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tce Pott I # (Re/>rin/rrf (rout Naiur*, Vol. 216, No. 5112, pp. 227-229, Ocwber 21, 1967) Chlorinated Hydrocarbons in British Wildlife Polychlorobiphenyl compounds have been detected In British wildlife. In birds' livers and eggs they are often in greater quantities than organochlorine pesticide residues. Polychioroblphenyts are known to be toxic, and their detection In wildlife raises the question of the adverse effect they may have. D. C. HOLMES, J. H. SIMMONS and J. O'G. TATTON Laboratory of the Government Chemist, London When wild birds' oggs or the liven of British wildlife are examined by gas-liquid chromatography (GLC) using oloctron-capture detection, poaks corresponding to such organoohloiino pesticides as BHC, dieldvin and DDT, and thoir metabolites and breakdown products, often appear on the resultant chromatograms. Those peaks sometimes represent amounts which are significant from n toxicolo gical point of viow, but the sensitivity of modem instru ments also enables completely insignificant amounts to be detected. Analysts eoxxeemed with the determination of organochlorine pesticides in wildlife, however, have long boon aware that it Is not unusual for an additional series of as many as ton peaks, corresponding to unidentified compounds, also to appear on these chromatograms1-4. Little is known of the structure and origin of these com pounds, but Robum* established that they are orgnnoohlorine in nature, with a substantial chlorine content, and consequently possess oloctron-capturing properties. On silioone or `Apiozon' GLC oolumns, the series of peaks for these compounds normally begins at the point at whioh pp'-TDE and pp'-DDT are emerging from the oolumns and extends, in terms of retention times, to four or five times that of pp'-DDT, which is the last of the commonly occurring pesticides to omergo. *0^ ? Before examination by GLC, samples liavo normally boon subjeoted to a fairly rigorous cloan-up procedure, suoh as that of de Faubert Maunder etal,1, which includes a dimethylformamido-hexane partition and passage through a column of prepared alumina. Because these unknown compounds are similar in nature and properties to tho organochiorine pesticides, they also pass through this and similar clean-up procedures; consequently, they orton interfere seriously with determinations of pp'-TDE and pp'-DDT in wildlife because of overlapping of the chromatographio peaks. Suitable procedures have been devised to overcome this difficulty of interfering peaks. Ono method is to offset a better resolution of those particular peaks by choice of a suitable stationary phase for GLC*. Another method, which also demonstrates the presence of these compounds in the sample, is the use of a preliminary thin-layer chromatographic treatment*. The clcaued-up extracts are examined on silica gel chromato plates using a 1 per cent solution of acetone in hexane as mobile phase*. Spots corresponding to the compounds can then be found at ftp values between 0*8 and 1*0, whoreas the commonly occurring organochiorine pesticides and thoir metabolites produce spots of lower itjp values. A similar separation can also be shown with reverse phase paper chromatography* whereby the unknown compounds move only a very short distance from the base line com pared with the pesticides and their metabolites. Gas-liquid chromatography using dual channel detec tion systems with separate elootron-capture and flameionization detectors has also, been used to demonstrate tho presence of large amounts of weakly electron-capturing material, in addition to known postioides residue, in the eggs of oyster catchers (Haemaloptu OBtralegus)9. , Experience in this laboratory has shown that these compounds ooour most frequently and in the largest pro portions in the livors, fat and eggs of birds, particularly terrestrial predators such as sporrowhawks (AccijriUr nisus) and kostrels (Falco tinnunculus) and marine feeders such as guillemots (Vria aalg) and kittiwakes (iiwsa tridactyla); they also commonly occur in freshwater Ash. We have detected spall amounts in human and animal fat samples, but less frequently and in much smallor proportions than in avian samples. A similar series of chromatographio peaks has been observed by Holden1*:11 when examining extraots of seals and Ash taken in Scottish waters. He found that the peaks given by the extraots of fresh and sea-water fish represen ted fairly low concentrations, but those given by extraots of seal blubber corresponded to much larger proportions, of the order found by this laboratory in birds' eggs. Despite considerable ouriosity about the identity of these compounds, little progress has been made in identifying them, but there has long been a general supposition that 2 MONS 083031 BctealloD time (mla) Pig. 1. Qaa-Uquld chromatogram of (a) extract of ktatrel liver Md <W a commercial potychtorobipheayt reals. they were either further breakdown or condensation product* of the organochlorine pesticides or possibly loose compounds of those with natural products such as protein matter. Jensen, however, succeeded in identifying a aeries of Muoh peaks os corresponding to polychlorobiphonyl com pounds. in 200 pike taken in different parts of Swcdon. and in an eagle11. We havo now been ablo to show that 3 HONS 0d3032 2 TftbU 1. RSbATIVR RETENTION TIJISS ON THXKIS DIW5RKNT OLO COLUMNS (Excluding timet for known iwstlciilci) Silloouo OK-S& 62 column KL1 PCD (Dieldrin-100) 'Aplczon V column KLE PCJI Cyanosilicone Ofi-XK 00 column KXK, I'tll 142 1-73 101 2-81 3 00 3-71 4-0S sot 710 1 42 1*73 100 2-81 3 &d 3-70 4-08 set 7-11 2-31 2-30 2-67 2 07 3-04 3-05 6-00 600 5 08 - 605 0-67 t 0-50 1-21 1 34 2-20 2 92 2-07 3-00 4-03 6 11 121 1-33 2-10 200 290 3 80 4-03 6 11 KLE. Kestrel liver extract; PCB, polychloroblphenyl reslo. the tong retention timo compounds occurring in British wildlife ere also polyohlorobiphenyl compounds. Polychlorinated biphenyl has been in common commer cial use for somo timo as a plasticizer in paints, rosins and plastics. It also has olectrical insulating properties and can be used for a number of other protootive purposes. Commercial preparations of this material are usually graded according to the degree of chlorination, but all of them are substantially complex mixtures of polychlorobiphonyl compounds that oould be expootcd to resomblo the organoohlorine pesticides in their chemical and physical properties. In the preeoribod extraction and GLC condi tions, these mixtures produce a series of chromatographic poaks of the type in question. The compounds occurring in British wildlife correspond to tho moro highly chlorinated biphenyl compounds rather than tho more lightly chlorinated types. These highly chlorinated biphenyls also ocour to some extent in the analogous polyohloroterphenyl preparations which have commercial uses similar to those of polyohlorobiphenyl. It is possiklo that some of tho compounds with vory long retention times ooourring in British-wildlife are lightly ohlorinatod polyohloroterphenyl compounds. Fig. la shows the gas-liquid chromatogram of an extract from a kestrel liver. The peaks corresponding to pp'-DDE (0*43 ng), beta-BHC (0*33 ng) and gamma-BHC (0*03 ng) are labelled. The other peaks form the series referred to above*and until now would have been regarded simply as "unknown organoohlorine compounds not corresponding to any known posticide or its metabolites'*. Fig. \b shows the analogous chromatogram of a commer cial polyohlorobiphcDyl resin. So far as retention times are conoemed and apart from the pesticide peaks, there is exaot matching for all but a few peaks in the two chromatograms. The chromatograms shown were obtained by use of a silicone gum {QBSB 52) column, but this precise corre spondence between tho retention times of tho peaks for the unknown compounds and those from the polychiorobiphonyl rosin has also boon demonstrated on `Apiozon L' .4 M0NS 083033 and oyunosilicouo columns. Tho actual retention times recorded for tho poaks on those three typos of column are givon in Table 1. Further confirmation as to the identity of those com pounds has been obtained by a study of thoir behaviour on thin-lnyor chromatoplatos, alumina and silica gol absorption columns and reverse phase paper chromato grams. In addition, those compounds have boon shown to rosomblo the polyohlorobiphenyl compounds in thoir ehemioal inortness. Thus they are not easily modified by simple ohemioal reactions to produce readily identifiable shifts in thoir retontion times on OLC as can bo done, for instanco, to pp'-DDT by hydrolysing it to pp'-DDE or to aldrin by oxidizing it to dieldrin. A number of tivors and eggs taken from birds in the British Isles or thoir ooastal waters have been examined in this laboratory with results similar to those obtained for tho kostrel liver referred to earlier. While the identifica tion of theso compounds in British wildlife may solve the mystery of theso peaks on OLC chromatograms, it raises, hosvovor, many other questions. Why, for example, do those compounds seem to be accumulated, most frequently and in tho largest proportions, in certain types of wild life such ns birds, seals and fish, and only at the most in vory small proportions in man or his domestic animals such os oows or shoop Y In this respeot they seem to differ markedly from the organoehlorine pestieidoe which soem to havo invaded nearly all aspeots of our environment and to whioli thoy bear a strong resemblance chemically and physically. Some of tho samples of birds examined in this laboratory havo soomod to oontain higher proportions of polyohloro biphenyl compounds than organoehlorine pesticides. For example, the peaks on the ohromAtogram for the kestrel livor shown in Fig. 1 were estimated to be equivalent to about 12 ng of polyohlorobiphenyl compounds. This must be contrasted against the organoohlorine pestieide residues listed above. These total only about 0*8 ng of which 0*33 ng is beta-BHC, an isomer of BHC which is generally regarded as non-toxic to wildlife. Great concern has often beon expressed by conservation ists about the effects of posticides on wildlife, particularly birds. The identification of these other organoehlorine compounds in wildlife prompts enquiries as to the possible toxio offeots they may be having. That these compounds oro toxio is generally agreed1*-14; the polychlorobiphenyls are in fact regarded aa an industrial hazard and threshold limits for them in air have boon advised1*. Wo thank the Nature ConsorvAnoy for samples of wildlife and Monsanto Chemicale Ltd. for samples of polyohlorobiphenyl rosins. IVerclvcd October 3,1W7. 5 MOMS 083034 * ftobum, J.. Analyrt, 10, 467 (1066). ' Harrison, R. B.. J. Set. Food Afrie., 17.10 (1066). * Report oftAo Oootrnmtni Chemist, 79 (HM Statlonirr Office, London, 1061). * Walker. C. II.. Hamilton, O. A.. And Harrison. B. B.. J. Set. Food Afrie., 13. IJ3 (1007). * do Paubert Maunder, M. J., Egon, H., Oodljr, B. W., Hammond, 8. \f,, Boburn. J., and Thomson. J., Analyrt, SO, 166 (1064). * Simmons, J. II., and Tattoo, J. O'O., J. Chromatoy,, 97. 299 (1067). ' Abbott, O. C.. Rgan. H.t and Thomson, J., J. Chromatoy., 16, 461 (1064). * Evans. H.. Anoint, 97,960 (I960). ' Report of the QooonmoM Chomitt, 100 (HM Stationery OAn, London, 1066), ' Holden, A. 7., and Marsdsn, K., J. Pros. In*. Sow. Pori/., 796 (1966). " Ifoldon, A. V., J. Appt. BeoL, $, suppl.. 46 (1966). 'Jensen, 8., N*$ Set., 30, 618 (I960). Baa, H. Irving. Dmnyorooo Proprritoe of Industrial AtoUrtoU, 507 (Rain- hold Publishing Corporation. New York, 1069). 14 Brown, &. M., Chomiet-Anoint, 96, IS (1047). ** DoenmonkUton of Threshold Limit Valmt, 41 (Commute* on Threshold Limit Value*. 1966). Printed In Great Britain by Fleher, Knltbt A Co- Ltd.. St. Albans. MQNS 083035