Document npbQ8L3o28zO2jqm45agOY5zX

Brussels, Belgium February 22, 1967 DOT Dr. K. Bmet Kelly St. Louis J.X. Buchanan, at Loui* V T J.K. Filer, Brussels ^';;'3CCpy^g^XD,V, Bardy, MCL " T^'Kugen* tfilde, 8t Louis Dear Dr. Kelly, I should like to refer to your letter dated the 10th of February and our telephone discussion of yesterday's date. I enclose a copy of SOren Jensen' a original paper which in fact was presented in English ~ Dr. Jensen apologises for the typing etc. but this copy is taken from his original paper which has not yet been re-typed end tidied up. I 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 areany small points outstanding, please let me know and I will try to get you further information. I am sending the only copy of Hr. 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. 1 would however emphasise the post script on page 3 of Ola Palm's letter in which he states Nr. Jensen has consented to send us the full copy of his paper on the understanding that this is used only internally within the Monsanto organisation. LEXOLDMONOOOQ34 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; AftOCLORS 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- v&rdskemmitt& - operating under Statens Naturvet-hoitapl.. Fcrskningsr&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 LEXOLDMONOOOQ35 *: i 3 t N G &. S T R A N O Mr. D. Wood - MCL Dr. Alan Holden - address fresh Water Flcheri.es Lciborai. ei',y, 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 mentioned 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, Sbren 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, h .-.A' HONS 090517 LEXOLDMONOOOQ36 rising *. strand Mr. D. Wood - MCL 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 090518 LEXOLDMONOOOQ37 Mr Chairman, ladies and ge> *ntl.o,Cnon, ' In honor to our Brittish goet I will try to hold this lecture in Bngliah* As tho titlo of this loeture states, I an today going to tell about tho dioeovory of some hithorto unobserved chlorinated hydrocarbons haring up to eight chlorine in the molooule and found'in reoiduo cnn- lyuio. Tha chonloftl nano of polychlorlnotod bifonyle ( In tho following oollod PCB). Io get fcnillar with PCB I will start with tho ohoniotry and Jbozloolbgi. ' - Ohoniotry `in The aln-oharabtorlstlo of PCB is 1. Their very high stability* As an example they eon be boiled with nitrin sold without being destroyed. 2, They aro h*dly netabollsed in living organism. 3. if more than 4 chlorine are^pre^eent they are non lnflamable. It is dear that these threo characteristics does it easy to understand that when they havo entorod the ^living organiso. the will have a low persistence . But it la difficult 'to explain how they find their way into the living organic?* Ono thing seeme to be clear, they don't ooao from agricultural use, but from a technical one and most probable it domes to tho nature via wastes that are tried to b# burnt up, because then we have them, at.onco in the air, beoauee of thalr non inflaaability. . . . .I^lClQEl "NS *90*8 * Tho PCB wero introduced in 1929 and ao early as 1936 Jonoe and Alden reported that 23 out of 24 man employed in manufacturing of PCB sufferod from an acno form eruption of the akin. Acne did not appear until 6 to 6 months after the matorlal was first usod. 1c 1937 Drinker reported that rats exposed to chlorinated biphenyls In concentration of approxi mately 1 mg/a? for 16 hours a day for 6 weeks chowdd damage of tho livor. After that time tho allowod concentration of PCB in air ie 0,5 ng/a3. (For DDt tha same value is 0,5 - 1 ng/a3). The somo authors finished thoir experimenta In 1938, and related that these compounds hove on lnjouriouo effect, manifested solely in the liver. Chlorinated bipohnylo appeared to.be the moat lnjouriouo chlorinated compounds of all tested. LEXOLDMONOOOQ38 Greenburg, Mayer and Smith 1939 reported that PCS and polychlorinated naphtalcnos are blamed for the death of three young workers,, mid that?* pregnant women and persona who have at any time had any livor dlecasos are particularly euspectible. t I Wedol, Hollor and Benton gave 1942 animals PCB including odminiotration by inhalation, ingostion and akin ebsorbtion. histological exoaination ef tho viscera showed important toxic street only in the skin and livor, and tho dogonoration effects in the liver are essentially the sane what- ovor was'tho mothod for the administration. Paribok (l955/ found no on occupational poison in the eleotrloal industry, mixed totra and posts ohlorobiphenyl cauoos folliculitis, oomodo, pyodermia and other okin affections, and that its principal toxic effect is fatty degeneration of the liver. ' Miller (1944) injected 69 mg PCB (4 and 3 ohlorine) subcontaneouoly In 32 gulnoa pigs. Sight to ten days after injection, fat droplets wero noted In the liver cells, and after 16 days they were present in modcroto or very large numbere. Rabbits and rats were also tested in this lnvooti- gatlon, as well as the PCB wae adminietated both continously, subcuntia- 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 laughlin 1964 reported a method to test the chemical toxicity and teratogenic effect by injection into the yolk Bac of fertile egge prior to lnoubatlon* PCB was found between the eight compounds among 100 tested having the highest order of toxicity. No hatch was found at a level of 23 mg ps egg. At a level of JO mg per egg, one chick hatched out of 20 Injected egge, but died 2 days later. Some embryos which were examined after they died, ehowed weak deformitlee (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 eobyos have showed exten give brain damage. Mercuric chloride showed no hatch even at a level of , 0,3 mg per egg. MQNS 049049 . As the analytical chemistry is a pronounced service science I have boen in contact with many scientists from other fields during the work with residue analysis, and X have always found this contact very stimulating for my own work. This co-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-dlvisione: . LEXOLDMON000039 77 I 1. Chemistry of PCB and their tocioology. 2. Analytical methods for residue analysis and proof of structures. 5* Behaviour of PCB in nature, differences in metabolising rate of the PCB components, potenoation in an ecological eerie, con centration levels and examples of samples which have boen 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 take away interferrlng substanoes, most often fate. e, 2. Identification analysis by moon of gas chromatography. Thlu- __ layer chromatography and mass epeotroaotry. J. Quontltatlvo analysis. 1. At an ecological laboratory in Blksmuadet in Stockholm 1-2 g of a samplo is cut 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. $ 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 eases 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 is added to the sampling tube, and the whole ls'hooogenieed with an intertable homogonizer. The resulting powder is transferred into a special Soxhlet extractor. After 4 hours of extraction the solvent is evaporated, leaving the fat in a small weighed test tube at the bottom *8ox.-tube) of the extractor. This fat is dissolved in methylene chloride In such a way that 100 ul (0,1 ml) contain 20 mg of fat. The 100 id solution is now transferred to a little object glass, 3 x 7 cm, covered with a si11cage1 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 tho bottom of which is covered by a few mm of methylene chloride, the solvent will be sucked up in the dry layer of Bilicagol, and at least reach the upper end of the plate. The fact is that the fat haB a greater affinity to the powder on the plate than the chlorinated hydrocarbon have. - and wo get a separation. The fat being more polar than the chlorinated (hydrocarbons will never go longer than 2 cm before thb MONS 049050 LEXOLDMONOOOQ4C / o 47 ziutioh tub $ a 6 a solvent roacboa the upper part of the glass. Tho front of the fat appoaro quito vioiblo against a lamp, and with tho old of a razor blado tho zone abovo tho fat is tranoferred to tho olutloa tubo and the ohlorlnatod biooldos absorbed on the powder can now bo oluted by one nl of other. The concentration is aufflciont for detection of tho ohlorlnatod hydrocarbons down to tho 10-12 g levol. Tho noxt stop In tho analytical procedure concerns tho separation of tho dlfforont ohlorlnatod hydrooorbons that tho sample may contain. Ao a nattor of foot, this is a troublesome task:. It Is easy to ostimato what 1b not prosont, but sore difficult to eay exactly on la present. Vo suffor from tho negative demonstration, as will bo shorn lator. . At first a fow words about tho separation of the components prsoont la tho sample and their visualization. , Tho separation is aooompllehod by mean of a gao chromatograph fitted to a dotoctor that transfers lto impulse to a recorder. o i i The system Is shortly .described: A spirally formed glass tube with an innor diameter of 2 mm and about 2 m in length is filled up by a support, eovored with an thin layer of an oil. The tube is hoated in the chromatograph to about 200. Through the tube a stream of nitrogen contlnously follows. When about 10 ul (1/100 of 1 ml) of the purified sample is injected Into the tube, the components of the sample will be evaporized and go forward through the column with the gas stream. As the constituents hare different affinity to the column filling thoy will pass the column with different spood and.lt will take different time for then to roach the dotoctor at tho other end of tho glass tube. 1$ tho temperature and the nitrogen flow are held constant this time, tho retention time, has a spoclflevaluc for a certain compound. This is true, but unfortunately it Is also a fact that two components oan have thO'S&mo retention time. This Is one of the bigger problems in gas ohromatographlc analysis of unknown samples, ao will soon be obvious. To make it possible to ostimato the retention time It Is necessary to visualize the chlorinated hydrocarbons. For that purpose more or loss 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 picogram ( 10 g of llndan). Unfortunately this detector is not specific for chlorine, bu gives answer alco for oxygencontalning cospounda. The response here le muc-: lower but can be counterbalanced If the concen tration of the oxygen containing is much higher. HONS 049051 Tho principle for tho electron capture detector is shortly: At the end of the gas chromatographic tube is placed a little tube con taining a foil of titanium tritido. This 1r An-radlant. The o- LEXOLDMONOOOQ41 particlea ora reacting with the nitrogen molecules coming from the column. Then we get + Ng -- " + Ng. Over the detector we 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 onv-mV rocordor aa a constant baseline. When now a chlorinated hydrocarbon Ioayoo tho column this compound hoc high affinity to the elootrono and this moans thnt 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 ae a peak on the recorder. The 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 aroa of an unknown component with the retention time and area of the known standard. As said before thiB detector is not speci fic for chlrlne but anyhow very useful, because of its high sensitivity. The systom described has, as we have seen, two disadvantages: 1. Two difforont compounds can have the name retention time and be detected as no peak. 2. A regiatrated peak doee not need to be chlorinated , because the detector ie 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 ie possible to concentrate the sample and analyse ,'it on a less sensitive detector uoh ae the mlerodoumetrlc one, which la specific for chlorine. The oompound la burned in a furnace and teh generated chlorine titrated directly. MONS 04 9052 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 a rather tiae-consumeing 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. U&py 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. If thlfl ramarlc in fftnnrt + T*n*. +h* wvbOiil + o n*f wnnw nwnwlftiis nnni-. LEXOLDMONOOOQ42 I. o I o ;o titabivo ana/;, As must be brought Into ques6\\ iinn.. In the present investi gation it is shown that nost of the unknown peak of chromatograms at reoidue analysis of chlorinated peoticides are due to polychlorinated biphenyls. X will show a chromatogram of human fat analysed on a so ca lled S? 96 column, the nost often used type in pesticide analyses. Early retention times were in agreement with DDE, EDI1op and EDTpp. Hoxt slide chows the same sample analysed on a QP-1 column. Now the former 2 DDT 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 components were at first thought to be metabolites of the insecticides. Against that spoke that neigther treatment nor conoontratod sulfuric acid in other. This treatmsnt made it rather sure that the compounds did not contain oxygen, In Swodon residues of organic nor cury have boen investigated rather intensively in the Swedish fauna. As those compounds give very high responses to the electron capture doteo- tor it wae 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,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 those belonged ' ' to the normal insectisldes. Therefore the unknown could hardly be mereu- riale or metabolites of them. MQNS 04905J As the eagle sample giving the chromatogram shown in fig. 10. could be estimated to contain EDT and EES up to 13 g/kg in extractable fat, the amount of unknown compounds also were suggested to be in tho 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 mclekular weight numbers of chlorine etc. This method is up to mow 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 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 LEXOLDMONOOOQ43 ** o ' cv o it ae much as possible and made an injection on the gaa chromatograph combined with the maso spectrometer. The result waB the chromatogram shown on the next olido. Every time the recorder showed that a compound ia leaving the oolumn, the effluent is led to the mass epeetromotor. Now juet a few words about the mass spec, '* The moleculoe leaving the column are bonded with electrons at K. Wo b&vo how got the molocule positive charged, but with the ease maso ao before. " This )i* is accolerated in a vacuum and will then gat a kinetic enorgi. - whero ia 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 o If we have a siove in the other end we can dlreotly 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. M DDi, M jjjjj, _ cc, Maee spectrograms from the different unknown peaks in the oaglo sample as shown. Tho mass numbers equal to the molecular weights of the unknowns could be read to 426,392, 338, 324. Astonishingly, the molecular diffe rence e were constantly 34 mass units. This difference shows a femilarity in origin of the unknown. Now the fact is that chlorine ezsiotc aa a mixture of two Isotopes with atom weights 35 and 37 in proportion 75525. If the molecule has one chlorine, this will give two molecule pc&ko, one for and one for Cl^. If there are two chlorine wo have the possibility of one with only one with both and 37 and one with 2 Cl^ and therefore MONS 0*9054 LEXOLDMONOOOQ44 r She relation of the peaks found on the different mass epee wore, Molecular weight . 324 358 392 426 Ohlorlno content 5 6 7B An explanation of the familiarity of the oompounds can he given if one eubstance is built from the former by substituting a hydrogen with chlorine . R?1 * HC1 M + 34 Ihon it is possib) to calculate the molecular weight of the parent hydrocarbon PHC. Mpjjg K - x + x Mjj , where M is the molecular weight of the component having x chlorine atoms. ?.ex. for m 426 and 8 Cl we will get . 426 - 260 + 0 - 154 and equal with the other molekylo. Iho moot probabls formula with carbon and hydrogen giving this moleoular weight la C^2 H^0 and 'this can only be satisfied when the parent-hydro carbon la biphony1, and the unknown being polychlorinated biphenyls. This explanation was later fully verified by injection of a synthetic PBC on the mass spec, Furthermore extensive gaa chromatographic investigations provod that the roc standard gave peaks with the same retention time as the unknown peaks from the sea eagle. * With the method just dssorlbcd 1 suppose that we have a naw possibility to study the residues in the air because the pine needles can nllways be We have had great difficult; in Quantifying the ?CB, but when getting a little more time it will be possible. We have dona a few calculations on a few species, and I suppose they are right within a factor 2. We have found the residue to be from a' It has been my statement here to-day to present this method for studies Of defiling of the nature, and with this method a naw typo of dofilin* agents has been found to be present In nature, and a few experiment have shown where they may be found. Now this method is going to be used in the first hadn to estimate how the situation is In nature sb 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 get news about the leaks, and to them 1 want to oay cone back in a year. MOMS 049055 LEXOLDMONOOOQ45 9 So much I think I can coy again that the PCB hardly can cone from agriculture. Aa support for thia suggeotion 1 can say that we have found PCB in eagle feathers from Rikeraueeot from 1944, where hardly any chlorinated poatioidea were used in agrlcultrue. One more thing that Z find important to eay la that in contrast to the mercury problem this does not ooom to bo a pure Swedish problem. I have just studied chromatograms takon from Xondoa air, and they cloorly contain PCB, and dr. Holden hun told mo that ho also find them in his fiahaamples. But finally in waiting at moro results Iiohould like to point8Se morething. It is proved that PCI eomos to n&turo, no dont know now where they are usod, but they are very persistant to chemicals and to fire. I think the poieon jury should try to state that a content of PCB shall always be found in an open declara tion. HONS 049056 / LEXOLDMONOOOQ46 (T CQ /. Mi Hurt 0/ l/HeJicic/e% and PC3. -frorn eayft ^ - .............. / *. O 2. Some sample &ifcr n< t trdien ., Ofl/y *PC& Mfnatn. B. pC3 - S /<3 nc!(ird* Cotuv.3f7: Q.FI 6 % on ^ f i *V <j&$cJirQrn ? too-flo tfifiih c*botioJ, fej*nttn&loci. f3oc* in . a//jbs IA - &***<t3`cm/set. x.g : lo 3 O ml/rn in bd. C0* C&t. ie/np'd* o ./\ 2 :\ l\ I\ A /1 /\ v,___ 1 iU 1n rvj 1 1i 1i . 1i 1 i1r 1i 1\ 1\ /3 /2 'y _____ V A s...y LEXOLDMONOOOQ47 LEXOLDMONOOOQ48 LEXOLDMONOOOQ49 &L QA/aj /A/0/)a 0906*0 SNOW i 3 9. 9. O 9. *0 Cum / $ } * SO *> ** $ SK <*. w *s Curve 2 ^ 5! * < Si N *N CuryQj C Ur^/r it 5* o 0 LEXOLDMON000050 HONS 0V?06l LEXOLDMONOOOQ51 N J//1/OA//7 Q -C-uatd CL <<W> *nM. ~C-ttrv4 4y 55 $ 5* -Cntr*c* O j 6-Uf~v <& o Ot *o >o v MONS 049062 LEXOLDMONOOOQ52 LEXOLDMONOOOQ53 LEXOLDMONOOOQ54 LEXOLDMONOOOQ55 LEXOLDMONOOOQ56 LEXOLDMONOOOQ57 LEXOLDMONOOOQ58 OS (S LEXOLDMONOOOQ59 I\, i 006*0 SNOW i I i LEXOLDMONOOOO6O Tm*0 SNOW LEXOLDMONOOOQ61 LEXOLDMONOOOQ62 ------- -- LEXOLDMONOOOQ63