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'i'J 1 A 52 1 Tcxi~i W E / o rr/o <? $eHrtCH - PolVu HETH 11 !'-< - w. Ed CowkL Toxicol. Voi 6, |)p. 32'J- 3K. i'cir,amon I'ioss |%8. l`i :nu\i in Ciica: !: i i::i The Migration of Lead from X5oIyriiers in (ha Rat Gastro-intcstinpJ Tract J, C. Gage and M. H. Litchfield Imperial Chemical Industries Limited, Industrial Hygiene Research Aldcrlcy Park, Cheshire, England {Received 20 October 1967) Abstract--The migration of lead from four polymer formulations during their passage through the rat gastrointestinal tract has been investigated. Diets containing 1% or the formulations have been administered to groups of rats for 4-5 months and measurements have been made of the lead content in blood, bile and urine, and in liver, kidneys and bone. As a reference, simitar analyses have been made on groups of rats receiving diets containing 2, 6 or 20 ppm lead as lead nitrate. Lead is poorly absorbed from rat intestine; with the diet containing 20 ppm lead it is not possible to detect a significant increase of lead in blood, urine or liver. A significant rise has been observed in kidney and bone, however, and bile is probably the best index of lead absorp tion. The greatest migration of lead into the gut from the polymer formulations was observed with PVC containing 2-3% lead sulphate, where the amount was equivalent to about 3 mg lcad/g polymer. A more realistic assessment of the ingestion risk is obtained from the amount of lead migrating per unit surface area of polymer under these conditions; with the PVC formula tion this was in the region of 25/ig/cms.The surface area of this formulation ingested daily would have to exceed 40 cm2 before the daily addition of lead to the diet would exceed 1 mg, and this area would be much larger in the case of the other polymers. It is concluded that the risk of lead poisoning from ingestion of any of the four polymers is negligible. INTRODUCTION From time to time over a number ofyears there have been reports of cases of lead poison ing in children, thought to derive from the ingestion of dried flakes of lead-containing paints from treated woodwork (MoncriefF, Koumides, Clayton, Patrick, Renwick & Roberts, 1964). These observations, and the allegation that young children are more sensi tive to lead than are adults (Greengard, 1966), have led to the suggestion that a tighter legislative control should be exercised on the lead content of surface coatings and of other materials that may be chewed or sucked by children. An excessively stringent limit on the lead content of such materials would present tech nical difficulties, as lead compounds provide the basis of cheap and efficient pigments, stabilizers and polymerization catalysts. It is desirable that before any new legislation is drafted, an attempt should be made to ascertain whether current formulations constitute a real hazard. Four typical polymer formulations containing lead have been investigated (Table 1); polythene containing a lead carbonate pearlescent pigment, polypropylene con taining a lead chromate-molybdate pigment, PVC stabilized with tribnsic lead sulphate and rigid urethane foam catalysed with an organic lead salt. As a direct toxicological study of the incidence of lead poisoning in experimental animals dosed with these formulations was considered unlikely to provide usclul results, an invcLtgation of the migration of lead from these materials as they pass down the gastro-intestina! 329 9" -V*- w o c: ca 05 O BFG043S9 330 J. ( . (iAC.I ;i I1<l M. II. 11 K III II I Tilbk'. 1. Particle size anti lca/1 content of polymer formula!> '-icorporalc 'it! diem Polymer PVC Polypropylene Rigid urethane foam Particles (%) retained by mesh 36 52 72 45 31 12-5 56 32 Lead component Lead carbonate Tribasic lead sulphate Lead cliromnte- molvbdatc Lead 2-clhylhexoatc Lead content ---------- polymer (%) In diet (ppm) 04 40 2-3 230 05 007 50 7-5 tract was undertaken. In general, siudies on the migration of components from polymers involve the use of simulated solvents under conditions which arc likely to accelerate the extraction, but as it was not possible to devise with any confidence extracting solvents which would simulate the conditions existing in the various regions of the gastrointestinal tract, it was decided to study the migration directly by administering the materials in a finely divided form to the diet of experimental animals, and then investigating the amount of lead liberated during passage along the gut. Rats were used in this investigation and the polymer samples were finely divided and incorporated in the diet at a concentration of 1 %. This was regarded as the highest concen tration which would not affect the nutritive value or palatability of the diet. It is known that the bulk of lead in the diet is not absorbed but is excreted in the faeces (Kehoe, 1961a), and as it was not possible to devise an analytical method for faeces capable of differentiating between the lead contained within the polymer and that migrating from it, it was necessary to use an indirect approach to the problem. A proportion of the lead in the diet is absorbed into the circulation; part of this is rc-excretcd into the gut tf ;h the bile re is stored in tissues and the remainder is excreted in the urine (Cantarow frumper, IV. ,;.Theamount of lead in the urine, while not a direct measure of the amount of lead migrating from the ingested polymer, should enable this to be estimated, if it is compared with the urinary excretion of lead by rats on diets with known amounts of added lead. This approach has been followed in this investigation, but as it is difficult to avoid contamination of urine by lead in the diet or faeces, more reliance has been placed on the analysis of tissues and bile in the comparison of groups of rats recebhng the polymers or lead in their diets. EXPERIMENTAL Preparation ofdiets. The four'polyrr.ers investigated were supplied by Imperial Chemical Industries Ltd., Dyestuffs and Plastics Divisions. They were finely ground and incorporated at a level of 1 % into standard rat diet, which, after the addition of 12% malt extract and 2% maize oil, was formed into pellets (Clark. McElligott & Weston Hurst, 1966). The lead con tents of the samples and of the diets are shown in Table 1. Three further diets were prepared by incorporating lead nitrate into the standard diet to give lead concentrations of 2, 6 and 20 ppm above the lead content of the control diet, which was found by the analytical pro cedure described l_low to have an n\-- ~e value of 1-5 ppm. This method was also used to check the lead content of the diets at intervals throughout the experiment. Animal experiments. Young male albino nits of the Aldcrfcy Park SPF strain, initial body weight 105-120 g, were used in these experiments. The diets containing lead nitrate mkiua'i ion or u-:ai; t xo.y, roi vunv-. 33; and the materials under investigation were fed for 4-5 months In gi t- o-r A; r.'is, -nd the same time a group of control rats was maintained on a norma! diet. At iL- mci f of (he experiment several control nils were killed, and the livers, kidney:,, bore (icmii:) and blood were taken Loin each animal for ana.Iysis. Similar measurements were made on a sample of the test and control animals killed at intervals throughout the duration of lire experiment. At the end of the experiment a pooled sample of 24-hr urine was collected from five rats on each diet, and from another pair of rats in each group a 24-hr sample of bile was taken by means of a polythene cannula exteriorized at the back of the neck. The weight of the rats was measured at weekly intervals throughout the experiment, and the food intake of the groups was also recorded. The determination of lead. The recommendations of the Analytical Methods Committee of the Society for Analytical Chemistry (I960) were followed for the destruction of tissues; the method of Browett & Moss (1965) was used for the oxidation of urine and bile. Liver, kidney and bone were ashed overnight at 500C, magnesium nitrate being used as an aid for the destruction of liver and kidney tissue. Blood was oxidized by a mixture of nitric and perchloric acids (3:2, by vol.). Diets other than that containing PVC were ashed by the method used for bone. With the PVC diet this procedure involved large losses of lead, but satisfactory results were obtained by oxidizing with a mixture of sulphuric, nitric and perchloric acids (2:1:1, by vol.). Lead was determined colorimetrically by the dithizone procedure (method A) described by the Analytical Methods Committee of the Society for Analytical Chemistry (1959). Checks for bismuth were made throughout the study and in no case was any interference detected. The smallest amount oflead which could be determined with reasonable precision was 0-3 pg, corresponding to an optical density of 0 01 unit. The amounts of samples taken for analysis and the limit of detection of lead in each are shown in Table 2. Table 2. Sensitivity of analytical method for lead in tissues, body fluids and diets Sample Kidney Liver Bone Blood Bile Urine Diet Amount taken . for analysis 2g 10 g 1-6 g 2 ml 20 ml 20 mi I8 Lower limit of lead detection C"g/g or /<g/ml) 015 0-03 0-20 015 0-015 0015 0-3 RESULTS The concentration oflead found in the tissues, urine and bile of the test and control rats throughout the experimental period are shown in Tables 3-S. The values for bone, kidneys and liver (Tables 3, 4 & 5) were subjected to a statistical analysis, The standard deviation of the control values was estimated by an analysis of replicate determinations, and the signifi cance ofthe differences between test and control measurements was assessed, using a control 20658002 A390 V>VG .1.12 j. c\ CAf.r. mid m. ii. UTCiiitHf.D sample taken at (lie same time as the test sample. Sip,, .ant dilYc. ~es (P <0-05) are marked in the tables with an asterisk. 'Hie significance of the differences between the test values and the overall control mean was also calculated, and values with PcO-OS are italiebed in the tables. This latter procedure does not take into consideration any systematic fluctuations in the control values, but it overcomes the error involved in having the control menu based on very few animals. On both the test and control diets the rats remained in good condition and no significant differences were observed between the different groups in respect of growth and food intake. DISCUSSION The absorption of leadfrom diets containing lead nitrate The figures in Table 6 for the urinary excretion oflead indicate that there is no significant difference between (lie control group of rats and those receiving the diet containing 20 ppm lead. This suggests that the additional lead in the diet either is not being absorbed, or, if absorbed, is being re-excreted into the gut. Urinary excretion cannot, therefore, be used as an index oflead released into the gut. The concentration oflead in the blood (Table 7) and liver (Table 5) also appears to be un affected by the concentration oflead in the diet or the duration of the experiment, although the figures for blood are near the limit of the method and are not very reliable. With bone (Table 3), there is an indication of an increased lead content from the lead-containing diets, though on account of the rather high standard deviation this only becomes definite with the 20 ppm diet. There is no evidence of an accumulation of lead in bone after the first week. Significantly high lead concentrations are also seen in the kidneys from animals on the 20 ppm diet (Table 4), and here there is a tendency for the figures to fall as the experiment proceeds, possibly due to a greater food intake/kg body v ' t with tlv mger animals and to the absence of accumulation of lead in the kidneys. The concentration of lead in bile, although lower than in blood, can be determined with more precision as the available volume is greater (Table 8). The 6 ppm diet produces an evident increase in biliary excretion oflead compared with the control, and the bile provides the roost sensitive index oflead absorption. The analytical results give an indication of the fate of dietary lead in the rat, at an average daily food consumption of 20 g. On the control diet the daily intake of lead was approxi mately 30 ng, and of this about 2% was found in urine and about 1-6% in bile. As there appears to have been no accumulation in the tissues, the remainder may be presumed to have been excreted in the faeces. With the diet containing 20 ppm added lead, giving a total daily intake of about 430 pg, the figures for urine and bile were about 0-1 and 0-2 % respectively. These figures suggest that the absorption oflead from the gut is very limited in the rat, and as it is not proportional to the dietary concentration, absorption may be due to a mechanism other than passive diffusion. The low blood concentration maintained is evidence against a higher absorption masked by re-excrction into the gut by a route other than the bile, and it may be deduced that absorption of lead in the rat is less than in man, where gut absorption is stated to be in the region of 10% (Kehoe, 1961b). The absorption of leadfrom polymers Polythene. . There is a significant increase in the lead content of bone towards the end of the experiment, but no indication of anv increase in the kidneys. The liver shows an early a 20658003 MIliitAHON or U!AI l-KO.M HJI.YM.'.KS fi T\ - 66oi r; o V s 8: S% s j ! 3 I f- oS66 s?ss o666 9 1 8 S ssss o6 oo 33 I1 S3 66 8B3 666 3 3"S3 o OS?O?OSCB 5333 S 'o O OoO 83SSS35 ooooioo 333 666 I (0-30 //y/a 02), SD 0-144 S ByG0439t 331 lor 3. c. CiAC.i. ;im! m. 1'. LVinuw.t.n SSS 22 = 8 oo6o SSBS -2=2 o 666 ! v 856 1 1 6 6 6 % s, s ssss sis! 3S ! ss h2 I 66 33S 8s2 I 1 I * I f !3 a I H s 8S3S 2 z <6 666 gsss o 666 SSSS3BS 2 a 2! ^ s s 6666 666 533 ^ XO C.666 2 I MIGRATION OJ I.l-Ai) F ROM I'OLYMJ-.K.S Table C. Lead content of mine of rats, fed diets containing lead !<"' /'/ .'. Diet Control With lead: 2 ppm 6 ppm 20 ppm With polythene With PVC Witli polypropylene With urethane foam Lead levels in terminal urine sam pics O'e/riit/2-1 1 >) 0-79 (14) 0-49(17) 0-27 03) 0-31 (15) 0-73 (20) 0-55 (7) 0-74(13) 0-47 (20) 015(11) 0-66(13) 0-36 (9) 0-42 (20) 0-31 (14) 0 71 (13) 0-26 (5) 0 07 (12) 0-39(13) 0 17(16) 0-61 05) 0-55 (18) 0-48 (JO) 0-47(19) 0 51 (13; 0-28 (6) 0-14(15) Each result is based on the pooled 24-hr urine from a group of five rats. Figures in parentheses indicate urine volume (ml/rat/24 hr). increase in lead content over the controls, and there is an appreciable excretion in bile at the end of the experiment. A comparison of these figures with those obtained from the diets containing lead salts, suggests that the lead released into the gut from the polythene corres ponds to a dietary concentration oflead between 6 and 20 ppm. PVC. There is a marked increase in the lead content of kidneys and bone, which appears at the beginning of the experiment and stays fairly constant. There is a slight initial increase in the content of the liver and an appreciable terminal increase in the biliary excretion. The figures suggest that the migration of lead from the PVC corresponds to rather more than 20 ppm in the diet, perhaps somewhere in the region of 30 ppm. Polypropylene and urethane. None of the figures in Tables 3-8 gives any indication of an increase in lead content over the controls. This is not surprising with the urethane diet, as if all the lead migrated from the polymer it would barely be possible to detect this by analysis of the organs or excreta. CONCLUSIONS These experiments have given some indication of the amount of lead which may be released from the polymers during their passage through the gut in a finely divided form, and these amounts are presented in Table 9 as lead/g polymer. It would not, however, be realistic to assume that this amount oflead would leave the polymer whatever the size of the piece ingested. The amount of lead migrating is clearly a function of the surface area of the polymer, and in assessing the possible hazard it is necessary to take this into consideration. The surface area obtained when 1 g of a solid is subdivided into spheres of diameter r is 3/rpy where p is the density. The approximate size analysis of the polymers incorporated in the diets (Table 1) leads to a conservative estimate of 120 cm2/g for their surface area; the actual area will certainly have been greater than this on account of the irregular shape ofthe particles. The last column in Table 9 indicates the amount oflead leaving 1 cm2 surface area of polymer during its passage through the gut. These figures provide a basis on which to assess the hazard arising from the ingestion of these materials, if it is assumed that the condi tions obtaining in the human gastro-intestinal tract are approximately the same as those which occur in the rat. It must also be emphasized that the results obtained apply only to the. particular lead compounds in the particular polymers used, and cannot necessarily be applied to other formulations. The observations that less lead is released from polypropy lene containing 0-5% lead as chromate than is released from the polythene containing 0-4% BFG04392 20558004 336 j. c. G/Uir :iik1 M. ii. i H'cui ii:U) 66 oo 5 >. U oCl >O. Values listed arc determinations on individual MIGKA'FJON (II- MAI) IkOM 1'Oi MILKS Table 8. Iawl content of i>i/< of tats fed dirts containin;: leadfor I'J ivk Diet Control With lead: 6 ppm 20 ppm With polythene With PVC With polypropylene With urethane foam Lead levels i n terminal bile sample-; (nt/'o/tnl) 29 (22) 49(13) 47 (22) 55 (20) 96 (20) 23 (23) 31 (16) 28(18) 43(14) 59(15) 42(16) 114(23) 35 (9) 20 (23) Values listed arc determinations on individual rats. Figures in parentheses indicate volume of bile collected (ml/rat/ 24 hr). Table 9. Extent of migration ofleadfrom polymers in the gut Polymer Polythene PVC Polypropylene Urethane foam Approx, equivalent dietary lead concn Q'g/g) 10 30 <6 <6 Degree of migration of lead from polymer (mg/g) 1 3 <0-6 <06 G'g/cnP) $ 25 <5 <5 lead as carbonate may indicate a difference in the properties of the lead salts or a difference in the polymers. According to the work of Kehoe (1964), the daily ingestion of 1 mg lead over the basic lead content of the diet for iong periods, does not lead to dangerous concentrations of lead in the tissues. With PVC, such a daily lead intake would require the daily ingestion of poly mer with at least 40 cm2 surface area, and with the other polymers a much greater area would be required. If ingestion does not occur every day, then the amount required on any one day would be correspondingly larger. As these polymers arc indigestible, and will not disintegrate in the gastrointestinal tract, it is necessary to decide whether an individual could daily masiicate enough material to produce a surface area of this order. It can easily be demonstrated that it is impossible by chewing to reduce these polymers to small pieces, with the possible exception of the urethane foam. This latter has, however, such a low lead content that it does not present a hazard. It must be concluded, therefore, that the risk of lead poisoning from these four formulations is negligible. Acknowledgements--Technical assistance in this investigation was provided by Mr. T. Green. We acknow ledge the assistance of Dr. D. G. Clark for the bile-canmilation preparations and of Mr. C. J. Clark (Pharma ceuticals Division) for the statistical analysis of the results. BFG04393 20S58005 cGAOHandM-H-UTCHHUUD 33$ REFERENCES AMlv'lical Methods Committee of the Socie-tf for Analytic.-,] Chemistry (1959). The determo,:,,;,,,, kad or the Society for Analytical Chemistry (I960). Methods for tire de-stroclion BroBTM|ilE<v"'&lMos" R (1965), Manual and scari-automatic methods for the tlstetmtnalioa of ti,lead lend P^ and (Vitheas Co., Mtimorc. n o 'MeEUiSflU.T.F.&Weston Hursl. E.(W66).lae tor.teitya' paraquat. Br.J. \fcd. 23, 126. rKrec-hnriy-aRrd aJ! ((119966611a).LTeahde pmoeistaobnoinligsminocfhlieldahdoiondm. Canliuinicahepacldthttrat.n5d,d2t6s9c.ase. 1. The normal nwiabolisn. of Keho* R A oiShTlK mcllboilsm ofiead in man in health and disease. I.The metabolism of lead under SJieff; A. A9, Kmunirics, O P., Clayton B. P A. Rehsv.clt, A. C. G. & Roberts.. Cr. E. (1964). Lead poisoning in children. Arch Dis. Chilah. 39, 1. --->3 nrm-enan* des polymeres dans Ic tractus Rdoienffseturmetneet--aesdOmdnainnaissterleetuadtraiaedc]etaussmggroiagusprtareosnintd.teesratitnsra.dl.edu4 ara5i.mDoeiss aeliuov.nerausdewte,,r,m,.i_n_e_la. teneur en op,j.o.,m,,,,b, dans 1c sang, la bile ct t'urine, ainsi que dans le foie. !es reins et tes os. SignaJons a tiire de reference qufc des analyses similaires ont cte effectuecs sur des groupes de rats qui rcccvaicnt une nourriture comcnant 2, 6 ou 20 ppm de plomb, sous forme de nitrate de ptorob. Lc plomb o'est que faiblemeitt absorfce par Pimestin do rat. Avec une noumuwe conteuant 20 ppm de plomb, il n'est pas possible d'obscrvcr un nccroissemeni notable dc la tc-ncur en plomb du sang, de Purine, ou du foie. On a toutefois remarque une augmentation significative dans les reins et les os. Le mciJJcur temoiti de Fabsorption du plomb est probablement la bile. La phis forte migration du plomb en direction dc I'intcstin, ci partir de ces polymeres, a ete observer avec du PVC (chlorure de polyvinyle) contcnaot 2,3 %,de sulfate de plomb. Dans ce cas, la tenevn en plomb est ['equivalent d'environ 3 mg par gramme de poJ\n:cr^. Une evaluation pivis realiste du risque de passage du plomb est dednite, dans ces mamas conditions, de la quantite de ~ plomb par unite de surface du polymerc. Avec la formule du PVC, clle sc situe aux environs de 25 fig par cm2. U faudrait que J'ingesfion quotidiesw.e de cette formula depasse la surface equivalent dc 40 cm2 pour que I'addition dc plomb au regime ddpasse 1 mg'jour, Cette surface scrait beaucoup plus grande dans le cas des aulres polymeres. On en conclut que le risque d'empojsonncmcnt par !c plomb est ncgligcable cn cas d'ingestion de I'un quelconque des quatreDpeorlytmJbercerst.riii von Blei arts Pdymoren in den Venlmmngskanal der Ratfe Zusanimcnfussung--Der Obcrtritt von Blei aus vier Polymerprapar.iten uahrend ibres Durchgangs (lurch den' Vcrdauungskanal der Rattc v-urdc uettersudu. Flitter mjt 1% der Praparatc wuide. 4-5 Manatc lang an Gruppcn von Ratten verabreiebt und dec Blcigeiialt in BJut, Gallc und Urin sowic in Leber, Nicrcn und Knochcn bestimrm. Zum Vergleich warden entsprcclicndc Analyscn an Gruppcn von Ratten durchgcfuhrt, die 2, 6 order 20 ppm Blei aJs Blcirn'trrtt im Fuller crhicltcn. Blei wild vom Darm der ilmie schRcht resorbiert; bei cinem eaFFnlsuutstlBltpeekrrreimnmclhiiicttrna22dl00cimppAppnlmm;aulUTBydseleecriinc1isTs<tht\uccicsshnnciincc.hhBtt lmmeibdwgglliiirccdhbv,, ncciimnneeDwwaeersmseemn(it.nl.ieiehhiee,sZZ,.uu..nn.aa.--hhmmcc ddeess BRlieciiggeehhaallttss imn BJiliuurt,, Urin urnrd Leber fcstizussttceHllcen. EBin wcessccnnlblli'chcr Anstiicg wurdec jedoch in Nicrcn iumnd Knochcn beob'--a-e'Ubtt/e.*t, ,u,nndj dftiec Gallc is!t wahrschbcminlich der beste 'Index Jde'r BRliefiirrccssoorrppttiioon. Der grodsisstlc Cbcrtrilt von Blei in den Dann wordc bci Polymcrprliparaien gefimdcn, die aus PVC mit 2,3 y Blcisulfat bcstnndcn, so class de..r.e..n.. lB``clci iPgoclivamltcrcpiwraap:3 mg JJJci/g Polymer enuprach. Eire r..e..a..l.is..t.ischcre iF.:imnssccbl:i;iit/uiing docerr Gefah..v...d..e...r..U...l.c..i.tu..ifnabmc: wurdcc aauuss der mBfccimenge crhaltcn die unt c-r d'Uicg.s./c',n, pBfcedbiungguunngfiecn jc F-.inhbcit der Polymnseerry'o.-jfi-ielic ubertritt; jbjCc;i dent PVCPOrtaiispsaler.i4t 0lacgn\2dieuslKeTl>scfci ii2je5i),^cgl/)ccmd'2ie. Die On\lKw.A.rrf0biii:gcirahhbcccddviieoesntesBljeiiigabucshdeamufgFcunlolemrmJ emneynubPeFrsiptcairJaetns wiirdc, und dieve Flticiic n'.'irc im Fajj der andcrcn Polymcre noch grbsscr. Fs ergibt sich daraus derScblvvss,class das RisilcP tier Weivcrgiftung (lurch VcrabrcicJmng cincsjeden'der vicr Polymerc vcniachiiissigt werden kmnn. Fd Cosntci. Toxicol. Vol. 6, pp, 339-340. Fcrgamon Press 1968. Printed in Great Britain SHORT PAPER A Note oil the Semi-quantitative Estimation of Aflatoxin M, in Liquid Milk by Thin-layer Chromatography B. A. Roberts and Ruth Allcroft Central Veterinary Lcdr.-raior.- . }>f'wisiry of Agriculture, Fisheries and Food, New Haw, Wevbridge, Surrey, England { Received ]0 April 1968) Introduction It is now well established that cows fed rations containing significant amounts of afiatoxin Bs excrete in their milk a metabolite, .afiatoxin Ms, file toxicity of which is of the same order as that of aflatoxin B4 for ducklings (AHcroft &Carnaghan, 1963; van der Linde, Freas, de longh & Vies, 1964; delongh, Vies & van Pelt, 1964; Holzapfel, Steyn & Purchase, 1966; Allcroft & Rrd-prts, I96S). Methods for extracting this toxic metabolite from dried milk been in jr some time and have recently been compared by Purchase & Steyn (1967). 'ljitf only recorded method of which we are aware for assaying the aflatoxin content of liquid milk is that described by van der Linde et of. (1964). In this method, methanol is used for precipitation of protein and initial extraction of aflatoxin, followed by extraction with chloroform and examination of the combined extracts by thin-layer chromatography. In our hands this procedure has frequently resulted in incomplete precipitation of protein and formation of persistent emulsions, especially when milk samples had been kept in the refrigerator or deep-freeze for several days, or had been reconstituted from dried milk. Moreover, filtration of the methanol-precipitated protein was very slow; the completion of one assay could sometimes take 4S hr. By using acetone instead of methanol we found that a satisfactory precipitation of protein and extraction of aflatoxin could be obtained. The acctone-precipitntcd protein allowed rapid filtration, gave little trouble with emulsions and permitted the use of 100 ml samples of liquid milk instead of the 50 ml recommended by van der Linde et al. (1964). The use of the larger sample was desirable as we wished to deteci the lowest identifiable traces of aflatoxin M excreted in the milk We have found that the following procedure afTords a relatively simple and rapid means of screening samples of liquid milk for the presence of aflatoxin Mt. Method Precautions should be taken at all stages to avoid undue exposure to light. Extraction. Mix 100 ml liquid milk with 200 ml acetone in a 500 ml beaker and bring to boiling point on a water bath. Cool for about 15 min to allow the precipitate to settle, and filter through a )5-cm Whatman no. 41 filter paper. Wash the precipitate twice with 50-ml portions of 70% acetone, combine the filtrates and remove the acetone by evaporation under rcdnc*,4 pressure r steam bath until only about 100 ml aqueous extract remains. When cold isfer the . uc to a 250-ml separating funnel with 50 ml methanol and shake wilh two successive 100-ml portions of petroleum ether to remove the fat. Extract the aflatoxin 339 O % coe o O'. BFG04394