Document xj6wjozgog6Ly7BO3B925ZO8J

Brussels, Belgium February 22, 1967 DM Or. R. Kswet Kelly St. Louis J.R. Buchanan, Bt Louie 'T s. . J,l. Filer* Brussels S' ;"s W^y?on\I>.V. Hardy, HCL J " /^"'"lugens Wilde* St Louis Pear Pr. Kelly* I should like to refer to your letter dated the 10th of February and our telephone discussion of yesterday's date. 1 enclose a copy of BOren Jensen's original paper Which in fact was presented in English - Pr. Jensen apologises for the typing etc. but this copy is taken from his original paper which has not yet been re-typed and tidied up. J asked our agent in Sweden to dig out the various pieces of information that you required and I attach a copy of his reply dated February 17th which X think fully covers the point raised in your letter. If there axeany small point* outstanding* please let me know and Z will try to get you further information. I am sanding the only copy of Mr. Jensen's paper to you and therefore if any of the other recipients of this letter needs to have access* they can take a copy from yourself. Z would however emphasise the post script on page 3 of Ola Palm's letter in which he states Mr. Jensen has consented to send us the full copy of his paper on the understanding that this is used only internally within the Monsanto organization. TOWOLDMONOOQ3200 RISING & STRAND OP/ao aktiebolao TELErOM; 3461 93 TELESRAM; HENRI* TELEX; 1424 roTOmo 3*443 kanhoiro 73-64*1 Stockholm va February 17, 1Qo7 SVEAVAGEN 47 Mr. David Wood Monsanto Europe BRUSSELS J Belgium Pear David, re; AROCLORS Reference is made to our telephone conversation ar.ci I have tried to dig out the information you asked for. Before going into any detail, however, I would like to refer to your letter to Mr. Soren Jensen , dated February 8. Mr. Jensen would be interested in samples of as many isomers as you can put at his disposal. The quantities cf course are very small indeed and he would need only about 10 mg of each. In theory you could put the lot behind a stamp and mail it to Mr. Sdren Jensen but in actual practice it is of course not as easy as that. Anyway, Mr. Jensen would be interested in both the lowchlorinated and the high-chlorinated types since this would help him to get a better picture of the metabolism. Now to the various points you raised. Enclosed you will find the original articles as published in "Dagens Nyheter" and "Svenska Dagbladet" on November 2;?, 'Jj6i. The people who organised the meeting wnere Mr. Jensen's paper was presented was a Committee - 190A &rs Natur- virdskcmmitt& - operating under Statens Naturvet-nsKapi.. Forskningsr&d (The Swedish Natural Science Hesearcr* Council). The Committee (for Conservation of Natural Resources of 196^) is working under the chairmansnip of a Dr. B. Lundholm. .. The meeting was organised under the auspices of Naturv&rdskommittfen and was called apparently only In order to give Mr. Stiren Jensen an opportunity to publish his findings. The timing was made so as to coincide with a visit of Dr. A. Holden from Scotland. HONS 090516 TOWOLDMONOOQ3201 *!`i a I N G &. S T R A N D Mr. D. Wgog - MCL Dr. Alan Holden - address fresh Water Fisheries Lciborai. cry, Pitlochry Pearcher, Scotland - is the coordinator for the twelve OECD countries, as far as the influence of biocides on Nature goes. Dr. Holden apparently is a specialist on such questions and particularly on fish. There is no intention that Dr. Holden, or the group of twelve OECD countries as such, should carry on any further investigations on the particular problem unearthed by Mr. Jensen. Any continuation will be carried on in Stockholm by Mr. Jensen or cooperating Swedish labora tories. The meeting of November 2J offered only one paper, i.e, that of Mr. Jensen. No decisions were made on any further action. The work done by Mr. Jensen was paid for by the abovementioned Committee for Conservation of Natural Resources. The further studies planned for on toxicology have not been started on yet since there is no money available. It is possible, however, that the same Committee of 196^ will be asked to subsidise even that work. If toxicological studies are to be made, these probably will be carried out at Karollnska Institutet, Division of Toxicology under Professor Bo Holmstedt. The full address of this institute is Kgl. Karollnska Institutet Avd. Toxikologi Solnavagen 1 Stockholm 60. Since no decision has been made on toxicological invest igations, obviously no information can be given as to the scope of the planned investigations. The LKB press release of January 10, 19uY mentionec tne research of twelve OECD countries. As was mentioned above, there is no question of any central investigations to be made under the auspices of the twelve countries. There is only the question of coordinated efforts by way of interchange of information. Enclosed you will find photo copies of the original paper of Mr, Sdren Jensen. We have taken this copy here and Mr. Jensen apologizes for the state of the paper which is his own typing. He has not yet had an oppor tunity to have it properly retyped for publication. I nope the above answers your questions. Yours sincere iy, ,i-i '< .VV.'-A' HONS 090517 TOWOLDMONOOQ3202 i.sing *. strand Mr. D. Wood - MCL +H..*OkAA P.S. David:For the sake of good order, I would like to mention that Mr. Sdren Jensen has sent us these papers with the understanding that they are only for internal use within the Monsanto organisation. Ola. MONS 0905 IB TOWOLDMONOOQ3203 ) Mr Chairman, ladies and gentlamon. In honor to our Brittieh goat I will try to hold this lecture in English. As tho titlo ot this loetura states, I are today going to tell about tho dlceovory of some hlthorto unobserved chlorinated hydrocarbono haring up to eight chlorine in the molooule and found'in reolduo analyclo. Tha chonlool nano of polychlorinatod bifonyle ( In the following oallod PCB). Io get fcnillar with PC I will start with the ohonietry and .toxloologl. * 9 fihowjetry h The reain-oharaoterletio of PCB is 1. Their very high stability* As on example they can be boiled with nitrin aoid without being destroyed. 2* They aro l)adly netaboliaed in living organism. 3. if more than 4 chlorine are t-pre^eent they are non lnflareable. It is dear that these threo characteristics does it easy to understand that when they havo ntorod the giving organism- the will have a low perDistance . But it is difficult 'to explain how they find their way into the living organic-re. Ono thing oeame to be clear, they don't one from egricultural uset but from a teohnioal one and reoet probable it domes to the nature via wastes that ars tried to be burnt up, because then we have then, at.onco in the air, because of their non inflaaabillty. . . . loxlcolod . . F* ' , Tho PC woro introduced in 1929 end ao early as 1936 Jones end Alden reported that 23 out of 24 non employed in manufacturing of PCB suffered from an acno fora eruption of the akin. Acne did not appear until 6 to 6 months after the notorial was first usod. In 1937 Drinker reported that rats exposed to chlorinated biphenyls In concentration of approxi mately 1 mg/m3 for 16 hours a day for 6 weeks chowdd damage of tho llvor. After that time tho aliowod concentration of PCB in air ie 0,5 ug/a3. (For DDt the saree value is 0,5 - 1 mg/a3). The somo authors finished ' their experiments In 1938, and related that these compounds hove an lnjouriouo effect, manifested solely in the liver. Chlorinated bipohnylo appeared to.be the moot lnjouriouo chlorinated compounds of all tested. TOWOLDMONOOQ3204 Greenburg, Mayer and Smith 1939 reported that PCB and polychlorinated nnphtalcnos are blamed for the death of three young workere, and thatT^ pregnant women and pereone who have at any time hud any liver diecaeoB are particularly euspectible. ( I Wedol, Bailor and Benton gave 1942 animals PCB including adminiotration by inhalation, ingostion and akin abaorbtion. HiBiological examination I of tho vieoera showed important toxic effect only in tho skin and livor, end tho dogonoration effects in the liver axe essentially the sane what- ovor wao'the method for the administration. Paribok (1955/ found ns on occupational poison in the eleotrloal Industry, mixed totra and po&ta ohlorobiphenyl eauoos folliculitis, oomodo, pyodermia and other okin affeotions, and that its principal toxic effect is fatty degeneration of the liver. ' Miller (1944) injected 69 mg PCB (4 and 5 ehlorine) eubcontoneoualy in 32 guinea pigs. Eight to ton days after injection, fat droplets wero noted in tho liver cells, and after 16 days they were present in modcroto or very largo numbers. Rabbits and rats were also tested in this lnvostlr gation, as wall ss the PCB was sdmlnlstated both continously, subcantla- ously or ingested in the food. In the feeding experiment Q guinea pigs received 2 doses of 69 mg of the chlorinated biphenyl 1 week apart. Death occurred in 11 to 29 days. Finally Me Z>aughlin 1964 reported a method to test the chemical toxicity and teratogenic effect by injection Into the yolk Bac of tortile eggs prior to incubation* PCB was found between the eight compounds among 100 tested having ths highest order of toxicity. No hatch was found at a level of 23 mg ps egg. At a level of 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 brek) and growth retardation. Lead acetate resulted as an example in no hatch . at a level of 1 mg per egg. Autopey of the dead eobyos have Bhowed exten elYe brain damage. Mercuric chloride showed no hatch even at a level of , 0,5 mg per egg. MOMS 049049 . As the analytical chemistry ie a pronounced service science I have boen in contact with many scientists from other fielde during the work with residue analysis, and I have always found this contact very stimulating for my own work* This oo-operation often demands that we are talking the same scientific language. Because 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 eub-dlvieione: < TOWOLDMONOOQ3205 1. Chemistry of PCB and their tocicology. Q 2* Analytical methods for residue analyais and proof of Btructurco. 3* Behaviour of PCB in nature, dlfferenoiee in metabolising t&to of the PCB components, potensation in an ecological eerie, con centration levels and examples of samples which have boon provod to con tain PCB* A residue analysis can be divided int ' 1, Extraction of the pesticides from the biological material, * followed by a careful cleanlng-up to tedee away interferring substanoes, most often fate* t> 2* Identification analysis by moen of gB chromatography. Thin- , layer chromatography and mass speotroaetry. 3* Quantitative analysis. . At an ecological laboratory in Biksmusiet in Stockholm 1-2 g of a samplo e is out out of the biological material and transferred into a weighed and oarefully cleaned test tube, and stored at -20 until analysis. Smaller O samples have been used, min. 3 mg of body fat, and with dry materials such as hair, feathera, pine needles 100 mg are sufficient to reach the deolred 10 ng/g level in residue analyais. In oases of water proofs 1 1. is used for reaching the 10 pg/g* level* B.l(homog) In order to facilitate complete extraction of the fatty materials from tho biological sample, the doulbe amount of finely powdered anhydrous ' magnesium sulphate ia added to the sampling tube, and ths whole is'homo genised with an intertable homogeniser* The resulting powder is transferred Into a special Soxhlet extractor. After 4 hours of extraction ths solvent is evaporated, leaving the fat in a email weighed test tube at the bottom 'fiox.-tube) of the extractor* This fat is dissolved in methylene chloride in such a way that 100 ul (o,1 ml) contain 20 mg of fat. The 100 ul solution is now transferred to a little object glass, 3 x 7 cm, covered with a silioagel layer 1 mm thick, in order to fora a line 0,7 cm from one end of the elide* Inserting this thin-layer plate into a vessel the bottom of which is covered by a few mm of methylene chloride, the solvent will be sucked up In the dry layer of silioagel, 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 than the chlorinated (hydrocarbons will never go longer than 2 cm before th& HONS 049050 TOWOLDMONOOQ3206 solvent roacbos the upper part of the class. Tho front of the fnt appoaro quito Yioiblo against a lamp, and with tho old of a razor blado tho zone abovo tho fat is transferred to tho olutloa tube and the ohlorlnatod biooldos absorbed on the powder can now bo oluted by one nl of other. The concentration is sufficient for detection of tho chlorinated hydrocarbons down to tho 10-12 g levol. Tho noxt stop in tho analytical procedure concerns tho separation of the dlfforont chlorinated hydrocarbons that the sample may contain. As a nattor of fact, this is a troublesome task:. It is easy to estimate what Is not prosost, hut more difficult to say exactly one Is present. We truffor from tho negative demonstration, as will be shorn later. . At first a few words about the separation of the components present la the sample and their visualization. , Tho separation is oooompllshod by mean of a gas chromatograph fitted to a dotoctor that transfers lte impulse to a recorder. The aybtom is shortly .described: A spirally formed glass tube with an innor diameter of 2 am and about 2 m in length is filled up by a support, covered with an thin layer of an oil. The tube is hoated in the chromatograph to about 200. Through the tube a stream of nitrogen costlnously follows. When about 10 ul (1/100 of 1 ml) of the purified sample la injected into the tube, the components of the sample will he evaporized and go forward through the column with the gas stream. As the coneiltuente have different affinity to the column filling they will pass the column with different apood and.lt will take different time for then to roach the dotoctor at tho othor end of tho glass tubs. If the temperature and the nitrogen flow are held constant this time, tho retention time, has a spoclfleualuc for a cortaln compound. Thio is true, but unfortunately it Is also a fact that two components con have the-same retention time. This la one of the bigger problems in gas ohromatographlc analysis of unknown samples, as will soon be obvious. To make it possible to estlmato the retention time it Is necessary to visualize the chlorinated hydrocarbons. For that purpose mors or lose specific detoetors are used. The detector most often used In posticido analysis Is the so called eloctron capture detector, which can detect down to one pieogram (* 10 g of lindan). Unfortunately thiB detector la not specific for chlorine, bu gives answer also for oxygencontalning compounds. The response here le muc-: lower but can be counterbalanced if the concen tration of the oxygen containing le much higher. MONS 049051 Tho principle for the electron capture detector is shortly: At the end of the gas chromatographic tube is placed a little tube con taining a foil HiArin of titanium tritido. This 1b an-rodlant. The o- TOWOLDMONOOQ3207 particles ore reacting with the nitrogen molecules coming from the column. Then we get + Ng -- " + Ng. Over the detector ne have a tenoion of 90 volt and by mean of the electrons wc will get a constant electrical eurront over the detector. This standing current is transferred to a onw-mV rocordor as a constant baseline. When now a chlorinated hydrocarbon lo&voo tho column thin compound has a high affinity to tho elootrono and this moans that the amount of elootrons will diminish, and they will diminish proportionally to the amount of ohlorine. The electrical eurront will also diminish and this is noted as a peak on the recorder. Tho area of tho peak will be proportional to the amount of substance in the sample. By moan of a standard injection it is now possible to compare the reten tion tlmo and the aroo of an unknown component with the retention time and area of the known standard. As said before thiB detector is not speci fic for cblrlne but anyhow very useful, because of its high sensitivity. The syetom described has, as we have seen, two disadvantages: 1. Two difforont compounds can have the same retention time and be detected as ono peak. 2. A registrated peak doee not need to be chlorinated , because the detector is not specific* If the sample 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 poseible to concentrate the sample and analyse,it on a less sensitive detector suoh as the mlerodoumetrlc one, which le specific for chlorine. The oompound is burned in a furnace and teh generated chlorine titrated directly. MONS 049052 As is seen from the two last mentioned possibilities it la anyhow possible to get a rather high degree of certainty in residue analysis, but it is a a rather tiae-oonsumeing work.When using this method just described, we vary ofton found that many chromatograms from residue analysis of most carefully purified samples still contain a large number of peaks. Uajay 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 ore no many unidentified that there also muct be an obvious rick of the . known penko to bo covered by unknown ones. If thlfl ramarlf in fftnnrt + T*n*. +h* r^otil + o n*f nun**-. TOWOLDMONOOQ3208 titabivo anaj^As oust be brought Into que>e&-'.\ \iinn.. In the present investi gation it is shown that nost of the unknown peak of chromatograms at residue analysis of chlorinated peotieldes are due to polychlorinated biphenyls. X will show a chromatogram of human fat analysed on a so called S? 96 column, the nost often used type in pesticide analyses. Early retention tinea were in agreement with DDE, LDTop and HDTpp, Hoxt slide ohowe the sane sample analysed on a QP-1 column. Now the former 2 PKT peaks have ' divided into 4 peaks,and two of them are still in agreement with DD2pp and op., tho two new wore unknown. Logically, those unknown oomponents were at first thought to be netabolltos of the insecticides. Against that spoke that neigther treatment nor conoontratod sulfuric acid in other. This treatment made it rather sure that the compounds did not contain oxygen. In Swodon residues of organic mer cury have boen investigated rather intensively in the Swedish fauna. Aa those compoundo give very high responses to the electron capture doteo- tor it was also investigated if the unknown peaks could have a mercuric origin. Xt was found that the water-ecologioal series had high residues of both mercury (Weotermark,Johnels) and the unknown ones, when the same indivi duals were analysed. Anyhow, the pheasant Buffering moat from mercury poisoning only contained low levels of electron capturing compounds and these belonged ' to the normal inaectialdea. Therefore the unknown could hardly be mercu rials or metabolites of them. H0NS 04905J As the eagle sample giving the chromatogram shown in fig. 10. could be estimated to contain HOT and DUE 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 dose sucoeoefiully 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. This method is up to now the method giving the highect 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 be very important in the future (when f.ex. a biologist has 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 this method. In the actual ease we took the extract from 20 mg eagle and concentrated TOWOLDMONOOQ3209  cy U o it aa much as poaalble and made an injection on the gaa chromatograph combined with the aaso spectrometer. The result waB the chromatogram shown on the next elide. Every tine the recorder showed that a compound is leaving the oolumn, the effluent is led to the mass epectromotor. Now juet a few words about the mas* spec. '* The moleculoe leaving the column are bonded with electrons at E. Vo b&vo how got the molocule positive charged, but with the same maoo ao before. This )i* is aceolorated in a vacuum and will then get a kinetic onergi. whero is 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 less for o If we have a sieve in the other end we can directly .read the molecular weight. Added to this parent molecule Id+ we will also got addition in formations, because of the fact that M+ may not be sthblo, a part of them will be broken down before they reach the sieve in the other end. ?.ex. U ^ M 2jj)j _ cci? Hass spectrograms from the different unknown peaks in the eaglo sample as shown. Tho mass numbers equal to the molecular weights of the unknowns could be read to 426,392, 358, 324. Astonishingly, the molecular diffe rences were constantly 34 mass units. This difference shows a femllarity in origin of the unknown. Now the fact is that chlorine ezsiolo as a mixture of two Isotopes with atom weights 35 and 37 in proportion 75s25. If the molecule has one chlorine, this will give two molecule pc tie, one for Cljj and one for Cl^. If there are two chlorine wo have the pooolbility of one with only one with both C1^ and 37 and one with 2 C1^ and therefore MONS 049054 TOWOLDMONOOQ3210 She relation of the peaks found on the different mass epee worai Molecular weight . 324 358 392 426 Ohlorlne content 5 6 76 An explanation of the familiarity of the compounds oan he given if one subetwice ie built from the former by substituting a hydrogen with ohlorlne KH M+ + 01, RC1 HC1 M++ 34 Thon it ie poeeib) to calculate the molecular weight of the parent hydrocarbon VHC. Mpj,c - K - x MC1 + x Mjj , where U la the molecular weight of the conponont having x chlorine atoms. P.ex. for m 426 and 8 Cl we will get Mpj.g . 426 - 280 + 6 m 154 and equal with the other molekylo. I ho moot probable formula with carbon and hydrogen giving this molecular weight 10 C12 *10 and 'this can only be satisfied when the parent-hydro carbon la blphonyl, and the unknown being polychlorinated biphenyls. This explanation was later fully verified by injection of a synthetic FBC on the mass epee, Furthermore extensive gas chromatographic investigations provod that the IQC standard gave peaks with the sane retention time as the unknown * peaks from the sea eagle. With the method just deooribed I suppose that we have a new possibility to study the residues in the air because the pine needles can allways bo We have had great difficult; in Quantifying the ?CB, but when getting a little more time it will be possible. We have done a few calculation# on a few species, and I suppose they are right within a factor 2. We have found the residue to be from It has been my statement hers to-day to present this method for studies Of defiling of the nBture, and with this method a new typo of dofiling agents has bsen found to be present in nature, and a few experiment have shown where they may be found, How this method is going to be used in the firct 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 are present here today to gat news about the leaks, and to them 1 want to say cose back in a year. MONS 049055 TOWOLDMONOOQ3211 So much Z think I can coy again that the PCB hardly can come from agriculture. Aa support for thia suggeotion 1 can say that we have found PCB in eagle feathers from Rikarauseet from 1944, where hardly any chlorinated postioides were used in ogrlcultrue. One more thing that 1 find important to say is that in contrast to the mercury problem this does not scorn to bo a pure Swedish problem. I have just studied chromatograms tedcon from Xondoa air, and they cloarly contain PCB, and dr. Holdon hun told mo that ho also find them in his fishsanples. But finally in waiting at more results Iiohould like to point)$e morething. It is proved that PCI eomos to naturo, no dont know now where they ore usod, but they are very persistant to chemicals and to fire. I think the poison jury should try to stats that a content of PCB shall always be found in an open declara tion. Q HONS 049056 / TOWOLDMONOOQ3212 .......... ................... ""'()................. .. . co /. Mi Hurt of tuseJicielet o,,d PC 3. 4ror^ e style JamfU 2.5Qr/>e SQmpIt A'Htr Kit * ^ {r&hen Only PC 3 Altnam. PC3-S fd ndarcf' Column'' OFl 6/i on ^<j&$ citron* ? ioo-tio meih C * ho?lcJt teji nttn&fccf. in i p. a//ybs$ At" /&cm/s?t. .$.'lo 3o ml/rnin $(/. CD. 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