ts. Residue Rev. 19: 135-149. 1967. phosphine has demonstrated that the level of residual 12. Disney, R. W. and Fowler, K. S., Pree.I. U,P.A.C. Second phos,phorus compounds after fumigation was ,less than 5 International Congress of Pesticide Chemistry, Tel Aviv, 1971. Personal Communication in Advance of Publicatrnn. ppm in the tobacco. This value is dependent on tobacco 13. Edmond, D. E., Preliminary Investigations of the Ability of Phosphine Gas ,to Penetrate Polyethylene Tobacco Case Liners.Tobacco Science, 1972, 16-39, p. 123-126, ISSN.0082-4523.pdf type, being lower for Burley than for flue-cured tobacco Tobacco Sci. 15, 11. 1971. at a given moisture content, and is increased by a rise 14. Edmond, D. E. et al., '.rhe Penetration of Phosphine Gas into Lined Tobacco Cases during Atmospheric Fumigation. To. in the moisture content of a given tobacco. bacco Sci. 15: 84~87. 1971. 15. Goodier, I. W. and Pritchard, D. H., The Half Life of 02p_ On smoking cigarettes made from each of these fumi­ Intern. J. Appl. Radiatio,n Isotopes 17: 121-123, 1966. gated tobaccos, the residual phosphorus, was retained 16. Harrap, F. E. G., The DeteC1tion of Phosphate E,sters on Paper Chromatograms. Analyst 85: 452. 1960. entirely in the ash, none being transferred to main­ 17. Haviland, R. T. and Bieber, L. L., Scintillation Counting of 32P without Added Scintillator in Aqueous Solutions and Or­ stream smoke. ganic Solvents and on Dry Chromatographic Media. Anal. Bio. There is no evidence from this work of the presence, chem, 33: 323-334. 1970. 18. Kuhn, H. et al., The Fumigation of Tobacco by Phosphine: after phosphine fumigation, of any phosphorus com­ Analysis of Residues and Taste Testing. Fachliche Mitt. Oesterr, Tabakregie 12: 191-203. 1971. pounds• not normally present in tobacco or ash. 19. Reynolds, E. M. et al., The Effect on Sitophilu.s granarius It is seen that the phosphine gas may not be regarded (L) (Coleoptera, C'urculionidae) of 1:Qxposure to Low Concentra­ tions of Phosphine. J. Stored Prod. Res. 2: 177-186. 1967. as -a "no-residue" fumigant; however it is shown that 20. Rob1nson, J. R. and Bond, E. J., The Toxic Action of Phos­ phine-Studies with J 2PHs; Terminal Residues in Biological Ma­ the residues identified in tobacco are non-toxic and are terials. J, Stored Prod. ReS. 6: 133-146. 1970. not transferred into smoke. 21. Todoro-vski, B., Phostoxin Fumigation of Stored Tobacco. Duvan 10: 198-211. 1960. 22. Vasilev, L., Application of Phostoxin to Control Ephestia elutella in Stored Tobacco, Tutun 13: 227-231. 1963. ACKNOWLEDGEMENTS 23. White, W. E. and Bushey, A. H., Aluminium Phosphide­ Preparation and Composition. J. Am, Chem. Soc. 66: 1666-1672. The author is indebted to Mrs. V. J. Smith and Mr. 194'1.

(Tobacco Science 126)