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I MO ft U l 20 November 1971 Chrmittry and /ndinrr The polychlorobiphenyls, their occurrence and significance: a review by R. Edwards H. J. Heinz Co. Ltd, Hayes Park, Haycst Middlesex Pollution of the global environment by man is rightly receiving considerable attention because of the increasing realisation of possible long term effects on both the physical environment and living organisms. Clearly it is necessary to distinguish between short term toxic effects and longterm ones (/.*. reproductive and carcinogenic effects), these latter being more difficult to assess. The chlorinated hydrocarbons used as biocides are well documented in their short term toxic effects, and with knowledge of their facile accumulation in the environment due to their considerable chemical stability, more work is being done on the long term effects coupled with tighter control of their use. With the latter the main points of entry into the terrestrial environment are through crop spraying and seed dressings with conse quent accumulation in the soil. From this point they can also reach tho aerial environment secondarily by evaporation or 'co-distillation* And carriage in fine particulate matter. They are also systemic in plants to varying degrees. Aqueous run-off carries them through the drainage system together with industrial effluent sources to reach the marine environ ment above the continental shelves. It is thus possible for these compounds to reach the lower members of the many foodchains. Man'sdomeslicandindustrial use of biocides for hygienic purposes leads to entry at various other points in tho food chains. The very large concentration factor (10*') possible in these food chains is well known and in the final members (e.g. marine mammals, predatory birds, man) the concentrations can reach alarming proportions. Lipid solubility means that a large percentage of these residues will be stored in the fat depots of the body. The polychlorobiphenyls (PCUs), with a very widely dispersed tisc throughout industry, have very similar pro perties to the chlorinated hydrocarbon pesticides, but are even more thermally and chemically stable. An important difference, however, lies in the routes through which they contaminate the environment.*Emphasis here is on indust rial effluents reaching the marine medium, and possibly the burning of industrial waste contributes relatively more to tho aerial distribution. Although recommended for use in conjunction with pesticides, there is no documented example of this known to the author, so that distribution through crop spraying should not be a contributory factor. This fits Ihe known pattern of distribution of PCBs in the environ ment, the highest concentrations coining from the heads of fresh water food chains, marine organisms and sea birds. High levels arc also known in terrestrial avian predators, but only recently has it been shown1 that in one region they can be found at levels comparable to those for organochlorinc pesticides. While relatively little toxicological work has been done on PCBs and some of the results are superficially con Dieting, this can be explained and there can be no doubt tha they have very similar effects on mammalian and avia? ' metabolic processes to those of chlorinated pesticides, am indeed that synergistic effects can occur between the tw< classes.2 Jf any control over the use of PCBs is ultimate!? attempted this will be much more difficult than with pesri cidcs because of the very large number of uses to w'hich the? arc pul. * There exists considerable difficulty in the analytical ftek because these compounds cannot be separated easily fron pesticide residues, and this has probably resulted in quite ; few reports in the past giving falsely high results for son* pesticide residues.* Unknown peaks in gas chromatograph? analyses were early recognised4-* but it was not until Jen sen7 applied mass spectrometry to the problem that son* of them were identified as PCBs. Properties and uses The PCUs received early notice back in 1881 and were it widespread industrial use by 1930.9 The biphenyls, terph enyls, naphthalenes, and mixtures of these are chlorinate* in varying degrees according to the use to which they ar* put,10 and arc marketed under trade names such as `Arochlor', `Phcnochlor', and `Clophcn*. The products ore comple* mixtures of the various compounds and isomers. They rung* from mobile liquids to crystalline materials or hard resins and while the lower members are readily soluble in non* polar solvents they become progressively less so with incrcas ing extent of chlorination. They are extremely stable com pounds, not hydrolysed by water, acid or nlkuli, and will a thermal stability such that they are used in fire-proofing They find use in elastomers, adhesives, paints, varnishes printing inks, putty and ns general fillers. The cxedlcn dielectric properties lead to widespread use for clcctri* insulators and ns coolant insulators in transformers. Thi stability and their low vapour pressures make them ideal a lubricants, hydraulic fluids, liquid seals, cutting oils am vacuum diffusion pump oils. Employment in packaging materials has led to the contamination of food.'* Thor* are a host of other uses, but finally it may be mentioned tha: both the Monsanto Company and the USDH have report:* that they net ns cflcciivc vapour suppressants in insccticidformulations and thus prolong the active life of an applicn tion. However, it has not been acknowledged that lCH have actually been used for this purpose commercial^ As an ancillary fact, a patent for the production of l`CH from lindane residues has been granted. This intiltiplicn; of usage has repercussions in the analytical area beenu* MONS GU65a1 SL s - JO tiovrmhcr IVl C'himlitry and In.linlry 1)41 miny solvents, tome adsorbents and other analytical materials, and even the atmosphere of a laboratory can be contaminated by PCBs. Occurfence in Ihc environment It it now well established that PCDs can be found in wild life In mony areas of the environment.1**-- -* The highest levels are generally associated with the industrial areas and the adjacent freshwater and marine food chains. The distribution is inevitably not quite so simple as this because of the migratory habits of some of the species involved*9*-4 and the fact of widespread aerial distribution.-* Cohen and Pinkerton37 reviewed the hitler in respect of pesticides, mainly over the North American continent, and found higher levels in dust than rainfall. Risebrough et a/.25 Studied the carriage of pesticides in the trade winds coming from Europe and Africa by collecting dust from the air over the Barbados Islands, and detected from 1-64 p.p.b. orgnnochlorine residues. Dust collected over the Californian ooast contained some thousand times greater concentration oforganochlorincs, but none could be found in dust collected from over the Central Pacific, although traces were inferred from the nature of the particulate matter. While no PCBs were detected in any of these samples, it was concluded that they were probably present jn trace amounts. Tarrant and Tatlon,*1 however, who found organochlorine pesticides in rainwater samples taken from all over the British Isles showed that levels were highest in the summer months (q/*. Risebrough et atP) and in populous areas, also found PCBs in all of them although they did not estimate the concentrations. The importance of aerial distribution of pesticides relative to the hydrospheric distribution has been stressed by Risebrough et a/., and it is inferred that PCBs have a similar pattern of distribution. However, it would seem that with PCBs more emphasis should be placed on the hydrospheric medium, because an important part in the transfer to the atmosphere, via dusts used as carriers in agricultural applications, can be considered as low in this case. If the vapour pressures of DDT and PCBs are taken as being of the same order, it would then be expected Chat atmospheric levels of PCBs would be low relative to DDT. Tho relative concentration of pesticides and PCDs in the hydrosphere and atmosphere have yet to be convincingly demonstrated. Tho outstanding feature of (he distribution is the occur rence in marine organisms on the continental shelves round North West Europe and North America, and also the extraordinarily high levels found in some freshwater avian predators, all associated with effluent from industrial aclivity.*J,,4,*^l7*20,2*,:56 Along the west coast of the USA PCBs have been detected in estuarine and coastal waters in San Francisco Bay. San Diego Bay and Puget Sound (Washington). In areas remote from industrial activity round the Bay of California and parts of the Panama coast for example, birds* eggs contained 20-30ug PCB each, but near Ihc above mentioned industrial locations the eyes con * tained up to several hundred and sometimes over a thousand pg PCB,'egg. The east coast of ihc USA has yielded similar evidence for contamination by PCBs. Holden and Marsden1* delected PCDs in seals from the Gulf of St Lawrence in shat- low waters (Magdalen Islands) and in the deeper waters of the Cabot Straits. They have also been qualitatively detected in fish and shellfish from Texas and Georgia, and in ihc south, in the Gulf of Mexico, in estuarine waters at Charle ston, S. Carolina, levels briess than 0-1 p.p.m. were found in fish and blue crabs.31* Duke et ai. also monitored the results of an industrial leakage into the Escambia River which was carried through several inner bays through to the Gulf of Mexico off Florida. Maximum levels at source ranged from 486 p.p.m. in sediment to 20 p.p.m. in fish, Crustacea and sediment in (he outer bays. The best indicators ofcontamin ant levels were static organisms like oysters (up to 3 p.p.m,). * Round the coasts of North West Europe the situation is similarly documented. Jensen et el.*7 investigated levels in marine ecosystems in Swedish waters. The closed nature of the Baltic area perhaps leads to somewhat higher con centrations than those generally found on Atlantic and American coasts. Levels tended lo be lower in waters o/T West Sweden and to ihe north of Ihc Baltic proper, in the Gulf of Bothnia, and higher going south from the Stockholm Archipelago to the South Baltic. I hc levels ranged from 0*002 lo 1*0 p.p.m. in mussels and fish up to 150-2*10 p.p.m. in white tailed eagles. The concentrations were equivalent to the total DDT levels found. Kocman et a/.*6 qualitatively identified PCBs along the Netherlands coast from the Wndden Sea southwards to, and up. the Rhine, in mussels, fish and birds and their eggs. The uniformity of GC patterns suggested (hat they all originated from pollution of the Rhine. Holmes, Simmons and Tntlon*3 reported the pre sence of PCBs in 'marine birds and their eggs collected from round the British Isles, and Holden and Mnrsden'1 showed them to be present in seals and porpoises round the coasts ofScotland, being highest in (hose from the cast coast (see below). Bonner,21 investigating the deaths of seal pups on the Cornish coast, found PCBs here (160 p.p.m. in blubber) higher than in comparable northern samples (34-48 p.p.m.), but less than in East Anglian specimens (350-520 p.p.m.). Presst, Jefferies and Moore, surveying PCB levels in British wildlife, reported an average of 4 (1-25) p.p.m. in (lie eggs of marine birds, and comparing the east and west coasts of Scotland in respect of two sets of twenty five eggs somewhat higher levels (7 as against 3*6 p.p.m.) in the western samples. No PCBs were detected in whales analysed by Wolmart and Wilson,39 and this is compatible with a distribution pattern confined to the continental shelves. However, this may be a rather naive use of the evidence because the .water solubility of PCBs. especially the higher chlorinated ones, is extremely low and the bulk of the PCB is more likely to be associated with sediment on the bottom and entering inlo the food chains mainly via bottom living organisms. Most whales feed on plankton, and thus might in any case be expected to showlow levels. A further point of perspective is also given by the fact that loss of lower PCBs from aqueous suspen sions when aerated has been observed.49 Holden3 has (raced aqueous pollution back to one type of source. The work on seals in Scotland having shown that 'levels were highest in the Finh of Forth region, water samples from the surface and at depth from ihe estuary and river up to Glasgow' showed no detectable amount* V............. ............. MQNS 066562 ( 1)42 20 November 1971 Chemistry ami huhiury And only trace quantities in trunk sewer effluents. The zooplankton contained less than 0 03 p.p.m., and fish up to 2*6 p.p.m. However, industrial diluents are usually pasted to the sewage works together with domestic sewage, and so the sludge, which is dumped offshore, was examined. The fifteen samples analysed ranged from 1-185 p.p.m. PCD on dry weight, with two sewage plants giving con sistently high levels(50-160 p.p.m.). Sludge from Manchester exhibited somewhat lower levels, and that from London lest again, but taking into account the varying outputs, each area was estimated to contribute roughly I ton/ year PCD contamination to the marine environment from sewoge sludge. In the case of inland areas sludge can be disposed of by spreading over the land surface, and so oiler a route to terrestrial food chains. The question of why it is that relatively low levels of PCB are found in man and his domesticated animals, posed in 1967* perhaps now has some answer. Distribution in terrestrial and freshwater ecosystems has largely been assayed in terms of the avifauna because of the effects of organochlorine pesticides and almost certainly PCBs on some bird populations. The DDT.PCB ratio has been used by Risebrough ct al. as an environmental index,i4'24.23 but when data is compared consideration must be made of the various biological factors, such as tissue distribution, species metabolism, migration, diet, health and age, which all afTect residue concentrations. These authors have shown that the DDT,PCB ratio is low, below 2, near industrial areas, and increases with the distance away to become greater than 10 in remote regions. The geographical distribution in terrestrial and. freshwater wildlife will be reviewed within the framework of these factors. Risebrough ft al.'* examined peregrine falcons and their prey, centring round the Bay of California. Falcons freshly migrated from the Arctic had low levels which increased with residence time in California; the levels in the Californian birds increased with age; and the indigenous adults showed the highest levels. The maximum Table I Tissue distribution of DDT and PCB In peregrines and (heir prey Total DDT BCP DDT/PCB p.p.m. p.p.m. ratio Peregrines: unhitched egg mueclo liver body fat Pwy: *s* whole body muscle 102 too 77 50 151 39 * 26-4 10-2 9% SI 32 45 98 0-098 100 10 1*3 15-6 33 5-5 270 values in Tabic I can be used to show the differences in tissue distribution. The same authors-* determined chlorinated hydrocarbon residues in breeding pelicans and cormorants in lakes to the north-west of the Great Lakes (Tabic I!). As the birds used the same food sources the clear differences in results demonstrated a species difference probably due to variation in residue levels in the migration areas, but possibly meta bolic differences in terms of rates of excretion of organochloriivcs into the eggs may be involved. Birds have also Tabic ll Chlorinated hydrocarbon residues in pelicans and cormorants TP'DDE PCB DDCPCB . Egg* p.p.m. p.p.m. ratio Cormorant Pelican Fish (whole) ' 104 1-7 008 8 06 05 I-3 29 0 16 been studied in Britain12,1* and the extensive survey (599 samples) by Prcsst, Jefferies and Moore19 illustrates some of the factors involved; firstly habitat and migration (Table 111). Secondly; the eggs from marine birds from two colonies widely separated from one another demonstrates very convincingly (Table IV) specific differences related to migration habits. The guillemot spends the longest lime in TiMe IV Specific differences related to nii^/atbu habits DDliPCB ratio St Abbs Head Guillemot Razorbill Shag Kittiwakc 0 213 0-500 0-625 0-666 Calloway 0250 0 455 0 727 0-875 coastal waters, the Kittiwakc the shortest time there, for breeding purposes only, and the Shag is essentially seden tary. The effect of health on relative and absolute levels is seen In the report by Bonner21 on seal pup deaths. (Table V). Table V Effect of chlorinated hydrocarbon levels an seal pup health Total DDT PCB DDT(PCB - p.p.m. p.p.m. ratio Liver: Cornish 2-4 46 Northern (well fed) 0-6 4 Northern (starving) 2-0 6 Blubber: Cornish 111 160 Northern (well fed; 9-8 34 Northern (starving) 18 1 48 005 0-15 0 33 007 029 038 Table HI Habit and mlgiationfactors affecting chlorinated hydrocarbon residues Liter Bird elans No samples (a) Terrestrial predators: suite partial migrants migrants (b) Freshwater (mostly static and mostly herons) (c) Marine 75 49 9 57 4 Mean p.p.m. 4-5 4*3 10 343 2-5 Egg No samples 115 6 5 124 96 Mean p.p.m. 17 2-0 10 4-7 40 MQNS 086583 tO November 1971 Clwtnhtry and Industry One or the most striking features of freshwater species is the extraordinarily high levels in herons at the head of a food chain where up to 900 p.p.m. have been found19 in British material. As the. British heron is sedentary, occurs nearly alt over the country, and there are few migrants from the continent, this species might be expected to be good geographical index of PCI) distribution. Unfortun ately nearly all the samples obtained in the survey of Prcssl el olS9 came from the Midlands and South East to that no clear pattern emerged. However, the age factor it well illustrated by this species ns the average liver levels for nestlings was found to be 5 p.p.m. while that for adults was 96 p.p.m. Diet is of course an important consideration, and means that different species from the same area can exhibit very different PCB concentrations. Thus, while birds feeding on insects have shown 0-1 p.p.m. (liver), raptors feeding on mammals yielded 0-50 p.p.m. It is necessary then, when comparing or interpreting data, to take into consideration all these factors. Terrestrial restriction of PCBs to nenr industrial areas would seem to be reinforced by (lie failure of Holden26 to find them in freshwater fish In Scotland, and by Davis-'1 who failed to find them in beetles, earthworms, slugs and grasshoppers of one agri cultural area in England. To what extent are PCBs found In human beings and foodstuffs? Jensen in 1966 first reported7 the presence of PCBs in human hair, and later reports17*23 indicated that levels in man and his foodstuffs were lower than those found in birds, and below 1 p.p.m. One exceptional case has been recorded,12 however. Recently a very extensive survey of foodstuffs in Sweden sampled between 1967 and 1969, and analysed for organochlorine pesticides and PCB's, has been published.1 In a wide range of foods and food materials, PCB levels were found to be below 0*1 p.p.m. (90 per cent of 1400 samples) and comparable to DDT levels. The higher con centrations came from freshwater and marine fish, especially those with a high fat content. Another case where relatively high levels were found was with milk. Cows excrete little DDT in their milk and here all eleven samples were below 0*03 p.p.m. DDT and 0'1 p.p.m. PCB on whole milk. In human milk relatively more DDT is excreted and the average over 22 samples was 0*107 p.p.m. DDT and 0*16 p.p.m. PCB. Other relatively high samples are eggs and butter. (Table VI) Table VI Human milk Em. wik Butler Total No DDT 22 0*107 20 0*21 12 01 * PCB 016 0*027 003 DDTjPCB (/*.p.m. whole material) ' 0*668 7*78 333 Toxicology Soon after the beginnings of industrial use of the chlorinated naphthalenes and biphenyls early in this century, human exposure was seen to lead to skin eruptions (chloracne) and fn animat inhalation and feeding experiments 0919), it was shown that liver damage occurred with the chlorinated naphthalenes. As a result of three human deaths, investi ng f gations were continued in the 1930s, and it was further demonstrated in work on rabbits that in the naphthalene series the higher chlorinated mixtures were the most toxic. The work was expanded by Bennett ctalV'** who also used mixtures including PCBs, and PCBs alone. A PCB con taining 6S per cent chlorine was used, and later one con taining 55 per cent. The substances were applied to rats by inhalation, subcutaneous injection. And oral means, and it was confirmed that the high chlorinated naphthalenes were more toxic than the lower ones, but that the PCB was more toxic still. Here, feeding at a lower rale (005g rat every other day) produced extensive mortality, and as with the naphthal enes resulted in yellowing and enlargement of the livers. Liver damage caused by these compounds had not disap peared two months after dosing had ceased. This damage caused the animals to be extremely sensitive to damage by other compounds, r.g. carbon tetrachloride (used as a solv ent for these mixtures), and much smaller doses than normal were rapidly fatal. Miller in 1944-'4 experimented with a PCB containing 42 per cent chlorine and worked on guinea pigs, rats and rabbits, using feeding, subcutaneous, skin and corneal applications. He used single doses ranging from 69 to 690mg and courses totalling up to 1380mg (over up to 54d) and found that guinea pigs were the most sensitive, rabbits less so, and rats least sensitive in respect of liver damage. Damage was largely confined to the liver but in rats there was also hypertrophy of the spleen. The occurrence of hyaline bodies in the liver was confined to the rat. Von Oettingen in 1955*$ found that even 0 05g,'d fed to rats caused liver damage. Skin lesions were similar to those found in man. * Man's exposure to these chlorinated hydrocarbons leads initially to dermatitis, then liver damage with resultant jaundice, and Greenburg,36 reviewing the situation in 1939, concluded that there had probably been at least six human deaths from this cause. Bennett ct alV had measured the air concentration at thirty industrial plants and suggested a maximum allowable concentration (MAC) of 0*5mg m3 for tetrachlorinatcd and higher chlorinated naphthalenes, with the observation that these levels had certainly been grossly exceeded over the previous twenty years. This work resulted in increased awareness of the hazards from vapours of these compounds and elementary improvements in industrial conditions has largely eliminated this source of danger (but see Biros22). The Amcricao Conference of Government and Industrial Hygienists in 195337 laid down an MAC for the lower chlorinatedPCBsof I Omg m'.andfor thcIiighcrchlormatcdoncs(`Arochlors'l254-)0*5mg nr.This appears to presume parallelism with the naphthalenes, and these figures are perpetuated in handbooks of industrial toxicology,3* but recent papers2*45 point lo the reverse situ ation (sec below). These MACs are to becomparedwiththose for 'Aldrin* and `Dieldrin' (0*25ingm-\ tentative) and para- thion (0-lmg'm'). The oxidised chlorinated biphenyls are more toxic than the parent compounds.3* McLaughlin in 1963 published the results of toxicological studies on a number of chemicals which might be found in food39 using a sophisticated method based on injection into fertile hens* eggs. 25,000 eggs were used oscr three \rars and strict controls set up. Among the compounds tested was the HONS 086584 1)44 , 20 November /97/ Chemistry ami Imhn/ry 'Arochlor 1242' injected at 0 025 and 0-lml giving, a 0 and S per cent hatch ns compared with over 95 per cent in the control proop. Teratogenic effects Mere also noted in some of the chicks which did hatch. These results may be com* patcd with those for `Hcptachlor', where 0 05 and 010ml of a I per cent solution in propylene gf>col (non-toxic) gave a 75 and 65 per cent hatch respectively. Flick et at*40 endeavouring to identify the factor causing the chick oedema disease, following icports that the factor was highly chlorinated and that a plasticiser (PCB) used as a cage wire coating fed at 0-01 per cent or higher caused oedema in chicks, investigated the effect of `Arochlor 1242'. Groups of twelve chicks were fed on diets containing 200 or 400 p.p.m. over a three week period, changes in weight being noted. Weight gain was lower with the 400 p.p.m. diet and indeed loss took place at the end of (he period. Three chicks on this diet died. Here the hydroperi cardium volume was ten times larger titan with the 200 p.p.m. level or the controls, the pancreas, kidneys and adrenals affected, and haemoglobin levels were lower. The PCI! produced similar effects to the oedema factor, but was not so potent, nnd gas chromatography demonstrated that it was not the same. Two groups of workers, Risebrough, PenkaH et ol, in the USi*.*4.41-3 (in(| Jefferies, Moore, Presst et al. in Great Britain,1,144 have actively pursued the effects of organochlorinc pesticides and PCBs on birds. The decline in certain bird populations, especially raptors, has been attributed to the presence of orgnnchlorine pesticides, but It is also possible that PCBs have played a part id this, not only because of their intrinsic toxicity but because it has been shown (in insects,2 and below) that there are synergistic effects between some PCBs and DDT and `Dicldrin'. Lethal toxicity to the adult bird is not the only factor because it has been shown14'41 that organochlorine pesticides at the order of one p.p.m. are effective in inducing steroid hydroxylases in avian liver, thus leading to lowered oestrogen levels and reduced reproductive capacity. Added to this DDH has an entirely different effect on the reproductive system, that ofcausing eggshell thinning, which causes added failures in reproduction. Teratogenic effects may also occur bceausc of the excrction/concentration factor in the eggs. It Is thus necessary to review the effects of the organo chlorine pesticides on birds in conjunction with those of the PCBs. Following work by PenkaH,41 which demonstrated that pigeons fed with DDT at 10 p.p.m. and/or 'Dicldrin* at 2 p.p.m. for one week had Higher levels of testosterone or progesterone metabolites than controls, Risebrough, Pcakull el e/.i* compared the effect of DDE, DDT and PCB ('Arochlor 1262') in increasing the levels of polar metabol ites in oestradiol metabolism, again using pigeon liver isolates in vitro. Their results arc given in Table VII. Able VII . Control DDK (-tOtng'kg) DDT (4Qmit;kgl PCB (20mp'ktf> Polar metabolites formed (mil males) 29 76 95 160 This PCB has about five times the capacity to reduce oestradiol levels than DDT or DDE. The effect of a single dose of DDT may last for over two months. This work was extended to compare the effects of two different PCBs in kestrels, again using liver microsomc isolates.4'* The kestrels were fed at 0-5 nnd 5 0 p.p.m. levels with `Arochlor. 1254' and `Arochlor 1262', and the results were as in Table VIII. Table vm Control 0-5 p.p.m. 5 0 p.p.m. Polar metaitoiifrs (nmoteslg microsomal fraction) *1254' '1202* 0 13*65 60-5 0 19-05 47-93 While these figures do not clearly indicate the relative activity of these PCBs, it has been shown that `Dicldrin' nnd PCB arc much more active in reducing oestrogen levels than either DDT or DDE.42 That pesticides reduce oestrogen levels in mammals has also been demonstrated. Presst, Jefferies and Moore44 investigated the lethal effects of `Arochlor 1254' on Bengalese finches. Food containing higher Icvch of PCB (440 p.p.m.) w-as refused by the birds (100 per cent mortality not occurring) but it was still possible to calculate the LDjo as 253*6mg/kg/d. Seven out of the 38 birds died. Enlarged kidneys was the main feature, with in two cases increased volume of peri cardial fluid..While there was a correlation between dose rales and'liver concentrations, there was also considerable varia tion in levels (3-697 p.p.m.). Whether a particular con centration will be filial will depend, as with organochlorine pesticides, on the condition of an animal. Considerable storage in fat is possible and until this reservoir is saturated, concentrations will probably not rise to lethal concentra tions in susceptible organs. So danger can arise in times of stress (e.g. starvation or with the breeding cycle) when the fut reserves are mobilised releasing large quantities of PCB or w hatever. The liver by itself in these birds may thus not be a good index because the liver fat reserves may vary considerably and will themselves be mobilised ultimately. The authors therefore suggest the brain/liver percentape ns a useful index, being high in those birds that died (83-7 per cent and low in the survivors (26-4 per cent). The I-Do for DDT is some thirteen times lower than for this PCB, but consideration has to be given to the fact that while the DDT mortality curve U steep (no deaths at 1000 and ail dead at 1200mg/kg/d), the PCB curve is shallow, so that at low levels PCB may be more toxic than DDT. The authors con clude on the basis of this experimental work, and reasonably assuming that no drastic change in useage of PCBs is likely to have taken place over the past two decades, that few bird deaths have been caused by PCBs. However they stale, without citing evidence, that the PCBs do not vary markedly in toxicity and this is at variance with recent work:*Jf -`Msec below), the PCB used here being of relatively low toxicity. Additionally the authors do not consider the possibility of synergistic effects.- U was recognised that the position with sublcthal effects may be different. MONS 036565  Jp NuvrtnPcr IV7I Chemistry am/ huhtstry ^ Very little work'0*41-9 has been done on the effect of PCBs (and tcrphcnyls) on aquatic organisms, despite -the fuel that it is known (hat some marine Crustacea and fish arc very sensitive to orpunochlorinc pesticide residues.44 For example Crangon vulgaris (brown shrimp) has an L.CV48h/ ISeC for DDT of 0.003-0*1 p.p.m., and levels ns low ns 0*0001 p.p.m. may be detrimental to some species of mollusc. One difficulty in the study of toxicity to aquatic organisms is the extremely low solubility of these com pounds in water and the difficulty of obtaining sufficiently Stable emulsions.41'9 The use of solubilising agents, of low toxicity, overcomes this. Zitko,49 using `Corexit 7664* to solubilise`Arochlor 1221* and 'Arochlor 1254*. carried out 192 hour tests with these compounds using Atlantic salmon parr. He found that concentrations above 2mg/l were lethal. Wildish49 using `Arochlor 1254' in emulsions, or solubilising with `Corexit 7664', carried out 30d tests with (iomntarus ocronirus and found lethal threshold values of 0-001 to 0*0lmg/l f'CorcxiO and 0*01 to 0*1 mg/1 (emulsions). Guthrie tt at.47 tested mixtures containing chlorinated tcrphcnylj and their oxidation products and found lethal threshold values of about 5mg/l and pertinently that low oxygen levels in the water enhanced the toxicity. Duke tt a/." who monitored FCB levels in biota and sediment in the Escambia Bay area of Florida as a result of an industrial leakage of PCB into the Escambia River, conducted both acute toxicity tests and 20d bioassays. 48h tests on pinftsh and juvenile shrimps at the 100 p.p.b. level was not toxic to (he flail but caused 100 per cent mortality in the shrimps. Oysters received a 96h test and the effect was measured in terms of Rrrest of shell growth. Here 1 p.p.b. resulted in 19 per cent decrease in shell growth and 100 p.p.b. 100 per cent. Resultant whole body concentrations for the three Organisms were 17,3*9 and 33 p.p.m. respectively. It should be noted that the oysters when replaced in PCB free water for three weeks recovered their normal growth rates. In the 20d assays using a concentration of 5 p.p.b., a 72 per Cent mortality occurred in juvenile shrimps and 5 per cent in juvenile crabs. It would appear then from all this work that some Crustacea and molluscs are as sensitive to PCBs as they are to orgnnochtorine pesticides. Some toxicological work has been effected using insects.2*44 Moriarty* dosed fourth instar nymphs of Chonhippus brumeus (grasshopper) topically with 'Arochlor 1254' at 12*5, 50 or 200;ig levels, but found that definite mortality was only associated with the 200pg group plus a possible sub-lethal effect associated with moulting (fat mobilisation). The muximum amounts of PCU in individuals of the latter group were I4pg (male) and 8tig (female). The work of Lichtenstein, however, is much more revealing as to the relative toxicity of the various PCBs and the synergistic effects with DDT and `Dieldrin'. Groups of 50 Drosophila tnclotiognslcr were placed in contact with vessels coated with a layer of the substance or mixture of substances, and groups of 15 Musca domcsiica were dosed topically. Test* of the individual substances showed that `Dieldrin' was some 25-30 times more toxic than DDT, and 1-8000 lime* more toxic than the PCBs. The higher chlorinated biphenyls, and the lerphcnyls, gave rise to no mortality in the 24 Of 4Vh tests, but toxicity increased with decrease in \us chlorine content in the iower half of the series. These results raise doubts ns to the relevance of the many papers which have used 'Arochlor 1254% which here has little or no effect under these conditions. The synergistic effects with 'Die!drin' were positive with some of lower `Arochlors* and uniformly positive with pp'DOJ up to 'Arochlor 1254*. However, with the higher chlorinated PCBs and PCTs a reduction of toxicity was observed. While it is improper to transpose results from one phylum to another and short term toxicity may have little bearing on long term effects, nevertheless this paper is a strong indication that consider able care should be taken in planning these toxicological ^todies. Work using mammals is equally sparse. A preliminary study by Sobotka,50 using 'Arochlor 1254' and 'Arochlor 1260' or DDT in single doses of 1-lOmg/kg ('Arochlor's) or 0-25mg/kg (DDT) carried out behaviour tests on mice over periods of 4 to Z4h after administration. There is a clear difference between the two groups of substances, DDT probably attenuating nnd PCBs probably enhancing central inhibitory systems. The paper by Bitman and Cecil4* surveying the oestrogenic activity of DDT analogues and PCMs in terms of the 18h glycogen response of immature rat uterus assumes considerable importance in relation to a lot of the work cited above. The stereochemical require ments are quite strict, and in general for diphenyl-methane and -ethane, and triphenylmethanc compounds activity is restricted to those where either the pp` positions are vacant or filled by hydroxy or methoxy groups, being inactive where halogen or alkyl groups are present. So the pp' compounds perthnne. kclthanc, DDE, DDD and methoxychlor are inactive. However, pp'DDT is active and the op'isomcr some sixteen times more so. while of the others mentioned only op'DDE gains a response. 'Arochlors 1221-48' arc about half ns active at pp DDT, but ihc higher members arc inactive, (c/. Lichtenstein). In perspective though these compounds only have something like 10*' times the activity of natural oestrogen*, but this fits well with the observed pattern of steroid hydroxylase induction in birds, where DDT and PCB are very active but DDE is less so. Perhaps another significant point is that the oo' and pp'biphenols have an equivalent oestrogenic activity. The lower chlorinated PCBs could be degraded in the atmosphere as has been shown by laboratory experiments where the greatest diminution in one gas chromatograph peak was found where PCIls on an aqueous surface were subject to UV irradiation.'1 At the same time it has been observed^-17-*1 that the lower chlorinated PCBs (end (o be absent in wildlife samples, particularly in birds, and it may be speculated to what degree there may be differential excretion or in vivo breakdown, and what effect this has. A recent study by Vos ct a/.J2 reveals another important aspect. They discov ered that two out of three 60 per cent chlorinated PCBs contained highly toxic oxvgenated impurities. These two commercial preparations (`Phcnoehlor DP6\ `Clophcn A60T when injected into hens' eggs at the 3*5mg level caused almost 100 per cent mortality whereas the third sample (`Arochlor 12<.0'> gave a relatively low mortality. A fraction containing no PCBs was isolated from one of them by `FlomiP column chromatography and shown to be toxic. MQNS 006586 / 1)46 20 November 197i Chetniury and lruinM< t H Gat chromatography and mus spectra showed this fraction lo contain telrathioro and pcntuchloro-dibcnzofurans which were, however, masked by hexachloronnphthaJenc and hep- tachlorobiphcnyl respectively, thus limiting the accuracy of quantification. The pcntnchlorodibenzofuran was estimated to be at about the 5 and 20 p.p.m. level in the two samples. That PCB exerted a similar ctVcct to that of the chick oedema factor (CEF) had been noted previously,40 and that toxico logical studies using PCBs iuid produced variable results. 1,2,3,7,8,9,-hcxachloro dibenzodioxin has now been identi fied as one of the oedema factors- and it may be recalled that the toxic factor in the weedkiller 2,3,5-T was shown to be a letrnchloro dioxin.'3 In fact these chlorinated dioxins and dibenzofuraits are very highly toxic groups of compounds. 0*2(1$ penlachlorodibcnzofuran is needed to cause 100 per cent mortality in hen eggs as compared to 0*05^6 ofhexa- chlorodibcnzo-p-dioxin. The occurrence of the dibenzo- furans In PCBs is associated with the use of sodium hydrox ide in the distillation of crude PCB. Pcntachlorophcnol (PCP) and 2,3,4,6- tetrnchlorophenol are possible precursors of CEF and have been found to be present in toxic oleic acids.94 * It is evident then that other compounds besides the PCBs themselves should be sought for in wildlife and that indivi dual compounds as well as commercial preparations should be used in toxicological studies. Unfortunately the indivi dual PCB compounds are not readily available. Holden and Mnrsden55 have already noted the presence of unidentified polar compounds in wildlife extracts. It is to be hoped that the current FDA programme'0 on the toxicology of PCBs takes into account all these factors. Analysis PCBs are extracted together with chlorinated hydrocarbon pesticides in routine residue analyses,37 and being complex mixtures of compounds with GC retention times on non polar columns almost identical to a number of pesticides, notably of the DDT group, present an analytical problem which is not completely soluble by gas chromatographic means alone. Chemical conversion of the pesticides, leaving the I'CDs almost unchanged, provides n partial but not completely satisfactory solution, but group separation is fundamentally sounder, this ofcourse providing additional evidence of identity. Both thin layer chromatography and column chromatography haw afforded quite good partial separation of PCB* from pesticides and the best scheme-8, claims to leave only `AMrin' with the PCBs, with which llscre Is very little interference by the PC'Bs. A number of workers1MM**eo have confirmed the presence of PCBs in living tissues by means of mass spectrometry, but apart from this means (he quantification of PCBs presents severe problems because of the large number of compounds involved and that the individual compounds arc not readily available, so that most authors have only been able to calculate approximate figures. . Gas chromatography retention data for PCBs is now adequately documented12,10*18,20-*0,01 (and also1*2,15*14,20, 24,is,)049) and on non-polnr columns the main region of interference of PCBs and organochlorinc pesticide residues with one another may vary from the point of emergence of PP'DDE that of pp'DDT. PCBs may continue to emerge with retention limes up to four or five times that of DDT. It has been noted in this laboratory that response to PCBs relative to organochlorinc pesticides can be small on some columns, and from the viewpoint of determining pesticide residues, can ameliorate the situation. However, using more polar columns, c.g. *QF-1' or `XE-607---'-9'-0-" PCBs emerge largely prior to the DDT group, which allows simple quantification of the latter, but may still leave an unsatis factorily complex chromatogram. Improved resolution of PCB compounds has been claimed03 using novel stationary phases. One alternative has been to alter the retention lime of pesticides by dcrivatisation, leaving the PCBs intact, and of course aiding the identification of the pesticides. Jensen-*9 used a mild nitration technique adapted from Erro el af. and originating from the Schcchtcr-HaHcr method, claimed that the PCBs remained unaltered while members of the DDT group were effectively removed, although other organochlorines (e.g. *Toxaphcne\ hcptachlor epoxide, lindane and BHC) are not removed. This scheme (1:1 HNO3/H2SO4 for 5min at 09c) was followed by Risebrough ef n/.,14,90 but Reynolds61 found that DDT derivative peaks ultimately emerged on the chromatogram making for an unprofitably long analysis lime. Moreover, some lower chlorinated PCBs appeared to have been affected by the procedure. Saponification with alcoholic potash15,14,?0 leads to the dchydrochlorination of pp'DDT, ppTDE, and Toxaphene', with the derivative peaks appearing earlier in the chromatogram, and the PCBs reportedly unaffected, BHC is destroyed by this procedure. PCBs have also been differentiated from DDT using carbon-skeleton chromato graphy.04 Separation prior lo gas chromatography is to be pre ferred not only because simpler chromatograms can be obtained but because this will give nearly as much evidence as to identity as derivation. Thin layer chromatography, reverse phase paper chromatography1- and column chroma tography afford means of obtaining partial separations of organochlorine pesticides from PCBs and fractions can easily be obtained where only `Aldrin', `Hcptachlor', and pp'DDE remain with the PCBs. The former two nrc not very important in respect of the gas chromatography separation because there is usually little interference between them and PCUs, but pp'DDE can emerge with the mnior PCB peaks using non-polar columns. Thin layer chromato graphy has given this sort of separation using silica eel1* and alumina,1*2 and in column chromatography Holden and Marsden20,49 claimed good separation of PCBs using either alumina, silica gel, or both in scries with long narrow columns. Reynolds01 documented a separation with *Florisil\ while Bevenue and Ogata0- commented on their failure to reproduce this work in terms of pesticide separations only, and noted the differences in activity between `regular' and *PR FlorisiP. This reviewer has found no difficulty in obtaining this separation using `Florisil'. Armour and Burke58 in a carefully documented paper showed that it was possible to carry out a separation leaving only Wldrin' with the PCBs. For this they used silicic aciJ p.tiih deacti vated with 3 per cent water mixed with cclilc and eluted the HONS 086587 2*20 November 1971 Chrmiiiry and Industry PCBs ('Atochlor's 1254 and 1260*) with petroleum ether. `Arochlor 1254' needed the larger volume or eluant and the separation was more critical. The cautionary note is added that any trace of polar solvent in the wimple will make the separation impossible and /yr'DDE will emerge with the pens. Also, the separation is only possible* provided that the column (20+5g) is not over londcd (/.r. not exceeding 20jig PCD). This then solves the main separation problem leaving on nonpolar gas chromatography columns only small PCS peak which may interfere with the estimation of`Aldrin'. These authors have also documented the separ ation of chlorinated naphthalenes.65 Confirmation of identity has been achieved by combined gas chromaIography/mass spectrometry,1 s-22?9,6(l but gener ally quantification is not simple because of the large number of compounds involved and because these are not readily available individually for reference purposes. If it can be assumed that a fraction contains only PCBs (i.e. in a column chromatography or gas chromatography effluent) and that the composition on a gas chromatography trace conforms lo that of a commercial mixture, then microcoulomctrie estimation will give an answer. Jensen ct n/.,n using a combination of mass spectrometry, ECCC and mkrocouiometry obtained results which were only accurate . to about a factor of two. With the same assumptions electron capture detection can be used, referring to commercial PCB mixtures26 either by estimating the total peak area19,59,63 or basing the calculation on one1*16*19 or more20 suitable peaks. pp'DDE or `Dicldrin' may be used as an internal standard.16*50 One cmpiricul approach calculated one PCB peak ns pp' DDT and multiplied by a factor of ten to obtain a result;24 another66 took the total nrca:wcight ratio to be equivalent to that for DDE; and another 19`50 assumed the response of each peak lo be equivalent to DDE and applied correction factor from the use of microcoulomctric detection (the authors did not have permanent access to a microcoulometric detector). Gregor)'62 studying the electron capture coefficients of mono- and dichloro- and methylbiphenyls showed that there is over a twentyfold variation between the chloro compounds. Zitko49 determined the concentrations of PCB in fresh and salt water by means of Ultraviolet spectrophotometry and fluorescence. White the lower chlorinated PCBs can be prepared easily60 and only simple mixtures formed when using the Ullmann reaction, the higher members are more difficult to make singly. Discussion * The decline of certain bird populations was associated with the use of 'Dicldrin* seed dressings and has been attributed to lethal toxic effects. With increasing awareness of these dangers, better monitoring and usage control, at least in developed countries, Icthul toxic effects arc progressively less likely to occur. Rather, the significance of PCBs (and pesticides) to man and other organisms must now be seen In the context of sub-lethal effects having long term influence on populations through the media of reproductive processes (reduction in fertility, delays in breeding), teratogenic and carcinogenic effects on both the parent and Inter generations. LDjio tests, even over a long period, arc irrelevant from this viewpoint. Certainly it is true that severe munumdian 1347 liver damage and death can result from high PCB levels but these arc only reached in .* few foodchain heads, and the normal levels found in man arc not of this order. To be see against this, however, is the finding (in LDjq terms) that while with DDT there appears to be a definite *no effect' level this is appnrcntly not true for PCB. Precisely whm compounds arc responsible for the toxicity of PCBs is not yet clear, but it seems certain that it concerns the loucr chlorinated compounds and probably the oxidation products in trio or ru vitro. It also emerges that impurities in some commercial mixtures arc certainly highly toxic, and if the fact of synergistic effects, though demonstrated as yet in one area on))', is taken into account then the picture becomes a very complicated one. Clearly it is extremely difficult to carry out strictly controlled toxicological studies at a feeding level of the order of I p.p.m. over a long period, and in terms of multigcncration tests. The need for multi generation tests may be related to DDT, where, despite many years study, it has only recently been indicated that, with only the original parents dosed, carcinogenic effects may occur in later generations (6lh generation mice). DDT may again be used to illustrate the sort ofeffect which may be looked for. In California, Oregon and Washington, genetic changes in the fruit fly population have been shown to result from heavy spraying programmes in these areas, and in the latter two states were tied to two specific forest spraying programmes. The pattern of these genetic changes can also be correlated with spread by winds over long dist ances. With PCBs the major dispersal medium is probably water, but otherwise there are no essential differences in the situation. The PCBs are spread all over the continental shelves which are fed by industrial effluents. The toxicology of the weedkiller 2,4,5-TJ? and of mercury residues69 has recently come to the fore and they present some parallels with the PCBs. While in contrast to PCBs there is a definite, but small, contribution to agricultural pollution, by mercury from seed dressings, the industrial contribution must lead to a similar marine distribution to PCBs, although as yet very little is known about this area. Mercury, in contrast to PCB, is only cumulative in living tissue to a limited extent, and there is a definite natural contribution from the sea floor. The massive aerial use of the weedkiller 2,4,5-T as a defoliant by the Americans resulted in teralologicnl as well as direct toxic effects due to traces of a chlorinated dibenzodioxin. It is as welt that PCBs arc not used in such fashion because it has now been shown that some commercial preparations contain traces of chlorinated dibenzofurnns which are also highly toxic. Water pollution is receiving considerable attention within the traditional framework of oxygen deficiency, reduced nnd oxidised nitrogen, temperature and so on, but the type of pollution problem presented by PCBs, DDT and so on is largely ignored. In a recent official report on toxic chemicals, which recommends further work lo appraise the situation in fresh, estuarine and coastal waters, there appears to be a large conceptual gap. Commenting on the presence of DDT in raw sewage, this is dismissed ns a sirmllcant source of riverine pollution because DDT is solid attached and largely remains in the sewage sludge, but what happens to the sludge is bliihciy ignored. The ironic situation is of "qns 0fl65as 1)41 20 November 1971 Chemistry nnd Industry course th;it (he sludge is commonly dumped in estuaries or even spread out over the kind so that pollutants such as the PCBs arc ircirculatcd through the food chains. These tire but a few aspects of environmental pollution which can ultimately lend to man's own detriment. In many cases the users of pollutant materials, industrial or domestic, have no conception of their own insidious contribution to pollution, nnd in the case of the very useful PCBs it is very difficult to sec how their disposal can be controlled to minimise pollution. From the point ofvicwofrcstrictingtypcs of use to those least likely to result in pollution, one prod ucer, Monsanto, has made a very responsible move70 in this direction, but as PCB manufacture is no longer covered by. patents this may not be effective. It is obvious that with their ubiquitous presence the effects of PCBs and associated compounds must be investigated with great thoroughness. It is not only to preserve bird populations or other wildlife but lo prevent long term changes in man himself that such searches arc directed. . The author is grateful to the H. J. Heinz. Co. Ltd, for permission to publish this paper. References * WestM, O., Norin, K. ft Andersson, M., Var I'iida, 1970,10 * Lichtenstein, 1!. P., Schulz, K. R., Puhremann, T. \V. ft Liang, T. T., /, free. EM., 1969,61, 761 . * Koeman, J. H. A Genderen, H. van, J. Appl. EcoL, 1966,3 (SuppL), 99 * Harrison, R. J)., J. Sci. Fd Ague., 1966,17, 10 Robum,Analyst, Load., 1965, 90, 467 * Walker, C It., Hamilton, G. A. 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W., EnvironmentalPollution, 1970,1,) * Duke, T. W,, Lowe, J. I. ft Wilson, A. J. Jr., Unit. Law Contain. A Toxicol., 1970, 5, 17] . 7i Bonner, W. N., 'Seal deaths in Cornwall, autumn 1969*, 1970, Natural Environment Research Council Publications Series C no 1 ** Biros, F. J.. Walker, A. C. A Mcd!*cry, A., Hull. Btv. Contain. & Toxicol., 1970,5, 317 ** Third Report of the Research Committee on Toxic Chemicals, 1970, `t/Sudan: Agru uliutol Research Council *4 Anderson, D. W.. Hickey, J. J., Risehtough. K. W., Hughes, D. F. ft Christensen, R. E., Can. I'M Sat., l%y, 83,91 15 Rtsebrouch. R. \V., Huggett, R. J. ft Griflln, I. J., Science, N. P.. 1968, 159, 123) Holden, A. V.. Nature. Land.. 1970, 228, 1220 *7 Cohen, J. M. ft Pinkerton, C,, in `Organic pesticides in (he environ ment*, cd. Gould, R. F.. 1966. Advances in Chemistry Series, no 60, Washington: Amerirun Chemical Society, p 16) i* Tarrant, K. R. ft Totten. J. O'G.. Nature, Load., 1968, 219,725 ** Wolman, A. A. ft Wilson, A. J. 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