Document Z8L9EyQvom4Rg6b6DqRqKw7K0

llr Chairman, Ladies and gontloaon. " In honor to our Brittieh 2***' I will try to hold this lecture In Snglioh* As tho titlo of this locture states, I an today going to tell about tho dlocorory of some hlthorto unobserved chlorinated hydrocorbono having up to eight chlorine In tho molooule and found In reolduo nna-- lyoio. Tho chonioal none of polychlorlnatod blfenyle ( In tho following oallod PCB), To got familiar with BOB I will start with the ohonletry and .toxicologi* - flhomistry 'xh . . The naln-choraoterietlo of PCB le 1. Their very high stability* As an ozuaple they can be boiled with nltrin aoid without being destroyed* 2* They aro h&dly metabolised In living organism* 3* If more than 4 chlorine are^pretjsent thoy are non Inflamable. It is clear that these threo characteristics does it easy to understand that when they havo entered the giving organism, the vill have a low persistence But it le difficult to explain how they find their way into the living organlcn* Ono thing oseme to be dear, they don't oome from agricultural use, but from a technical one and most probable it domes to the nature via wastes that are tried to be burnt up, because then we have them at.onco In the air, beoause of tholr non inflamabillty* ICXlCOlOgl a*90*8 Tho PCB woro introduced in 1929 and no early ag 1936 Jonoo and Alden reported that 23 out of 24 non employed in manufacturing of PCB eufforod from an acno form oruptlon of the skin. Acne did not appear until 6 to 8 months after the matorial was first usod. In 1937 DrinJcer reported that rats sxpossd to chlorinated biphenyls in concentration of approxi mately 1 mg/m^ for 16 hours a day for 6 weeks chowdd damage of tho liror After that time tho allonod concentration of PCB in air la 0,3 mg/a7. (For DDt the same value ie 0.3 - 1 mg/m^). The somo authors finished tholr experiments in 1938, and related that those compounds hove an lnjourlouo efroct, manifested solely in the liyer. Chlorinated blpohnylo appeared to.be the moet injouriouo chlorinated compounds of all tested. O' 0- Orenburg, Mayer and Smith 1959 reported that PCB and polychlorlneted naphtalcnoe are blamed for the death of three young workers, and tha-T* prognant women and persons who have at any time had any livor dlseoBoa nro particularly suspectible. Wodol, Hollor and Benton gave 1942 animals PCB including administration by inhalation, ingoBtion and slcin absorbtlon. Histological examination of tho viscera showed Important toxic effect only in tho skin and livor end tho dogonoratlon effects in the liver axe essentially the oanc what ever was'tho method for the administration. Paribok (1955/ found as an occupational poison in the electrical Industry, mixed totra and ponta ohlorobiphenyl causos folliculitis, comedo, pyodermia and other skin effeotlone, and that ltB principal toxic effect is fatty degeneration of the liver. ' Miller (1944) injected 69 mg PCB (4 and 5 chlorine) subcontaneoualy in 52 gulnoa pigs. Sight to ten days after injection, fat droplets wero noted in tho liver oells, and after 16 days they were present in modcrato or very large numbers. Babbits and ratB were also tested in this lnveotl- gatlon, as well as the PCB was admlnietated both continously, subcuntln- ously or ingested in the food. In the feeding experiment B guinea pigs received 2 doses of 69 mg of the chlorinated biphenyl 1 week apart. Death occurred in 11 to 29 days. Pinally Me Daughlin 1964 reported a method to test the chemical toxicity and teratogenic effect by injection into the yolk sac of fertile eggs prior to lnoubatlon. PCB was found between the eight compounds among 100 tested having the highest order of toxicity. Mo hatch was found at a level of 25 mg po egg. At a level or 10 mg per egg, one chick hatched out of 20 injected eggs, but died 2 days later. Some embryos which were examined after they died, showed weak deformities (often a short upper brak) and growth retardation. Lead acetate resulted as an example in no hatch at a level of 1 mg per egg. Autopsy of the dead embyos have showed extea slve brain damage. Mercuric chloride showed no hatch even at a level of , 0,5 mg per egg. MQNS 049049 As the analytical chemistry is a pronounced service science I have buen in contact with many scientists from other fields during the work with residue analysis, and I have always found this contact very stimulating for my own work. This co-operation often demands that we are talking the same scientific language. Bocause of this need 1 will today try to give a lecture in low level analytical chemistry for biologists, illustrated by the residue analysis of polychlorinated biphenyls. The lecture will be divided in the following three sub-dlvielons: o o 1. Chemistry of PCB and their tocicology, 2* Analytical methods for roeidue analysis and proof of structures* J* Behaviour of PCB in nature, dlfferencles in metabolising xato of the PCB components, potenoation in an eoologlcal oerie, con centration levels and examples of samples which have boen proved to con tain PCB* A residue analysis con be divided ini 1* Extraction of the pesticides from the biological material, followed by a careful cleaning-up to take away interferring substanoes, moat often fats* * 2* Identification analysis by moan of gas chromatography* Thin- __ layer chromatography and mass spectrometry* 3. Quantitative analysis* - At on ecological laboratory in Riksmuedet in Stockholm 1-2 g of a sample is out out of the biological material and transferred into a weighed and carefully cleaned test tube, and stored at -20 until analysis* Smaller samples have been used, min* 5 mg of body fat, and with dry materials such as hair, feathers, pins needles 100 mg are sufficient to reach the desired 10 ng/g level in residue analysis* In cases of water proofs 1 1. is used for reaching the 10 pb/$* level* S*U(hoaog) In order to facilitate complete extraction of the fatty materials from tho biological sample, the doulbe amount of finely powdered anhydrous magnesium sulphate Is added to the sampling tube, and the whole is'homogenlecd with an lneertable homogenlzer'* The resulting powder is transferred into a special Soxhlet extractor* After 4 hours of extraction the solvent ie evaporated, leaving the fat in a small weighed teat tube at the bottom 8x-tube) of the extractor* This fat 1b dissolved in methylene chloride in such a way that 100 ul (0,1 ml) contain 20 mg of fat* The 100 ul solution is now transferred to a little object glass, 3x7 cm, covered with a elllcagel layer 1 mm thick, in order to form a line 0,7 cm from one end of the slide* Inserting this thin-layer plate Into a vessel the bottom of which is covered by a few mm of methylene chlorido, the solvent will be sucked up in the dry layer of silicagel, and at least 3 reach the upper end of the plate* The fact is that the fat has & greater affinity to the powder on the plate than the chlorinated hydrocarbon have* - and we get a separation* The fat being more polar than the chlorinated hydrocarbons will never go longer than 2 cm before the HONS 049030 o JSIBTIOH tubs 5 ao 6 |i o o ) solvent roachoe tho upper part or the class. Tho front of the tot appears quite violblo against o lamp, and with the old of a razor blado the zone above the fat is transferred to tho olutlon tube and the chlorinated blooldos absorbed on the powder eon now be oluted by ono ml of ether. The concentration is sufficient for detection of tho chlorinatod hydrocarbons down to tho 10--12 g levol. Tho noxt atop in tho analytical procedure concerns the separation of tho dlfforont chlorinatod hydrocarbons that the oamplo may contain. Ao a nattor of fact, thio ie a broublosome task. It is easy to estimate what is not present, but more difficult to Bay exactly one is present. We ouffor from tho negative demonstration, as will be shown later. . At first a Xow words about the separation of the components preoont In the sample and their visualization. Tho ooporation ie aoooaplishod by mean of a gas chromatograph fitted to a dotoctor that transfers its impulse to a recorder. Tho syBtom is shortly .described: A spirally formed glass tube with an inner diameter of 2 mm and about 2 m in length is filled up by a support, covered with an thin layer of an oil. The tube is heated in the chromatograph to about 200. Through tho tube a stream of nitrogen contlnously follows. When about 10 ul (1/100 of 1 ml) of the purified eample is Injected into the tube, the components of the eample will be evaporized and go forward through the column with the gas stream. As the constituents have different affinity to the column filling they will pass the column with different spood and it will taka different time for then to roach the detoctor at tho other end of tho glass tubs. If the temperature and the nltrogon flow are held constant this time, tho retention time, has a spoclflcvaluc for a certain compound. Thio is true, but unfortunately it Is also a fact that two componoate con have thesame retention time. This is one of tho bigger problems in gae chromatographic analysis of unknown samples, ao will soon be obvious. To make it possible to ostimato the retention time it is noceso&ry to visualize the chlorinated hydrocarbons. For that purpose more or loss specific detoctors are used. The detector most often usod in postlcido analysis is the so called electron capture detector, which can detect down to ono picogram (* 10"12 g of lindan). Unfortunately this detector is not specific for chlorine, bu gives answer alco for oxygencontaining compounds. The response here is muc lower but can be counterbalanced if the concen tration of the oxygen containing ie much higher. HONS 049051 Tho principle for tho electron capture detector is shortly: At tho end of the gas chromatographic tube is placed a little tube con taining a foil made of titanium tritido. This In A-n-rodlant. The o- JJ o 3 particles are reacting with the nitrogen molecules coming from the column* Then we get + Ng -- e + Ng. Over the detector we have a tendon of 90 volt and by mean of the electrons wc will get a constant electrical ourront over the detector. This standing current is transferred to a one-mV rocordor as a constant baseline. V/hon now a chlorinated hydrocarbon loavoo tho column thio compound hao a high affinity to tho oleotrono and thle moans that the amount of elootrons will diminish, and they will diminish proportionally to the amount of chlorine. The electrical current will also diminish and this is noted as a peak on the recorder. The area of tho peak will be proportional to the amount of aubstance in the sample* By moan of a standard injection it 1b now possible to compare the reten tion time and the aroa of on unknown component with the retention time and area of the known standard* As said before this detector is not speci fic for chlrlne but anyhow very useful, because of its high sensitivity* The ayetom described has, as we have seen, two disadvantages: 1. Two difforont compounds can have the same retention time and bo detected as ono peak* 2. A registrated peak does not need to be chlorinated f because the detector is not specific. If the eample is injected in two different columns with different chemical properties we have increased the chance for a good separation* If two compounds have the same retention time on one column they may not have It on another. When a result seems doubtful, - if the compound being responsible for a certain peak contains chlorine or not - it is possible to concentrate the sample and analyse,it on a less sensitive detector such ae the microdoumetrlc one, which ie specific for chlorine* The oorapound is burned In a furnace and teh generated chlorine titrated directly. MONS 049052 As is seen from the two last mentioned possibilities it is anyhow possible to get a rather high degree of certainty in residue analysis, but it is ( a rather time-consumeing work.When UBing this method just described, we very often found that many chromatograms from residue analysis of most carefully purified samples still contain a large number of peoko* Many of these have retention times that do not agree with any known chlorinated pesticides, or their metabolites. This chromatogram con serve as an example* It was obtained by residue analysis of a sea-eagle found deed In the archipelago of Stockholm. In the range of the known peaks, there are so many unidentified that there also muct be an obvious rick of the . known penko to bo covered by unknown ones. Tf thl R remark in trn. +h n-f **nrw titabivo anal;, *5.8 must be brought into quesv in. In the present investi gation it ie shown that most of the unknown peak of chromatograms at residue analysis of chlorinated pesticides are due to polychlorinated biphenyls. I will Bhow a chromatogram of human fat analysed on a so called S? 96 ooluran, the moet often used type in pesticide analyses. Early retention times were in agreemont with DDE, DDTop and DDTpp, Next slide shows the sane sample analysed on a QF-1 column. Now the former 2 DDT peaks have ' divided into 4 peaks,and two of them are still in agreement with DDTpp and op., the two new were unknown. Logioally, those unknown components were at first thought to be metabolites of the insecticides. Against that spoke that neigther treatment nor concentrated sulfuric acid in other. This treatment made it rather sure that the compounds did not contain oxygen. In Sweden residues of organic mer cury have boon investigated rather intensively in the Swedish fauna. As those compounds givs very high responses to the electron capture deteo- tor it was also investigated if the unknown peaks could havo a mercuric origin. It was found that the water-ecological aeries had high residues of both mercury (Westermark,Johnelo) and the unknown ones, when the same indivi duals were analysed. Anyhow, the pheasant suffering most from mercury poisoning only contained low levels of electron capturing compounds and these belonged to the normal insectlsldes. Therefore the unknown could hardly be mercu rial, or metabolites of them. mGNS 049053 As the eagle sample giving the chromatogram shown in fig. 10. could be estimated to contain DDT and DDE up to 13 g/kg in extractable fat, the amount of unknown compounds also were suggested to be in the same range, and tehn sufficiently high to do a run on the combined gas chromatograph mass spectrometer. If this could be done successfully it would be possible to get very Important informations about the chemical nature of the unknown, for ex. the molekular weight numbers of chlorine etc. ThiB method is up to now the method giving the highest degree of certainty in the low level analytical chemistry, amounts of 100 ng substance being enough. Ae this method for identification of totally unknown residues surely will be very important in the future (when f.ex. a biologist ha3 found that fishes in a river die) it may^possible by mean of this method to find out exactly what compounds are responsible for the death. For this reason, I will go into come details with thlG method. In the actual case we took the extract from 20 mg eagle and concentrated ' (Y O it as much as possible and made an injection on the gas chromatograph combined with the mass spectrometer* The result was the chromatogram shown on the next olldo* Every time the recorder showed that a compound is leaving the column, the effluent Is led to the mass spectromotor. Now just a few words about the mass spec* * The molecules leaving the column are bonded with electrons at K. V/o h&vo now got the molocule positive charged, but with the same mass ao before* r- This U* Is accelerated in a vacuum and will then get a kinetic energi* whero la the speed* Next comes the magnetic field that . trios to bend the direction of the molecule* This will be big for a small molecule and lees for If we have a sieve in the other end we can direotly read the molecular weight* Added to this parent molecule M+ we will also got addition in formations, because of the fact that U+ may not be sthblo, a port of them will be broken down before they reach the sieve in the other end. r.x. x M jjjjj _ Coi^ Mass spectrograms from the different unknown peaks In the eagle sample as shown* Tho mass numbers equal to the molecular weights of the unknowns could be road to 426,392, 358, 324* Astonishingly, the molecular diffe rences wore constantly 34 mass units* This difference shows a familarity in origin of the unknown* Now the fact is that chlorine assists aa a mixture of two isotopes with atom weights 35 and 37 in proportion 75:25. If the molecule has one chlorine, this will give two molecule peuke, one for C1,e and one for C1,_* If there are two chlorine we hav the poooibility 35 37 of one with only one with both C1^ and 57 and one with 2 Cl^ and therefore MGNS 049054 o 2he relation of the peaks found on the different maae epee wares Molecular weight : 324 358 392 426 Ohlorino content 5676 An explanation of the familiarity of the oompounds can be given if one eubetance is built from the former by substituting a hydrogen with chlorine KH M+ + Cl, RC1 HC1 M++ 34 Thon it is poasibD ;o calculate the molocular weight of the parent hydrocarbon FHC. - K - s C1 + x Mjj , where H la the molecular weight of the conponont having x chlorine atoms. F.ex. for m 426 and 8 Cl v/e will get ** 426 - 260 + 8 a 154 and equal with the other molekylo. Tho moot probable formula with carbon and hydrogen giving this molecular weight Is 0^2 Hjq and this can only be satisfied when the parent-hydro carbon is biphenyl, and the unXnovm being polychlorinated biphenyls. This explanation was later fully verified by injection of a synthetic 7BC on the mass spec. Furthermore extensive gas chromatographic investigations provod that the roc standard gave peaks with the came retention time as the unknown peaks from the sea eagle. With the method just described I suppose that we h&ve a new possibility to study the residues in the air because the pine needles can allr/aye bo Ve have had great difficult; in Quantifying the ?CBf but when getting a little more time it will be possible. We have done a few calculations on a few species, and 1 suppose they are right within a factor 2. We have found the residue to be from It hae been my statement here to-day to present this method for studies of defiling of the nature, and with this method a new typo of dofiling agents has been found to be present in nature, and a few experiment have shown where they may bo found. Mow this method is going to be used in the first hadn to estimate how the situation is in nature as a whole, and In the other hand to find the leaks throug which they find its way to nature. Soem maybe ore present here today to get news about the leaks, and to them I want to say come back in a year. HONS 049055 So much I think I can cay again that the ?CB hardly con cone from agriculture* As euppoft for this suggestion X can say that we have found FOB in eagle feathers from Riksmuseot from 1944, where hardly any chlorinated pesticides were used in ogricultrue. One moro thing that I find important to say is that in contrast to the mercury problem this does not Doom to bo a pure Swedish problem, X have just studied chromatograms takon from London air, and they cloarly contain PCB, and dr. Holdon hun told me that ho also find them in his fishsamples. But finally in waiting at moro results Xnshould like to point$Xe morething. It is proved that PC] comoo to naturo, wo dont know now where they are uood, but they are vory persistant to chemloalB and to fire. X think the poison Jury should try to state that a content of PCB shall always be found in an open declara tion. 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