Document DMwq2eXdXwp9LGNpOo0dpoXN

Polyclilorolip!iciiy!t4 (PCR's) and ihcir Interference with Pesticide Residue Analysis by Lincoln M. Reynolds I Ontario Reiearch Foundation Sheridan Park, Ontario, Canada I The PCB's Introduction (X indicating the possible chlorine poeltions) were studied as early as 1881 (1) and by 1930 (2) were in wide use. They are known to be quite toxic, especially to liver cells. As early as 1936, Jones and Alden (3) reported that sen employed in the production of PCB's developed acne-type skin eruptions. Three years later, Greenburg and coworkers (A) reported that PCB's and polychlorinated naph thalenes were resposlble for the deaths of three workers. Residue chemists, especially in Europe, have recently become interested in these PCB's as well as the polychlorinated triphfgn- yls, naphthalenes, terpene6, and other related compounds, since NOTEi This paper was presented at the Eastern Canada Sem-i inar on Pesticide Residue Analysis, November 18-19, 1968, at Guelph, Ontario, Canada. ACKNOWLEDGMENTS! This research was supported by funds from the Pesticide Section, Canadian Wildlife Service, Ottawa, and from the Province of Ontario through the Department of Trade and Development. The technical assistance of Mary Coleman, Terry Cooper, and other members of the ORF Pesticide Laboratory is gratefully acknowledged. us Bolletia < EarinaiMaUl CaaUaiaatka A Toxkalnq, Yl a N*. A, IMS, ptUlM hr Spriapr-Verkc N*w Ycck lac. i ( \1 I DSW 332257 STLCOPCB4078833 I Jensen (5) in Sweden reported their presence in wildlife tissues about two years ago. The FCB's and related compounds (although in the rest of this paper reference will be made to the PCB's only, the other related compounds are also quite important) have very numerous and impor tant industrial uses, but are not used as pesticides. Because of ! their similarities in structure and properties to the DDT pesti- ( cide group, the FCB's, if present, are carried through the usual pesticide extraction and screening procedures, and since they { possess electron absorbing properties, will interfere with gas liquid chromatographic electron capture (GLC-EC) analysis of the . organochlorine compounds. t ^ Before any discussion of the type of interference encountered j it would be appropriate to mention briefly some of the properties and uses of the FCB's. They are produced and marketed under a number of commercial trade names e.g. 'Aroclor', 'Clophen A50', etc. The PCS'a are i available aa liquids, resins, or solids) insoluble in water; thermoplastic) non-drying) stable on long heating at 150*C.) electrically non-conducting) not affected by boiling with NaOH * solution) do not support combustion when alone above 360*C.) are 1 easily soluble in most common organic solvents and drying oils. They are used in protective coatings, as plasticizers and i extenders, as sealers in water-proofing compounds and putty, in , asphaltic materials, printing inks, waxes, and synthetio u adhe: therr grint < seal< ( tretm Cons' or ni mabic Jens* f ind i ceivi resu) -also when that tion Comp* that makir dual DSW 332258 STLCOPCB4078834 tissues of this related 1 impor:ause of pestii usual :hey gas of the ! \ : ' , >unt erect forties ' ?rcial i are rrj i I ' NaOH l are oils. ' and y. in I f adhesives. Liquid PCB's are used as dielectrics, as hydraulic fluids, in thermostats, in cutting oils, as extreme pressure lubricants, as grinding fluids, and as heat transfer media. Solid FCB's are used to impregnate carbon resistors, as sealers or impregnating agents for electrical apparatus. Obviously, the stability of these compounds makes them ex tremely useful and versatile for a great number of applications. Considering their stability - not affected by boiling with NaOH or nitric acid, not metabolized in living organisms, and nonflam mable if containing more than four chlorine groups, it is as Jensen (6) pointed out, difficult to explain how these compounds find their way into living organisms. However, with the numerous applications, it is not incon ceivable that fish and other wildlife could be polluted as a result of the flushing of wastes into rivers, lakes, etc. It is -also possible that contamination could proceed via the atmosphere when wastes containing these compounds are burnt. However, a third and more likely source is the possibility that some companies might be using PCB's in pesticide formula tion to increase the kill-life of insecticides. The Monsanto Company, which manufactures the Aroclors, stated back in 1965 (7) that the Aroclors can "trap" and hold more volatile ingredients making volatile insecticides and repellents last longer in resi dual activity. The most pronounced effect for increasing the isa * DSW 332259 j i i I i 1 | { ! \ I tI \ i l* STLCOPCB4078835 I kill-life of Insecticides was obtained with lindane, chlordane, and benzene hexachloride (BHC). A ten-fold effectiveness for lindane was reported by the U.S.D.A. by includins 5-25Z FCB's 1q the fornulstion. Attempts to determine whether this ides had \ 1 i[ been put into practice by soae companies have so far been unsuc cessful. But there is no doubt that, if the FCB's are being used in pesticide foraulation, then this would certainly explain their I f presence in wildlife tissues and other saaples. ' Jensen (6) has used a nitration procedure in order to differ- * entiate the FCB's fron the pesticide residues. He treated the cleaned-up extract with a mixture of concentrated HNO^ and con- centrated H^SO^ (111) for 5 ain. at 0*C. After the addition of crushed ice, he extracted the reaction mixture with hexane and i reinjected the extract. He states that the method should leave FCB's, lindane, and BHC unaffected. Our attempts to repeat this reaction have not been fully successful. There appears to be soae loss of the more volatile (early emerging) FCB's, heptachlor epoxide is not affected, and peaks with longer retention times appear. Although Jensen did not elaborate as to the fate of the pes ticides, we have demonstrated that apparently, nitration does occur. This was shown for DDT when a large peak (probably due to , the tetranitro derivative) appeared on the chromatogram about 2 ' hours after Injection of the nitrated extract. Of course, this reaction is a modification of the old ill * Sc (f we lc an st tl of dc c< 0 n: n< m t P f d b P DSW 332260 STLCOPCB4078836 chlordane, eness for 25? FCB's ln Idea had been unsuc re being used otplain their er to differ ent ed the 2 and conaddition of exane and Juld leave repeat this :s to be heptachlor on times of the peson does ably due to a about 2 Schechter-Haller (8) DDT method In which more drastic conditions (fuming HN03 and concentrated H^SO^ with heating on steam bath) were used to ensure oxidation and removal of interfering biolog ical materials. The nitrated pesticides were extracted with ether and a colorimetric method was used in the final determinative step. Erro et al. used this technique to determine toxaphene in the presence of DDT, on the basis that the chromatographic pattern of toxaphene is not affected by nitration while the nitrated DDT does not chromatograph under the specified conditions. Obviously, nitration does not appear to be the answer for complex mixtures of pesticides and FCB's since some pesticides (lindane, BHC, toxaphene, 'Strobane*, etc.) apparently will not nitrate while some of the FCB's might nitrate. Although we have not used Jensen's column packing (the liquid phase SF-96 is a methyl silicone), it is impossible to avoid complication and in terference from the nitro derivatives formed, especially when the pesticides are present in large amounts. There are three main reasons why we prefer an approach dif ferent from Jensen'si 1. It is preferable to separate the two groups rather than destroying one, especially when it is the pesticides that are being destroyed. 2. The nitration approach tends to complicate the inter pretation of the chromatograms, since the nitro derivatives us dS\N 332261 i I } J I '1 I i STLCOPCB4078837 CHROMATOGRAM OF STANDARO MIXTURE OF 0R 6A N 0C H L0R IN E PESTICIDES - possess greater electron absorbing power and with their longer retention tines, should emerge and interfere with subsequent injections. 3. We have been unable to repeat Jensen's clear-cut dif ferentiation, apparently partly because of the nitration of bom of the PCB's. Interference of PCB's We have attempted a more ideal approach to differentiate the f two groups by separation followed by the separate analysis of I each group. The GLC work was carried out under the following conditionii Gas Chromatograph! Varian Model 1200, fitted with tritiumelectron capture detector} column! glass, spiral, 6' x 1/8" O.D., packed with 6Z QF-1 and 4I SE-30 on Chronosorb W (AW). Ho. of theoretical plates for DOT * 2227. I I I \ Operating Conditional Column temperature 190C.| injector temperature 245*C.} detector (base) temperature 240*C.{ H^ flow lI rate, approximately 40 ml./min.; volume injected, 5pl. Recorders Varian Aerograph Model 20, 1 mV, full scale deflection. Chart speed! 2/3" per min. Fig. 1 indicates the degree of separation of 8 pesticides in a standard mixture. The excellent^soperation obtained for DDE and dieldrin in this column which was first used by McCully and McKinley (10) should be noted. I 131 f t \ \ dSVM 332262 STLCOPCB4078838 It longer sequent i r-cut difLon of soae rentiate the lyaia of conditional th tritiuaK 1/8" O.D., a No* Of j injector Nj flow . Recorderi n. Chart esticidea ined for by McCully j l ( \ 9 M t.ma sS f ' t-0l I ll t.0l** a<c Xoo OX US **0) * r* WOHOVldSM jjii ? o 58 *-0l 2 s *2 a-W 11 y n NOUVMXN29MO; T 1M t { i j i I / i ii i i DS\N 332263 STLCOPCB4078839 Figure tained for a demonstrates pesticides ai It Is li found pestici Interfere If with Jensen' i Separatior With thi the FCB's ('/ front on the our cleanup p usually invol the FCB's cou knowing that specific cond Four pre feasibility o of standard F x 2.5 cm. O.D height) Flori until ready f Na2_SO4.. Elut. percentage ret } I DSVV 332264 STLCOPCB4078840 Figure 2 shows the number of peaks and the separation ob tained for a sample of PCB's ('Aroclor' 1254) while Figure 3 demonstrates the degree of interference encountered when the pesticides are mixed with the PCB's. It is interesting to note that the peaks of the commonly found pestioidts all havo a corresponding FCB peak that would interfere if present in the same extract. This is in agreement with Jensen's work. Separation of PCB's from Pesticides by the Use of Florlsll With thin layer chromatography (TLC) it was observed that the PCB's ('Aroclor* 1254) tended to run towards the solvent front on the TL plates. Bearing this in mind and the fact that our cleanup procedures for pesticide residues in animal tissues usually involve a final Florlsll step, we experimented to see if the PCB's could be eluted from the Florisil column with n-hexane knowing that most of the pesticides are not eluted under these specific conditions. Four preliminary experiments were carried out to test the feasibility of this separation on Florisil. In Expt. I, 5 ml. of standard PCB preparation was added to the glass column (30 cm x 2.5 cm. O.D.) packed with 40 ml. (ca. 19 gm. or 10 cm. in height) Florisil (60-100 mesh, Floridin Co., stored at 130*C. until ready for use) and topped with an 1/2" layer of anhydrous Na^SO^. Elution was carried out with 100 ml. n-hexane, and the percentage recoveries were determined. This experiment was 136 T 13$ i i i i STLCOPCB4078841 repeated but the elution was effected with 200 al. hexane (Expt. II). The sane experiments were carried out with the standard pes ticide mixture eluting with 100 (Expt. Ill) and 200 al. (Expt. IV) hexane respectively. TABLE I Percent reoovery of KB's and Pesticides from Florisil columns by elution with hexane (*) FCB peak Expt. I Expt. II FesticideiDJExpt. Ill Expt. IV no. (GLC) 100 ml hex 200 ml hex peak 100 ml hec 200 ml hex 1 80.1 92.2 Lindane None None 2 86.7 103.1 Heptachlor None 92.7 3 65.6 100.0 Aldrin 62.8 94.1 4 98.2 101.0 Hept. epox. None None 5 42.1 100.0 DDE 20.5 97.5 6 44.9 98.7 Dieldrin None None 7 64.0 101.2 DDD None None 8 96.8 105.2 p,p'-DDT None None 9 60.4 105.8 10 72.6 103.8 11 76.9 99.9 12 57.2 100.0 13 100.0 100.0 14 71.4 100.0 a. Recoveries are based on peak height comparisons and each value represents the average of duplicate determinations. b. Under the experimental conditions, 250 ml. of 202 ethyl ether in hexane is used normally to elute the pesticides although 200 al. can quantitatively remove them. , . The experimental results which are shown in Table I indicate that separation on a Florisil column is feasible. Almost quanti- tative removal of the PCB's is effected'with 200 al. hexane, while under the saae conditions only three of the 8 pesticides tried ' showed evidence of elution (heptachlor 92.72, aldrin 94.12, and DDE 97.52). Itis interesting to note that these three pesti- 1ST j j ; I [ j i cides showing sthe PCB*sf quit Two furthe aration was sti (Expt. V) and w m*l tissue (Exp hexane, the rec carried out wit the pesticides. The result and confirm our aldrin, and hep pesticides can The fact t can be used to . of DDT by dehyd: of DDT in the penhanced if DDE drochlorination originally pres amounts of DDE, The result of Jensen indieresidue analyst w DSW 332266 STLCOPCB4078842 me (Expt. landard pes, (Expt. IV) | S i l Expt. IV 200 ml hex None 92.7 94.1 None 97.5 None None None I I iI id each lations. ethyl icides I indicate 'St quantixane, while ;a tried .12, and : pesti ' ii i i . t ; 4 cides showing some elution from Florisil with hexane, are, like the PCB's, quite mobile under our TLC conditions. Two further experiments were carried out to see if the sep aration was still effective when PCB's and pesticides were mixed (Expt. V) and when they were present in the extract from an ani mal tissue (Expt. VI). The first elution was made with 200 ml. hexane, the receiver was changad, and the second elution was carried out with 250 ml. of 202 ethyl ether in hexane to remove the pesticides. The results of the two experiments are shown in Table II, and confirm our earlier finding that with the exception of DDE, aldrin, and heptachlor, a clear-cut separation of the PCB's and pesticides can be made by the use of a Florisil column. The fact that DDE is eluted with the PCB's by pure hexane can be used to advantage in the confirmation and quantification of DDT by dehydrochlorination. The estimation of small amounts of DDT in the presence of interference (for exanple, a PCB) is enhanced if DDE is previously removed. The DDE produced by dehy drochlorination can then be used to estimate the amount of DDT originally present. In the presence of comparatively large amounts of DDE, this approach is not very dependable. Discussion The results of the above experiments coupled with the work of Jensen indicate that there are serious problems confronting residue analysts. However, as far as the writer is aware, there ua i 1 i i I Ii i ! 1 \ f t \ i ii DS* STLCOPCB4078843 TABLE II Percent Recoveries of PCB's and Pesticides from a Mixture ' after Separation on Florisil (Expts. V & VI)* Eluted with 200 ml. hexane With 250 ml. 20Z ether in hexane PCB aiid/or z ,* Pesticide pest, peak Recov.*' Recov.^ peak dRecov. () Recov/b) Heptachlor PCB1 PCB2 + Ald.C PCB3 PCB4 PCB5 DDE0 PCB6 92.7 104.0 102.1 100.0 104.2 97.8 101.3 PCB7 PCB 8 PCB9 PCB10 PCB11 PCB12 PCB13 PCB14 . 97.8 100.0 91.6 104.7 100.0 100.0 100.0 96.2 98,1 101.7 96.6 106.0 102.6 102.4 100.0 105.1 100.0 97.0 104.3 105.5 100.0 100.0 100.0 Lindane 93.6 Heptaohlor None Aldrin 1.3 Hept. epox . 96.4 DDE None Dieldrin 100.0 DDD 102.3 DDT 99.8 98.5 None 4.0 102.2 None 100.0 98.9 * 92.5 * The peaks are arranged in order of their emergence (increas ing retention time) from the GLC column, and where a PCB and a pesticide peak appear in the same line (horizontally) they have similar retention times. a. A standard mixture of PCB's and pesticides in pure hexane was placed on the Florisil column; first elution was made with 200 ml. hexane, receiver was changed, and the column eluted with 250 ml. 20Z ethyl ether in hexane. b. Same as in (a) except that the PCB's and pesticides were first mixed with an extract from an animal tissue which was known to be essentially free of pesticides. c. Since a single peak was obtained, the recovery was calcu lated by a comparison of the peak height against that in the combined standard mixture of PCB and pesticide. In all other cases the peak height was compared to that in the standard Injected separately. has t) wildl leave is ac all o lites phenoi to be 1 conder 1 sibili 1 only v 2 first might DDT us PCB's confin remain if PCB H are PC) obtaint DS\N 332268 STLCOPCB4078844 m a Mixture . VI)* ;ence (increasrhere a PCB horizontally) pure hexane on was made l the colussi cides were sue which * was calcuist that in cide. In :o that in has been no positive confirmation of the presence of PCB's in wildlife tissues by techniques other than chromatography. This leaves doubts that the presence of the unidentified peaks (UIP's) is actually due to PCB's. There is the possibility that some or all of the peaks are due to condensation products of the metabo lites of pesticides like DDT. For example, 4,4'-dichlorobenzo- phenone (DCB) Cl^ ^C1 ls known to be a metabolite of the DDT group. The presence of the keto group makes it quite feasible for condensation to take place. There are at least two points that lend support to this pos sibility. 1. The UIP's (being called PCB's) are usually observed only when large amounts of the DDT group are present. 2. Jensen checked eagle feathers collected since 1880 and first detected PCB (not confirmed) in an eagle from 1944. It might be a coincidence, but this is approximately the time that < DDT use came into prominence. It should be noted also that the PCB's were in wide use as early as 1930 (2). Thus until positive ; confirmation (e.g. with mass spectra) is obtained, there will remain some doubt that these UIP's are due to PCB's - especially t , ' if PCB's are not used in pesticide^fqrmulations. It is certainly true, however, that whether or not the UIP's are PCB's thgir presence leads to difficulties. The results obtained for some samples of fat recently analyzed in our labora- 140 DSW 332269 STLCOPCB4078845 -Cory are typical of the problem. The sample containing the highest levels of residues contained the following pesticides la p.p.m.t DDE - 1.42, dieldrin - 2.13, DDD - 5.61, and p,p'-DDT 2.60. I Even prior to subjection to TLC confirmation, the DDD value appeared unusually high when it is considered that its presence la tissues is usually accounted for by three main routesi j a) It is used as a pesticide, butnot extensively. , b) It is one of the metabolites of DDT - however, the DDT--ME j pathway is much more prevalent than DDT ~*DDD, with the latter ], usually occurring in the liver, hence the fat tissue is an unlike ly location for large amounts of DDD. c) It is a frequent contaminant of technical DDT used in spray programs. ^ When confirmation of the pesticides was attempted, the JLC plates showed no DDD, although the apparent amount present should have given a distinct spot on the plate. However, a spot was observed running near the solvent front, a considerable dis tance from DDD. When this spot was scraped off the TLC plate, eluted, and reinjected into the gas chromatograph, a peak having retention time identical to DDD was observed. Although this in terfering material has not been identified - it could be a PCI since its retention time coincides with one of the PCB's - it is obvious how easily one could report false results, especially if use is made of the GLC-EC results without further confirmation. 141 i TLC t are times, pesticides with this The d tionary ph has pointe ter of ide pesticides related. Bearii ing infrart confirmatic sis and rel pesticides retention t With o N - Modific tion of the we have obs> one pesticit plus some ot With TLC as whole as be! depending or DSW 332270 STLCOPCB4078846 aining the pesticides in nd p,p'-DDT - the DDD value its presence in test Y, the DDT--*DDE h the latter ue is an unlike* used in spray pted, the Jl present awever, a spot siderable dise TLC plate, a peak having hough this inuld be a PCB PCB's - it i* especially if confirmation. ! i TLC continues to be our main confirmatory method, but there are times, especially with smaller (but significant) amounts of pesticides, when it is impossible to make a positive confirmation with this technique alone. The determination of GLC retention times on two or more sta tionary phases is quite useful in some cases, but as Robinson (11) has pointed out, it cannot be regarded as an independent parame ter of identity since it stay be shown that various organochlorine pesticides on different stationary phases are significantly cor related. Bearing in mind these problems and the difficulty of apply ing infrared, mass spectra, and other spectroscopic methods for confirmation of small amounts of pesticide residues, more empha sis and reliance should be given to chemical modification of the pesticides and reinjection into the gas chromatograph, using the retention times of the products as means of confirmation. With our SMI technique (S = Separation of PCB's on Florisil, M Modification of the pesticide by chemical means, I =* Injec tion of the extract containing the product into the GLC apparatus we have observed some cases where a single GLC peak indicating one pesticide was in fact a mixture consisting of the pesticide plus some other PCB-type unknown having the same retention time. With TLC as the sole confirmatory method, one could quantify the whole as being due to the pesticide and be out by many factors depending on the ratios of the two compounds giving rise to the 3^ STLCOPCB4078847 ) single GLC peak I I References 1. H. SCHMIDT and G. SCHULTZ, Ann. 207, 338 (1881) 2. C.H. PENNING, Ind. Eng. Chem. 22, 1180-2 (1930) . 3. J.W. JONES and H.S. ALDEN, Arch. Dermat. Syphilol. 33, 1022 1034 (1936) 4. L. GREENBURG, M.R. MAYERS and A.R. SMITH, J. Ind. Hyg. Toxic. / 21, 29-38 (1939) , 5. S. JENSEN, New Scientist, p. 612 (15 December 1966) 6. S. JENSEN, Private communication (1967) j Dieldrin 7. 'The Aroclor Compounds', Monsanto Chemical Company Bulletin, p. 17 (1965) , 8. N.S. SHECHTER, S.B. SOLOWAY, R.A. HAYES, and H.L. HALLER, Ind. Eng. Chem., Anal. Ed., 17, 704 (1945) ` 9. F. ERRO, A. BEVENUE and H. BECKMAN, Bull. Environ. Contarn. | I and Tox. 2, 372 (1967) t The pre I 10. K.A. McCULLY and W.P. McKINLEY, JAOAC 47, 652 (1964) ; carbon lnsec 11. J. ROBINSON, Chemistry and Industry, p. 1974 (25 November 1967) in controll! menta, howev \ effects of c insecticides I There a system and t chronic expo the most not changes beln respectively even at low I tissues has '"Th Billotia ( Eavirou VoL 4, N*. J, 1969, f DSW 332272 STLCOPCB4078848