Document 3QzOQXYbwaOa0Ly54ybbEYyNa

Q c/L</v> ^/Vv^iSi G ?v\ . Mr Chairwon, Ladles and gontlenen, % V't In honor to our Brlttioh fceot I will try to hold thlo lecturu In Bnglloh. ^ \x As tho title of thlo looturo states, I an today going to toll about the diooorory of oone hitherto uoobeervod chlorloatod hydroearbooo haring up to olght ohlorlne in the noleoulo and found In residue ana lysis. She choaioal nano of polychlorlnatod blfonyls ( In tho folioring sailed POD), So got faalllar with PCB I will start with the ohonlotry and .toxleologi, Ohopistry rv. 6 She main choraoteriotlo of PCB Is 1. Thefr very high stability, j.o on osaaple they oan he boiled with nitrin sold without being destroyed. 2. Tboy axe hardly nstabollssd In liring organ!an, J. If noro than 4 ohlorlno are present they are non lnflamablc. It Is clear that thote three eharaoterlstloe does it easy to understand that when they have entered the living organism. the will have a low perelatonco Bui It is difficult to explain how they find their way Into tho living organic* One thing seems to be olear, they don't oone from agricultural uoj, but froa a teohnloal one and aost probable It donee to the nature via waetoe that are tried to bo burnt up, beeauee then we have thou at onoo In tho air, because of their non lnflaaablllty. y.Toacloolosl HONS 035760 y\ 1 *he PCB woro lntroduood in 1929 and as early as 1956 Jones and Aldcn reported that 23 out of 24 aen employed In manufacturing of PCB euffc.od froa an acno form eruption of tho skin. Acne did not appoar until 6 to 6 nonthe after the material was firot uood. In 1937 Drlnkor roportod that rate oxpoaed to ohlorlnatod biphenyls in concentration of approxi mately 1 mg/a^ for 16 hours a day for 6 nooks ohowdd damage of tho llvor. After that tlae the Allowed ooneontratlon of PCB In air la 0,5 ug/u^* (For DX>t the value le 0.5 - 1 ng/n5). Tho ramo authors finiohcd their experiments In 1938, and related that thooo compounds havo an lnjourlouo effect, manifested solely in tho liver. Chlorlnatod blpohnylt^l appeared to bo tho moot injurious ohlorlnatod oospounde of all tested. ( o o 2. I Oroenburg, Mayor and Smith 1939 roportod that PCB and polychlorinated naphtalonofl arc blamed for tho deoth of three young workers,, and thot pregnant women and pereone who have at any time had any liver dioeaoon aro particularly euepeotible. Vtdo]., Halldr and Banton gava 1942 animalbPCB Includingadalnlatrnuon \ j j | | by Inhalation, ingeotion and ekin aboorbtlon. llletologioal exaainatiorx Of the viscera showed Important tozio effect only in the okln and llvor, and th* dogenoratlon effeots in the liver arc essentially the came what- ; I j ever was the method for the administration. Porthole (1955/ found aa an oooupatlonal poison In ths eleotrioal Industry, mlzsd tstra end poata ohlorobiphsnyl oausos folliculitis, comedo, pyodcrmla and other akin affeotlons, and that ite principal tozio effeot is fatty dcgoaeratlea of the liver* Ulller (1944) injeoted 69 g PCB (4 and 5 chlorine) aubcontaneouely in 32 gulnon pigs* Sight to ten days after injection, fat droplets wsro noted In the liver eelle, and after 16 dayo they were preeent In modcruto or very large numbers* Babbits and rata were also tested in thle invootlgatlon, as well as ths PCB was adminlststsd both continouely, eubcantlnouely or ingested in the food. In the feeding experiment 0 guinea pigs received 2 doses of 69 mg of thechlorinatedbiphenyl 1 week apart. : ( ! j ! ! j : ] Death oocurred in 11 to 29 days. Finally Me Laughlin 1964 reported a method to teat the chemical toxicity and teratogenlo effect by injection into ths yolk sac of fertile egge prior to inoubation* PCB was found between the eight compounds among 100 tested having the highest order of toxicity. Mo hatch was found at a j J' \ j level of 25 mg po egg. At a level of 10 mg per egg, one ehlck hatched | out of 20 injected eggs, but died 2 days later. Some embryos whloh vers j examined after they died, ohowsd weak deformities (ofton a short upper brak): and growth retardation. Lead aoetate resulted ae on example in no hatch at a level of 1 mg per egg* Autopsy of the dead embyos have showed exton lve brain damage* Merourlo ohloride showed no hatoh even at a level of | 0,5 mg par egg. As the analytical ohemistry is a pronounced service science I have boon in contact with many scientists from other fields during the work with ' residue analysis, and 1 have always found this contact vory stimulating for my own work. This oo-operation often demands that we are talking the same scientific language. Because of this need I will today try to give a leoture in low level analytical cheaiotry for biologists, illustrated by tho reeldue enalyolo of polychlorinated biphcnyle. The leoture will be divided in the following three eub-divloionet . 1 HONS 035761 Q3 O I I 1. Chemistry t PCB and their to--icology. 2* Analytical methods for rooidue analysis and proof of structurco, 5* Behaviour of PCB in nature, differences in metabolicing rato of the FOB components, potonoation in an ecological oorie, oon- oentration levels and examples of somplse which have been proved to con tain PCB. A residue analysis can be dlvldod lnt 1. Extraction of the pesticides from the blologloel aaterlal, followed by a careful oleaning-up to take away lnterforring subetanoee, noet often fate. 2. Identification analysis by mean of gas chromatography. Thln- _ layer ohromatography and mass spectromstry. 3. Quantitative analysie. 1 At an ecological laboratory In Blksmuedst la Stockholm 1-2 g of a sample is out out of the biological material and transferred Into a welgbod and carefully oleanod test tube, and stored at -20 until analysis. Smaller samples have been ueed, min. 5 mg of body fat, and with dry materials such as hair, featbsrs, pine needles 100 mg are sufficient to reaoh the deslrod 10 njy'g level in residue analysis. In oases of watsr proofs 1 1. is used for raaehlng the 10 pg/g. level. B.l(homog) In order to facilitate eomplete extraction of the fatty materials from the biologioal sample, the doulbe amount of finely powdered anhydrous magnesium sulphate is added to the sampling tube, and the whole is homo genised with an lnsertable homogeniser. The resulting powder is transferred into a spoolal Soxhlet extractor. After 4 hours of extraction the solvent is evaporated, leaving the fat in a small weighed test tube at the bottom Sox.-tube) of the extractor. This fat ie dissolved in methyleps chloride In eucb 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 ca, oovered with a sllloagal layer 1 ma thick, in order to fora o line 0,7 cm from one end of tbs slide. Inserting this thin-layer plato Into a veaeol the bottom of whieb Is oovered by a few mm of methylene chloride, tho solvent will be sucked up in the dry layer of sillcsgel, and at least reach the upper end of the plate. The fact is that the fat has a greater affinity to the powder on the plate than the chlorinated hydrocarbon have. - and we get a separation. The fat being more polar then tho ohlorlnuted hydrocarbons will never go longor than 2 cm bofore tho HUNS 035762 / EL0TIOH tubo 5> & go O O 4- solvent roachos the uppor part of the glass. Tho front of the fat appears quite risible against a lamp, and with tho I i aid of a razor blado the sons above the fat Is transferred to the olu.lom tube and the ohlorlnated bloeidoe absorbed on the powder oan now be olutod by ono ml of other. The concentration is sufficient for deteotion of the ohlorlnated hydrocarbons down to the 10**12 g level. The next step In tho analytical procedure concerna the eepAr*ii>v.t ,\t th* different chlorlnatod hydrocarbono that t)<> */ ... *. mattor of foot, this is a broubleoome task. It is easy to ewnot Id not prosont, but more difficult to eay exactly one le present, te cuffor from the negative demonstration, as will bs shown lator. It first a fsw words about the separation of the eompononts present In the sample and their vieuollaation. The separation is aeoompllshod by msan of a gas ehroaatograph fittsd to a detector that transfers its impulse to s reoorder. The system is shortly dssoribedi A spirally foraod glass tube with an lnnor 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 the tubo a stream of nitrogen eontinouely follows. When about 10 ul (l/lOO of 1 ml) of tho purified sample Is injected Into the tubs, the components of the sample will bo evaporlsed and go forward through the column with tho gaa stream. As the constituents have different affinity to the column filling they will pass the column with different speed and it will tako different time for then to roach tho detoctor at tho other end of the glass tubs. If the temperature and the nitrogen flow are held constant this time, tho retention time, has a spociflcualue for a oertaln compound. Thle is true, but unfortunately it Is also a fact that two components can have the same retantion time. This is one of tho bigger problems In gas hromatographlo analysis of unknown samples, as will soon be obvious, to make It possible to ostimato the rstontlon time it Is neoessary to visualise tho ohlorlnated hydrocarbons. For that purposo mors or loss spselflo detectors are used. Tho detoctor most often ueod la postleido analysis is tho so oalled oloctron capture detector, which can detect down to ono ploogram ( 10**12 g of llndon). Unfortunately this deteotor is not specific for chlorine, bu gives anowor aloo for oxygonoontainlng eeapounds. Tho rosponoo hero lo muojj lowor but can bo oountorbalancod if tho concen tration of the oxygen oontalnlng is much higher. The prlnelple for the electron capture doteotor in shortly: At the end of tho goo chromatographic tubo is plaeod o little tubo oor.- talning a foil made of titanium tritldo. This le ap-radlant. Tho p- MQNS 035763 o 5. o particles are reaoting with the nitrogen molecules coming from the column. Than we get Kj Ovor the dctoctor we have a tenolon of 90 volt and by mean of the electrono wo will got a oonotant oloctrio&l ourront ovor the detector* Thlo standing oiurront. is transferred to a one-mY recordor as a oonotant baseline. V/heo now a ohlorinated hydrocarbon leaveo the oolumn thlo oompound hao a high affinity to th elootrono and this means that the amount of elootrons will d&ninlsh, and thoy will diminish proportionally to the amount of chlorine. Tho electrical current will also diminioh and thie ie noted as a peak on the recorder. Tho area of the peak will be proportional to the amount of substance in the sample. By mean of a standard injeotion it Is now possible to compare the reten tion time and the area of an unknown component with the retention time and area of the known standard. Aa eaid before this detector is not apeoi- fie for ohlrlne but anyhow very useful, because of its high sensitivity. The system described has, as ws have seen, two disadvantages: 1. Two different compounds can have the some retention time and be detected as one peak. 2. A registrated peak does not need to be chlorinated , becauoo the detector le not epeoifio. If the aample ie injected in two different columns with different chemical properties we have increased the chance for a good separation. If two compounds have the eame retention tlmo on one column they may not have It on another. When a result seeao doubtful, - if the compound being responsible for a oertaln peak contains chlorine or not - it Is possible to conoentrats the sample and analyse It on a lees senaltlve detector suoh as the mlcrooloumetrlc one, which is speclflo for chlorine. The compound Is burned In a furnace end teh generated chlorine titrated dlreotly. As is seen from the two last mentioned possibilities It is anyhow poooible to get a rather high degree of certainty in residue analysis, but It is a rather time-oonBumelng work.then using this method just described, we very often found that many chromatograms from residue analysis of most oarefully purified samples still contain a largs number of peaks, -any of these have retention times that do not agree with any known chlorinated pesticides, or their metabolites. This chromatogram oon serve as an example. It was obtained by residue analysis of a sea-eagle found dead in the archipelago of Stockholm. In the range of the known peaks, there are so many unidentified that there aloo must be an obvious rick of the . known peoko to be covered by unknown onon. If this remark io found true, the reported results of many provfouo quoa- MGNS 035764 oo tltabive analysis must be brought into question. In the prooent inveoti- gatlon it la ehown that moot of tho unknown peak of chromatogramo at residue analysis of chlorinated peotioidco are due to polyehlorinotod biphenyls. i X will show a chromatogram of human fat analysed on a so called SN 96 column) the most often used type in pesticide analyses. Early retention times woro In agreement with DDE, Mm op and M/Tpp. Next slide ohewo the same sample analysed on a QF-1 column. Now the former 2 DM peaks havo ' divided into 4 peaks,and two of them exe still in agreement with Mftpp and op., the two new were unknown. Logically, these unknown components were at first thought to be metabolites of the insecticides. Against that spoke that nelgther treatment nor concentrated sulfuric sold in other. This treatment made it rather sure that the compounds did not contain osygen. In Sweden residues of organic set cury have been investigated rather intensively in the Swedish fauna. As these compounds give very high rssponsos to the electron capture detoo- tor It wae also investigated if the unknown peaks could have a aercurio origin. I. It was found that tho water-ecological series had high residues of both meroury (Weatenaark,Johnele) and the unknown ones, when the sane indivi duals were analysed. Anyhow, the pheasant suffering moot from mercury poisoning only contained low levels of electron capturing compounds and these belonged to the normal ineectlsidee. Therefore the unknown could hardly be mercu rials or metabolites of them. As the eagle sample giving the chromatogram shorn in fig. 10. could be i estimated to contain BUT and DDE up to 17 g/kg in extractable fat, the amount of unknown compounds also were suggested to be in the earns range, and tehn sufficiently high to do a run on the comblnod gas chromatograph - i mass spectrometer. If this oould 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 otc. This aethod 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. As this method for identification of totally unknown residues surely will bo very Important in the future (whon f.ex. a biologist has found that fishes in a river die) it mayb$oaelble by mean of this method to find out exactly what oompoundo are responsible for the death. For this reason, I will go into cone details with thlo method. In tho actual coco we took the extract from 20 mg eagle and coucontrutcd HONS 035765 o o 7. it ft much fts poeolble and made an injection on the gas chromatograph eombinod with the mass spectrometer. The result woe the chromatogram shown on tho next elide. Every tine the recorder ehowod that a ooapound is loaving tho oolumn, tho effluent is led to tho nase epeotromotor. No.* Just a fow words about the mass spec* Tho molecules leaving tho column are hooded with electrons *t M. Vo bavo now got tho molecule positive charged, but with tho same mass as before* ' This M+ Is aooolorated in a vacuum and will thon get a klnotio enorji. where la the apood. Host comos the magnetic field that . tries to bend the dlreotlon of the molecule. Thle will be big for a small molaoula and leas for If wa have a aieva in the other end we can directly read the molecular weight. Added to this parent molecule \t we will also get addition in formations, because of the fact that U* may not be etbble, a part of them will be broken down before they reach the sieve in the other ond. U tom " DM - OCIj Mass spectrograms from the different unknown peaks in the eagle eaaple ao shown* The mass numbers equal to the molecular weights of the unknowno could be read to 426,392, 358, 324. Astonishingly, the molecular diffe rences ware oonatantly 34 maea unite. This difference shows a faoilarlty In origin of the unknown. Row the fact is that chlorine exeists as a mixture of two Isotopee with atom weights 33 and 37 in proportion 75*25. If the molecule has one ohlorine, this will give two moleoule peaks, one for Cland one for C1J7. If there are two chlorine we hav the possibility of one with only C1j5 one with both C1J5 and 37 and one with 2 and therefore MGNS 035766 oo Tho rolatlon of tho poako found on tho dlfforont masa spec woroi Uolecular weight Ohlorino contont 324 3 358 6 392 7 426 a An explanation of the familiarity of the oompoundo con be glvon if one eubotanoe le built from the former by substituting e hydrogen with ohlorino JtH RC1 HC1 *+ 54 Then it le poeelble to calculate tho molecular weight of the parent hydrocarbon IHC. UpjjQ M - x UQy + * Mg , where M le the noleoulor weight of the oomponoat j having x chlorine atone* P.ex. for m - 426 and 6 Cl we will get . 426 - 280 + 0 - 154 and equal with the other molokyle. The noat probable formula with carbon and hydrogen giving this noleoulor woight le Cjg H10 and thie can only be satiofied when the paront-hydrocarbon le biphenyl, and the unknown bolng polychlorinated blphenyle. Thie explanation wae later fully verified by injection of a eynthetlc ISO on the mass speo. furthermore exteneive gee chromatographio Investigations proved that the ISC standard gave peaks with the oame retention time as the unknown peaka from the eea eagle. I With the method just described 2 suppose thot we have a new possibility to study the residues In the air because the pine needles can allways be We have had great difficult; in quantifying the FCB, but when getting a little more time it will be poeelble. We have i done a few calculations on a few species, and 2 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 tho nature, and with this method a new typo of doflling agents has been found to be present in nature, and a few experiment have shown where they may be found. I Vow thie method is going to be uaod in the firat hadn to estimate how the situation le in nature ae a whole, and in the other hand to find tho leaks throug whioh they find its way to nature. Soem maybe are prooont here today to get news about the leaks, and to then I want to oay cone back in a year. 1 HONS 035767 9. OO So much I think X can Boy again that the PCB hardly con como from agriculture. Aa suppoft for this euggeotlon I con soy that we have found PCB In oaglo foatharo from Bikorauceot from 1944, whoro hardly any chlorinated pesticides were used In agrioultrue. Ono moro thing that I find Important to say Is that In contrast to the meroury problem thla doeo pot soon to be a pure Suodish problom. I have just studied chromatograms takon from I*ondon air, and they clonrly oontaln PCB, and dr. Holdon bao told mo that bo aleo find them in hia floheamploo. But finally in waiting at moro rosults Xiahould llko to pointKSe morething. It ie provod that PCB pomoe to naturo, wo dont fanow now where they are usod, but they are vory pereletant to chemlcale and to fire. X think the poison Jury should try to etate that a content of pCB shall always be found In ao open declara tion. MONS 035768 MONS 035769 n n // K O O w vr o o f /'/ ./$ /* fl /2 P'P'dot 9 7 ' 6 3 / to 3 !o o PP'OOS Pit 9 HONS 035771 ft c> c to 13 ;i it O t*o> ?o tj f2 tt /ff /e 99 // 77 O 2 U> 6 6 9 o u> u *J *d X ls> / 3> 9 3 1 f DIELDRin j>'-ocr+ 7 o p-p'-Dbe 3 RLDRiM U/JDAH MOWS 035773 H n t l 73 ` nn a // // // o 9 6 V 2 / f 3 to 9 9 to / o.p'-oor y 9 j l / l ia/om/p X / jV J 7 lO+pp'-DOT. ftp. doo o otaoR/A/ Pp-Odf PL DA/A/ IWOK/SS * , u SCO SNOW S tiS C O SNOW 0^ '/ 0 p 0 0} 0 HONS 0 3 5 7 7 6 l- MQNS 0 3 5 7 7 7 MONS 0 3 5 7 7 8 MONS 0 3 5 7 7 9 /}<ro 7^0 /7/"- ^ H.-iU I 'Or */- 3a 2w *-ec^. .*r-ylt. 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