Document B5DqRxeEV0a8GZBvkpjzxnqmL

Polychlorinated Biphenyls: Distribution and Storage in Body Fluids and Tissues of Sherman Rats1 Aim.ust Ouiu.iy, Vhu.yn W. lUmsi:, Mauy li. Cuim, IUumi W. Jknnincs, am) Hai.pii ji. Ij.vdkk Cluimhlrc Tuxirnlu^ij l .alunahny, }.m imnwcnlal Prolrriimi Anaict/, 4770 Hujard Hiul.wui/, C/tf/iiiMiv, (.303-JI An aiml\timi iiii-jImmI (nr ilclrnnmajinn nl (Amdur 1251) in blood, tninc, (rn s. ,uk! lisMii-s <i{ Sim man .straiit r.tl.x tl(cr pnilmjyrd dietary intake (100, f5CK). .mil 1000 ppm) is presented. The method imolws hnniojjenr/ation and e\tiaetion will) liev.me, elriinup on a silica yel mieioetdunm and analysis Itv elerh un-eaplme yas- |i(|iinl clmniinlnipaplvv. Itrem ery data arc shown at 0.1. 5. II, and 100 ppm. Mass spci tiusc.jpir t I uotetei islics <{ 1*('H in tissues arc ntv-en. Distributum, storage, and twcM'tifin rales dnrinu leeduril and alter discontiimanee nl PC'.W in the diet arc pie'anted. I'ulyclilniinatrd biphenyls (l'CBs) were first described in 1881, by Schmidt mil Shultz. ( 1 ). (mnuncrcinl production sens begun in 1930 (2). These compounds luive been :iml still me widely used its phislici/ers mid resins mid in chlorinnted nihlier (3. 3). They me known to enlimtee (he iiisccticidnl properties of lindane (5), 1)1)1'. and dieldrin (6). They may be used as stationary phases in ('as-liquid chiuiiuilugrnphy (7). Their widespread use may have n-siillul in contamination of the cco-system as snggi sled by many recent reports. Aroclor' and DOT were both used during World War II. In tD48. total organic chlorine analysis provided data for the first (Irtet initial ion nl DDT storage in man (8). Since this method has no specificity lor DDT. the total organic chlorine content would not preclude the presence of other oro.mii i himine-iuntaiiiing moieties. In terms of halide specificitv, the Doliimmin miciocoulometrie gas chromatographic method for detecting pesticides was well established by I960 (9), and electron-capture was in use by 1961 (10), Unbuilt (II) first observed extraneous peaks in 1965; lie used a potcutUnnetric mode ol analysis lor total chlorine and found disagreement between this method and total chlorine content calculated from gas chromatographic results in which Vrgnn ionization and electron-capture were used for the analysis of tissues and `Tlir Monsanto (aimpmiv mmmfaetmes PCtis chiefly in tin- United States under die iriaie name of Aim lor. They me iiimaif.alined in I'rmico liy I'rmletee, and in (iernimiy liy llayer, with trade names, I'lienuehlm and (hilphen, respeetively. 'these enmpmmds me designated liy mimliers. The lirsl tun dibits represent the molecular ly|>e: 12, ehlormaled Ilipheip ts; 25 mid t I, blends nf ehlminnleil biphenyls and chloimated leiphenyls (75V biphenyl amt fi()V biphenyl, lespeelisely ); 51, chlorinated terphenyls. The last Ian dimls jjiee tin* \eeirdtl pereenl nt ehhnine. Tims, Aiuelor 1251 and I2fi0, with whieh tliis jv,i|>er is enneerned, are eblnvinnled biphenyls emitamini' 5-1 and (it)V eldtn ine, respectively. `0 |t)72 hy Academic Press, Ine. 481 DSW 026031 STLCOPCB4009993 482 cum.r.v l.r ai.. eggs of wild birds. The samples .showed .significant amounts of unknown clccti im-captm mg compounds. Holnirn postulated that these were metabolic inter mediates ol one or more ol the organoi hlorine pesticides. The differences could also have heeo indicative ol PCB contamination. It was nol until I960' that S. Jen sen (12) usin'.' eleetron-eapture and mass spectrometry was able to identity as PCBs unknown peaks found in the analysis of fish ami a bird carcass. Undoubtedlv, the time Ini' between initiation use and detection of PCB in the en vironment is a matter of accumulative concentration and/or instnunentatiun development. The presence of PC IBs in environmental samples has been well established (!:}, U). Acute and subacute feeding studies to determine toxicities and some residue levels have been done with rats, birds, and guinea pigs ( 15-18, 22). Tol erance studies have been done with fish and other forms ol marine life ( 19, 21, 22). Pathological changes in the liver and other organs have been shown in guinea pigs, rats, and rabbits at high concentrations (22). I Ivdropcricardium and growth depression proportional to dietary levels ol the toxicant have been pro duced in the chicken (23). Polychlorinated biphenyls are known to alfect estrogen levels in birds which in turn reduces calcium reserves and results in thin eggshells. Chemical porphyrin from oral administration has also been shown in chicks ( IS). This paper presents distribution, storage, and excretion rates of Aroelor in cer tain tissues, body fluids, and excreta of rats following (1) a single oral dose. (2) repeated dietary intake, and (3) after discontinuance of PCB in the diet. METHODS Aroelor 1254 and 1260 were supplied by the Monsanto Chemical Company with respective lot numbers, AK-38 and AK-3. Dietary formulations were prepared according to the methods described by Gabies and Kimbrough (24). Isxrr.niMKNT A Aroelor 1254 and Aroelor 1260 were administered at IG(X) mg/kg anil 3200 mg/kg, respectively, as a single oral dose, by stomach tube to three female. 173to 18.3-da\-old. white Sherman rats weighing 260-318 g. I'our control rats, two in each group, were given peanut oil only. Twenty-four hours later the animals were killed, and blood (oxalated), tat, muscle (abdominal), liver, kidney, lung, and brain were taken lor analysis. If.VelUlIMKNT B Aroelor 12.54 at 1000 ppm was fell lor 98 days to 29- to 34-day-old Sherman strain male and leniale weanling rats, in groups of 10. Controls were led only plain chow. I'ood consumption was measured at intervals as described prcvinuslv Iw Kimbnmgh and Caines (4). All rats were weighed on a weekly basis hum experiment initiation until time of sacrifice or death. The rats consumed an aver age of 72 7 mg/ kg / Aroelor/da v. Blood (oxalated ), lal, muscle ( abdominal). liver, kiduev. lung, and brain were taken from each rat at time ol sacrifice. Tissues were preserved in 10V Immalin and held for anulys s. DSW 026032 Shermai chow nr i days. I'url rccoien | of 8 days, viving rat rats were aver a 16 collect ionkidney, ai feces wer plasma n maii/fd filed on I diet, two Sherma dietary ic initially ; measureii anil 36.4 pies w ere Pol veh higlilv li| this prop Plasma in the sti No signil with the Samp], homngi a Bram trai ts wi Urine to a voh pies wei The In x with 1.5 I'eies pul verw on an m STLCOPCB4009994 I'oi.vcm.rv.uY v r n mi'ii '.vyls 4S3 12xei.ruMi:Nr C Sherman strain male weanling jats (61- to 66-days-old } war fed n diet of plain chow or chow fortified witli Aroelor 1254 at ;i concentration of 100 ppm for 58 days. Fortified clrovv was discontinued, and samples were collected during the recovery period. Food consumption was measured at 2-day intervals for a period at S days, alter -12 days loud consumption was measured daily for 4 days. Sur viving rats were weighed once a week. At various times during tlic experiment rats uere ind vidually housed in metahol sm cages. Urine and fetes were collected over a 16-hour period during which time plain chow was foil. Following excreta collections, rats were sacrificed. Blond (uxalatcd), fat, muscle (abdominal), liver, kidney, and hrain w< re taken from each rat at time of .sacrifice. Tissues, m ine, and feces were frozen at time of collection. Whole hlood was centrifuged and the plasma refrigerated. The outline of the experiment as aforementioned is sum marized as follows: These annuals (two on the diet and true control) were sacri ficed cm Day 4, 9, 13, 17, 25, T4, 42, and 58. After discontinuance of PCB in the diet, two animals were sacrificed on Day 8. 16. 24. and 71. Fxi'kiomi-'nt.s D AM) 12 Sherman strain male weanlings (36- to 41-days-old) were fed Aroelor 1254 at dietary levels of 100 ppm or 5(H) ppm for 252 days. Body weights were determined initially and weekly thereafter for 121 days. On the basis of food consumption measurements, the average rates for intake of Aroelor 1254 were 6.8 tng/kg/day and 30.4 mg/kg/day for 100-ppm and 500-ppm dietary levels, respectively. Sam ples ucre collected in the same manner as Experiment C. Samclk Fiuu'akatiox Polychlorinated biphenyls like DDT, arc not water soluble but are, in fact, highly lipid soluble. Solvents for tissues and plasma extractions were selected for this property. Plasma ( at least 2 ml) was extracted by the method of Dale el til. (25). Later in the study, the 111 vant-Thmnpson modification of this method was used (26). Sti significant dillcrcncc has Ix'cn found between these methods in tin's laboratory with the exception that convenience favors the Hryant-Thompson modification. Samples ol fat (250 mg), liver, muscle, kidney, and lungs (500 mg each) were la.... igciiizcd two times in 4 ml of hexane, and the extracts were combined. Hrain (500 mg) was homogenized two times in 4 ml of acetone, and the ex tracts were eomhhred (27). Urine (mean volume of 10 ml) at pH < 7 (using pllydriun paper) was diluted to a volume ol 25 ml by the addition of water and 1 ml isopropanol. These sam ples were extracted in 125-mi separatory funnels three times with 10 ml hexane. The lu xaiic extracts were combined, washed once with 3 ml of water, and dried with 1.5 g of anhydrous sodium sulfate. Feces were desiccated over calcium chloride for 1 week. One gram was then pulverized in a mortar and pestle and extracted for 1 hour with 25 nil of act tone on an automatic shaker. The acetone extract was decanted ami placed in a 50-ml 484 cunuiY m al. ft!itiilugc till<. l';iitifiilatf matter in tlie extract was removed by centrifuging and/or decanting. The extracts were evaporated witii a .stream of dry nitrogen to a volume of 0.5 ml in a water bath (40). The acetone extracts were evaporated just to dryness and made to a volume of 0.5 ml in hexane. The samples were dried with anhydrous sodium sidfate and held for cleanup anti analysis I))- election capture gas-liquid chromatography. I'nuPAiiATioN ok Silica Cli. ami Mh.ho-Coi.umn Silica gel (27) from Woclm (Activity grade 1) was activated at 130 for 4 hours and desiccated over calcium chloride tor IB hours before deactivation at :]`i (v/w) bv the addition of water. The silica gel was allowed to equilibrate at least 2 hours before use. After comparing the weight ol the silica gel belore and alter activation, it was found to be activity grade ! as specified by the manu facturer and (urthcr activation was unnecessary; however, each new batch was routinely activated. One gram ol the deactivated silica gel was placed in a disposable pipet, which had been loosely plugged with silanized glass wool, and gently seated. Columns were prewashed with 10 ml of hexane. Each sample was placed on the column in a volume of 0.5 ml. At least 1 ml of hexane was used to quantitatively trnn.sler the sample to the head of the column. The column was eluted with 10 ml ol a J:1 benzene:hexane mixture (27). Samples were chromatographed in sets of eight with duplicate column blanks. Aroclor 1254 standards were eluted1.simul taneously in order to ascertain the variance in silica gel activity and to detect anv possible contamination from solvents, glassware, or silica gel. (.'as (.'iiiioma IOCltAI'llV Sampler from experiments, A, C, D, and E as treated above were analyzed by electron-capture gas-liquid chromatography under one of the following condi tions using a MicroTck MT-220 gas chromatograph: pulse Mode (using dual columns and dual detectors); cell voltage, 36.5 soils: pulse width. 5,5 / see, pulse rate. 120 ftsec; deteelor, 'll, parallel plats- design, No. 1 205, No. 2 I (OS"; column. 6' X o.d. U-shaped pvrex glass packed with 5/ OY 210 on SO/100 nu-sli Gluomosorb \V, high performance, 170"; inlet 23,S"; carrier gas, argon-methane (95-5/), .50 psi; flow-through Detector No. 1, 50 cc/minotc: Deteelor No 2, B0 cc/ininnto. The DC mode was as follows; cell voltage. 15 volts; detector, II, par allel plate design. 210; Column, 6' X )i" o.d. U-shaped pyrex glass packed with 1.57 OV-17/1.95'/ QF-1 on 100/200 mesh Chromusorb W, HP, 200"; inlet, 230 . carrier gas, n-tiogeo, 40 psi; llow-through defector, 75 cc/minutc. Samples from Experiment H were injected directly a.s hexane extracts ami analyzed by electron-capture gas-liquid chromatography under the following 'conditions using a MicroTck MT 220 gas c-hminalugi aph. cell voltage, -1(1 \ulK DC; detector, '-'Ni, 250; column, 6' X K" o.d. U-shaped glass column packed with 1.5/ OV-17/1.95/ QF-1 on KK1/200 mesh Chromosorb \V, 1IP20()0C; iulel 210; carrier gas, nitrogen, 40 psi; (low-through detector, SO er/miuute, 20 cc/mm utc scavenger plus 60 cc/minutc currier gas (low. DSW Q26034 I'OI-YCUl.OlUNATen till'I HAY US 4S5 Uk<:ovi:iiy S'i'i;i)ii;s llccuvcrv studies were completed using (lie billowing methods Inr control tis sues, plasma, iind hS-lioor urine and leccs collections. The furlilitution level lor tucli type of siunple wis (lie me:iii concentration, with (lie exception of hit, of Arodor 12-5-4 found for that siimple in Experiment C. Fat samples were fortified will] 100 ppm. Tissues (f;it, liter, kidney, lrruiu, and muscle): Aroclor was added directly to llie hexane extract prior to licpiid chromatography. Plasma: Aroclor was added directly to fresh plasma, thoroughly mixed using ,i mini-mixer, and allowed to equilibrate overnight under refrigeration. Samples ivere then treated as mentioned in methods. I'Yees: Aroclor was added directly to pulverized dry feces and treated as men tioned in methods. Urine (three slightly dillerent approaches were used): \o. ]--Aroclor was added to I ml of isopropanol, then mixed with a solution of mine and water (10 nil + In ml) and analyzed as mentioned above; No. 2-- Aroclor was added to a hexane extract of 10 ml of urine which had been prepared .is mentioned above; No. 3--L'rine (5 ml) was added to a known quantity of \roclnr in a 15-m) ceiilriluge lube, mixed on a niiniinixer, and allowed to equili brate at loom temperature for Ifi hours. Urine was extracted three times with > ml ul hexane; the hexane extracts were combined and evaporated to 0.5 ml. IIIMU'S' A.\D DIX'CrSSlON limit nely. this laboratory operates dual channel solid state electrometers at a sensitivity ol 4 X 10 amps lull stale which permits the detection of ]() to 30 |iieogvams of the more common chlorinated hydrocarbons. There was a .significant ilillerenee between the response characteristics of tilt' Aroelors as compared to the chlorinated hydrocarbons. To achieve a representative gas chromatographic trace, at least 350 anti 100 pg of Aroclor 125-1 anti Aroclor 1260 were required. A gas chromatogram illustrating the tlidcrenee between the electron-capture re sponse characteristics ol these compounds as compared to chlorinated hydrocar bons is shown in Fig. !. lsxeeitisMA'T A Data resulting from electron-capture analysis of plasma and tissues in lixperiinrnl A are presented in Table I. (Quantitation is an approximation. In all cases, the peak heights (in nun) of all I'Cill components were summed for the respec tive standard at a gisen picogram quantity injected. A response factor was deter mined lor each standard in pg/uim. For each sample, a summation was made of the total response (in nun), and the product of this times tile response factor cal culated to give picograms ol FOB or PCB-drrivcd material in the sample. When the gas chromatograms for the samples were compared with those of the Aroclor Uandard, there were no recurrences ol the general Aroclor pattern. Some peaks had completely disappeared. This was also true lor each of the succeeding studies. Although the dosage level ol Aroclor 1260 was twice that of Aroclor 1254, the OSW 026035 486 CUULliY i-:t At.. n a ft S' tl I'm. 1. c:s cliromalngraim for Aroclor I25-J ami chlorinated hydrocarbon standards. (A plof of detector response vs time.) TA1U.I', I DisTinntiTioN os Aitouums 24-iioUiis akti.ii Dual. 1\oi:stion ity Stomach Tune Aroclor 12.*i4 (tiosttxe. level--MiOt) nif',k(') Avnvluv 12t>0 lcvt`1 -- iJ'JOU n>n kjj) 1'lnsnm Mean SK Hiuin Mean SK Km Mean SK Liver A lean SK Kidney Mean Sis l-iiiiI M<\'U si-; Muscle Menu SK 24(Kl s (IS MS.(Ml :i(i 41 1140 <1 .-,71 r, J 4 I . 20 47 SO 274.IW till 17 (i:-) . 1P I jii as SO20 07 As u 70 <U*0 u: 17 2:! AO !i:to u -121i (i *j:uv l I Mi :i2s i i4 o lOfj 17 21 i:: :(7 on 22 07 <1 tl 3 A 3 c a ft T P A DSW 026036 I i STLCOPCB4009998 l,(>I.YCIM.OIllNATi:i) llll'IIKXYl.K 4,ST mean storage levels niter 24 hours ueiv essentially the same. Considerable vari ation in the storage levels was found in caeh Rrmip. Dillcring absorption rates' Imin the not might aeeounl lor the variation in levels lonnd; variation was highest in the lat and lowest in the plasma. According to Haves (35), acute dosages, such as tlie one given here, do not result in storage, lmt rather in a flooding of the organism. lsxeuuMCNT B Only surviving rats were analyzed in Experiment lb Five of the ten male rats died--one on the last day (day 9S), and one each on days 55 and 56 ami two on iluv 30. flight ol the ten lemale rats died--one each on days S9, SO, 47. 41, and .35. and three on day 55. Tucker and Crabtree ( 15) fed male albino rats 1000 ppm Aruclnr 1251 and reported lethal elicits to none ot 6 by 14 days, 1 of 5 bv 25 days, 3 of 4 by 43 days and 4 of 4 by 53 days including a 21% reduction in food con sumption. All survivors in Experiment H appeared to be normal; however no in crease in weight w as observed in surviving males alter S4 days of dietary intake and alter 77 days for surviving females. Food consumption for dosed males and females was 66 and 64!f, respectively, of consumption by controls. Data from electron-capture analysis ol tissues and plasma are presented in Table II. The variation in the concentrations ol PCB in this experiment, as in Ex periment A. is least in the serum and brain and highest in the muscle and fat. A significant dillercncc between males and females was not found for storage of IWItl.I; II I >istnun tm>\ up KI> Mm-.invt. I,'ni,i,m\ imi 0.s-I)\y Kxrosrm: in v lln.'i \iiy Li.w.l ijf 1000 ITM Aimci.oif I'J.VI Mule ijipai I I'Viniilcs Males vs Keinnlo I'ltiMim Mi-mu SK K;ii Me.'in SK MlN'il' Meal, SK l.lllll! Menu si-: lirnni Mean SK Kiiliirv sr:' Liver Mi'Mii si-; 17 (i 1 1 27.s .')7 12 i 117 7s *2.j i 11 11 .'ti; o 1 .M IS .1 Mill .17!* 7:>:\ 721 ,V2 _M 01 Ml 7.1 MM Min i:; j> > // > O./jO /> > 0 20 p > 0 ."ill /; > o :.u p > o ;>o /. > 0 20 t A t - i. DSM 026037 STLCOPCB4009999 -4 S.S Cl) I ILLY li'l' AL. Aroclor 1254. 11 ayes (35) and Dale ct <il. (30) report that storage of DDT in female rats' exceeds (lint of mail' rats when hotli are maintained mi the same diet and that the enhancement in storage exceeds the greater food intake by females. The absence of certain peaks in (lie Arcelor standard when compared to l'GISderived material in the samples became more dramatic in this experiment as compared to Experiment A. Generally, the percentage of matching Aroclor peaks found in each tissue was as follows: plasma 81V, fat 74V, brain BOV, liver 61V, lung 03V', muscle 62!f, and kidney 53V. These differences could he attributed to: ( 1 ) selectivity in storage for each tissue; (2) enzyme activity resulting in some form of metabolism; (3) excretion favoring some isomeric forms as opposed to others. Exokuimi'NT G The data from the analysis of tissue, plasma, and excrement obtained according to lbe schedule outlined in Experiment G are shown in I'igs 2 and 3. There is a slcadv buildup of Aroclor in all tissues, while the excretion trend is quite erratic. Kin. 2. Distrilmliun of I'C.'ll-ilerivecI material in tissues mill plasma ivnm lats on a ilictaiv level n1 l()[) ppm Arnelnr J25I. DSW 026038 mu I'oi.Ycm.omN'Ti i:i> diphenyls 489 l-it.. .V (aiuceiili alien ul t'CH and I't.'H-dcrivrd uiulcniil in frees and urine ficmi nils mi u ilirt.nl Incl ui 10!) ppm AiiK-lnr 1251. The excretion trend may Ik- due in part to the discontinuous collections; there fore. these results reflect only the excretion pattern at specific times. The residue lei els observed in Experiment B created some douht that lev els observed alter the oS-day dietary intake were representative of steady state values for Aroclor 1251. liases (.15) states that DDT fed to rats at a constant rate is increasingly stored in their lat until it reaches a plateau, and on a diet containing 2(H) ppm or less this plateau is readied within 90 to 140 days. Dale ct (it. (.JO) found mean concentrations of DDT and DDE in the fat of male rats led 200 ppm in the diet for 90 days to be 523 and 51 ppm, respectively, while male rats on the same diet for HO days had mean concentrations of 300 and 42 ppm. A maximum concentra tion in liver, kidney, and brain is achieved within a few days for a dietary level of 1000 ppm (35). lixmilMENT D AM) 12 'Insults of Experiment D showed that rats on the same dietary levels stori d more Aroclor in their tissues alter 240 days than at the end of 58 days. The 240day j;rnup showed no significant dillevence in the iptantity of the PCB-derivcd material (omul in the urine but showed significantly more I'C.B excreted in the feces. Analysis of tissue, plasma, and excreta from Experiment 12 indicates that '.t, ,r '* 'if* It,' .4 MS ' $} ' i\ l 026039 STLCOPCB4010001 190 CUIILKY T AL. Aroclor storage, like DDT, is directly related to tile daily dosage. However, tinpoints at vvliic ii equilibrium storage is aeliievcd arc yet to be determined. At dietary leads of 100, -100, and 800 ppm lor 2 years (37), die mean values nl DD T stored in the fat ol rats at equilibrium were: reported as 9.5, 1028, ami -42(K) ppm. la sportively. 'Die concentrations of Aroclor 125-1 found in the fat alter 210 days at dietary leads of 100 and 500 ppm weir .1101 and 10.021, respectively CencraUy, the le vels ol DDT stored in the lat of male' lats after 180 clays arc equivalent to levels alter 2 years (37), 'although there is some reduction in DDT storage beyond 2 yesns. These levels in the fat illustrate' the vast dilleiraces in storage at the steady stales lor these two compounds, and the vast clilleirnccs in tlu ir relative toxicities and/or lipid solubilities. (The brain concentration, vatlu-r them fat concentration is a better indication of toxicity) (38). DDT storage can be reduced if exposure is reduced or discontinued. Lang and t''it/.!mgl) (37) inline) retentOn ol 50-757 and 251 of DDT in the- fat. .30 and 911 days alter discontinuing diets containing 5 to 50 ppm. After 58 days on a dietary level ol 100 ppm Aroclor and a recovery time of 71 days, 807 of the concentration in (lie- fat remained, Elimination seems to parallel that ol DDT. These data indicated the- concentration of Aroclor found at time of sacrifice to In' in the following order: fat > liver > feee's > kidney > brain > plasma > mine. The order of storage for tissues and plasma seems to parallel lipid content. Ecca! excretion exceeds urine excretion with Amelias as it does with DDT and other chlorinated hydrocarbons. Evaluation ok Mktjjod The- efficiency of the silica gel microcolvnvm and the extraction techniques em ployed were evaluated by determining the1 recovery of Aroclor 125-1 from in vitro fortification. Recovery data for all samples ehiteel from a silica gel mierocoluinn are prcscntc'd in Table 3. All in vitro recoveries, whether from liquid chromatog raphy or a combination of extraction and liquid chromatography, were at an acceptable level with the exception of urine. Recoveries of DDT from human urine using liquid-liquid extraction techniques have been reported !>)' Cuelo and Rims (28) and Cranmcr at til. (29) with recoveries of 72 and 987, respectively. In hoth instances, their extracting solvent or combination of solvents was more polar than hexane. The recovery in approach No. 1 and possibly 3 might be attributed to the partition coefficient ol hexane in an aqueous system. In order to evaluate tin's, reciprocal p-values were determined using the technique of Howman and Hcro/a (30). The average of triplicate analyses gave a reciprocal p-valim ol 0.761, or 76.-17 of tile Aroclor was- partitioned into the hexane layer from tinaqueous system. This value agrees favorably with approach No. 3 hut dues not account for the 177 didcronec in approach No. 1. Zitko (20) states that he found fractionation taking place1 when emulsions resulting Irom mixing Aroclor 1231 with water were broken by centrifugation. Cuts chromatographic analysis ol a hexane extract of the supernatant showed it to be richer in the lower ehloiinated biphenyls than the original preparation. 'This varied with batches o( Aroclor. When a peak-for-peak comparison was made, whether in vitro fortification or /--value studies were being considered, recovery was generally low for all con- QSW 026040 TOl-YCIimil IN ATI-11) Mil'll LWYI.S 491 T.MU.K ill 1! 1 I'llVIillV OK 1.'si VlTIMt Kt >I<TI Kit `\TNl\ Ml ('nvntor, Swirl.kk with Aitocuiit 12.V1 Tittle WciKlil I'tirlified (ppm; Al! 1*2'>4 Lcm-nl. remvery' Hu.siim Inn. Liver MiiM-lt* Hriiin Kitlnrv Kc-c.-s 1 Vim* (A|)|rtjtrii 1,2,-li) 2.(1 .251) . r>(it i Aim :fjo ..">(](> | IK HI ()..> too <).<> au ;;.o 2.U III! 0. 1 nt ni MS' 0 S2.2 04 . S 11)4 1 ion 4 VIKl d t)!l S 02. 0 114 1) 77 0 ' I'ln-nm, f;il, liver, itmsilr, lii-ain, kiilney, ft-i-i-.s--mi-mi of 4 ili-lmnimilioii.s, mid iirini- .V luc.-ui ill 11ii|>Iicn 1 (- tli-u-nuiiHtlintis. stilucnts, and tlx- average recovery was reported. Low recovery from the urine was probably due In tin- extracting solvent. Tlie values reported were not correete:l for in vilrn rccuv erii s, Although p.p'-DDT mill p.p'-DDL wen1 determined at (lie 0.01 ppm .sensitivity level in liver from a control rat, the decree to which silica gel removes interfering moieties Irom (issue or excreta extracts cannot he determined at this time. Before and after elution Irom silica gel there usually was a marked diflcrcncc in the ap pearance of the extracts dependin'; on the weights of tissue and excreta used. (Law and Goi-rlitz. (39) report the success of pigment removal with silica gel from water samples.) Muscle and plasma were the least allected hy liipiid-solid chromatography and could have been analyzed very easily without this treatment. In fact, for tile con centrations of Arodor found, most of (he samples could have been analyzed without any cleanup, taking into consideration the problems associated with column and detector contamination. Alter elution patterns ol Aroelor 1254 were established, 10 ml ot benzine: lu x.ine were nut required lor tptanlitative elution of PCB, in fact, only 4 ml were required. This saved time in subseijtient analysis. The elution pattern of Aroelor 1254 was unchanged at the fortification levels mentioned in Table III. The Aroelor 1251 standard that was chromatographed simultaneously with each batch ol samples analyzed had a mean recovery ol 104If with a SL of 3.1 for 23 samples. In i i:niT-;m;.\c:i-: or DDT with PCI! Analysis 3 lie presence of PCBs, DDT, anil DDT-like moieties in samples presents n problem both qualitatively and quantitatively during analysis. Initially, samples of liver and fat from control rats lor each sampling period were analyzed for DDT mul its metabolites; samples were taken from the oldest control rat in the study. These tissues usually showed measurable amounts ol DDT, DDD, and DDL. Thivalues for liver and fat, respectively, wen- as lollows: p.p'-DDT, 0.046 and 1.366 ppm; p.p'-DDI), 0.036 and 0.546 ppm; pp'-DDL, 0.016 and 1.095 ppm. Since preliminary evaluations showed liver and fat storage of PCBs to be a >;k -' Mi SW 026041 STLCOPCB4010003 192 CUHLEY I'.T AL. minimum of If) to J00 times the above mentioned values, interference was amsidered In he nonexistent. However, three possible approaches to separation uf I'CB and DDT-likc materials without any chemical degradation of the pesticides concerned were evaluated. Thin-layer chromatography was considered first. Usmji silica gel (I and essentially the techniques as outlined hy Walker am! Rcru/a (31). hoi It Aroclor 1251 and Aroelor 1260 in the presence of the chlorinated hydro, carbons. p.p'-DDE, p.p'-DDT, o,p'-DDE, o.p'-DDT, dieldrin, and heptachlor epo.side, moved with the solvent Iront. Using polar and nonpolar moliile phases, the 111 values for RGBs and p.p'-DDE wen- identical and not resolved. This can possiblv be list'll in confirmation and identification of Amelias. Tin- two other approaches utilized column chromatographs'. Florisil was used, as proposed bv Reynolds (32). PC Its arc eluted Irom the column with hexane, while other chlorinated hydrocarbons are eluted with a diethylcthcr; hexane mixture. Reynolds evaluated Florisil ss'itli a mixed standard of Aroclor 125-1 and a chlorinated hydrocarbon mixture containing lindane, licplachlur. aldriu, heptachlor epoxide, p,p'-DDE, dieldrin, p.p'-DDD, and p.p-DDT. Alilrin. p.p'-OOli, and beptaeblor are eluted in (lie PCR liaction. Although these pes ticides were not .separated, this approach might still be acceptable since aldriu and heptachlor are seldom found in environmental samples in their unepoxydated lorms. Him ever, whin an attempt was made to reproduce the wink uf Reynolds using PR grade Florisil, a mixture uf Aroclor 125-1 plus the chlorinated hydro carbons, lindane, heptachlor epoxide, dieldrin, p.p'-DDD, p.p'-DDE and p.p' DDT, measurable quantities ol p,p'-DDT, p.p'-DDD and lindane were found in Fraction I, i.e., 60, 10, and 32/ of the total, respectively. Reynolds does not men tion the quality ol the Florisil although he does mention how it was handled. The problem, however, seems not to be related to tire grade of Florisil. Revenue ami Ogafa (33) base presented data using both qualities of Florisil and were nnahle to reproduce the work of Reynolds. There is some disagreement between the fractioning pattern of Revenue and Ogata (33) anil the one observed in this work. This could probably be due to the quality and treatment of the Florisil. These problems suggest that the elution characteristics of Florisil should hr evaluated Indore use for separating 1'OB and DDT. . Armour and Burke (3-1) used a column of silicic acid (treated) and flelite Sir. The column is eluted first with petroleum ether for PC Iks and then with a mixed solvent system of diehloromethane-acetmiilrile-liexane (80:1:19) lor the chin, rinalcd hydrocarbons. This system was evaluated using standards at levels below those that would be expected in environmental samples, i.e., 0.5 ppm Aroelor 1251 and Aroelor 1260 and 0.1 ppm y- and /if-isomer of BIIC, heptachlor epoxide, o,p' DDE, p.p'-DDE, dieldrin, p.p'-DDD, and p,p'-DDT. Separation has been a problem with the p.p'-DDE; Fraction I contained 205 p.p'-DDE with the remain ing 80'/ in Fraction 2. Although some recoveries were less than 85'/-, all other pest'eides evaluated were only found in Fraction 2. According to Armour and Burke (3-1) the elution pattern can be altered by the water content ol the silicii acid. Tire first pesticide to lie alleeted in the group ol compounds that were used In show the changes was p.p'-DDE. Tire fact that there was not a complete peak for peak mate)) between stored ami DSW 026042 STLCOPCB4010005 494 cumxY ivr ai.. e.xerel ed l'CB and (lie dietary standard is not new (Fig. -1). Whether this is evidciur of metabolism is yet to Ik- determined. These dilhrenees were siiiwssfully demonstrated by electron capture gas-liquid chromatography and gas dimmatugraphy-inas.s .spectrometry in that there were changes in the relative abundanees ol some of the components of Aroclor 125-4 in fat and the urine samples. This has not yet been demonstrated in any other tissue. The mass spectra showed molecular ions at m/c 2S8, 32.'3, 35-S, and 292 con taining Cl,, Cl., Cl,,, and Cl; isotopic clusters in the fat and 254, 288. 124, and 35S in the urine. Two additional ions which wen1 observed in the urine only, were m/c .901 and 405. These ions contained the Cl, isotopic cluster The presence of unaltered Aroclor in the feces may he indicative of lack of absoiptou; this was the only sample that showed no alteration when compared to standard Aroclor 1254. Tin- neutral urinary excretion pattern was quite the contrary to feces. This mav in part he due to a .solubility phenomena which Zilko (20) encountered when working with I'CH and water. The situation ol Zitko with either urine or alcoholwater mixtures under in vitro conditions was not reproduced. Under in uiuo con ditions. there may he selective solvation of certain constituents of Aroclor. Selected samples of urine were analyzed hv gas chromatographs- usuig the clcctinhti-.: conductivity detector in the reductive modi-. The presence of chloride ion was further substantiated. The retention times of these components compared favorably with peaks in the Aroclor standard. coxci.rsiovs It is hoped that these data will elucidate some of the problems inherent in studying the polychlorinated biphenyls: and that sonic correlations can he found between the residue levels reported and the Ll),,, detcrnenalIons including the pathological findings to be reported in a later publication by Kimbrough ct <rf. (10). There are some basic generalizations that can be drawn born the data presented: ( I ) Following dosage with Aroclor 1234 or 1209, whether acute or chronic, residue amounts can be delected in all body tissues, fluids, and excrement. (2) At the same dosage level rats store more I'CBs than DDT. ( ')) No sigiclicant d llerenee is apparent in the storage of PCIBs by male or h male rats when led the same dietary levels. (4) Polvchloi mated biphenyls are stored primarily in adipose tissue. (3) S nee there was no recurrence of the general Aroclor pattern in the gas chromatograms or total ion current traces of the- mass spectrometer between the Aroclor standards and components observed in the fat and urine, pos sible metabolism or diflerential absorption is suggested. ACKNOWI.KDCMKXTS \W Piank Mis. K.stolfe Crav for statistical analysis, Mr. Tlmn.is II. (aiincs and Dr. 11. D. Kinilnni'jli lor assistance ith (lie animal studies. . KKKKKKNCKS 1. S< HMiivr, H,, \si> Sik's.ia, (IKK!). Ann. C/irm. 207, ASK--l 1. 2. Pi;\i.i.. D win It., ami I.ivnu, J. I.. (lijTO). Hio. Sri. 2(1 ( 17 ). . DSW 026044 3. Mijiis;iu(ri Trrlmical Hulicliits 0/10,*31 1A, ()/ld.-300, and O'I' h/l. I. MniiNiiiilti 'Irtliiiir.il llnllrliiis O/I'L-Al IA. 20. 5. 1., and Sui.i.tvAN, \V. N. (V)53). /. Krnu. Entomol. 4(t, 037. 0. |,u:ini.\Mi r\, K. 1*. ( 1000). J. Eetni. Entomol. 62, 761. 7. Saprlco, Inc. (attain^ (1070). 8. I Inwn.i., 0. K. { 1948). IVoc. OU</. Acad. Sri. 20, 31. 0. Cot ixiv, 1). M., Caywacii, L. A., Di.Vnn.s, ). L., ami Wolijicm, IV (J'JOO), it uni Client. K 300. 10. (-iiodwin, K. S.. Cdvmmin, IV. am) Miivnoi i>s, ]. (J. ( 1001 ). Analyst (London) 81, 007. 11. Iliim iix, J. ( 1003). Analyst (Louden) 00, (1073) 4 13. 12. Aullinr unknown ( 1000). ,Ynr Sri. 32, 012. 13. HiM.imm <.ii, IV \\ ., Him mi;, I*., Piaku.i., 1). IV, IIchmay, S. and Kjiim .n, M. ( 1009 ). Xa/uie 220. 100.S. . i \m y S. ( 1000). Xatuir 224. 217. Tu ki:ii, H. k., \mi (jiurnii:);, I). (1070). "J [andhook of Tovicity of Pesticides |<> \\*iI<fIift.'` l'. S. Dept, nl Jnlnior, llnirau ol Np:>rl Kislirncs ami Wildlife. Mrsomves PhIi- liralioii No. 8 I. I,'ki;ak, 1). J1!. II. (1008). "Pesticide llamlbo.ik Kuloina.'' CnUr^c .Science PnLlislirr.s, Stale (.'olirnr, Ha. 1 080 I. 17. Hid s11', I,, fi.i Mill-:-*, IX J.. and Mmuiii-:, \. W. Kimum. Pollnt. 1(1), 3-20. IS. I)i; Vos, J. (7, asm Kdi:m w, J, If. ( 1970). Toxicol. A{>i>l. Phumifu til, 17, 050-08. 10. IXm, T. \\\, Lou i , J. 1., ami Wii.ma. A. |., Ju. (1070). Hull. Km inni. Contamiu. Toxittil. 5. (2). 20. Ziiko. V. ( 1070). Hull. Einiiou. Contain. Toxicol. 5 (3), 270-2S5. 21. MTumsii, I). I. t 1070). Hull. Enriinn. C.untnm. Toxicol. 5 (3), 202-201. 22. Mh.uk, J. \V. ( I9UL Huh. Health Hep. 50 (33), 1085-03. 23. I i.ic k, IX K. ( 1003). Poult nj Sti. 44. ] 100. 21. (i \isj;s, T. IV. xmj kiMunnvu.M, IV I). ( 1001). Hull. \V. II. O. 31, 737-715. 25. Dm.i., Wsi, K.. (!i m.iA, A., ami (ci.u), ( 1000), Life Sri. 5, 15-5 1. 20 I* in am, A. (... ami Tiiomimiv. J. (to lie pnLlislietl). 27, JiK. W. (1008). Hjolojiitaf Sample Cleanup lor Chlorinated Hydrocarbon ami ()inantiplinspSorns Ana! > sis Vsim LlrclmnAiaplnre C.as-Li<|iiid Chromatography. Paper pirsrirlrd Nat l. ACS Mtetinn, Atliinlic Cit\, \J. 28. Cu id. (!., ami Minns, K. (1007). Toxicol. Phannol. 10, 201-209. 20, (lii vwii ii, M, l'\, ('.Amoi.L. ]. J., and Coio.i. \M), M. K. ( 1909). Hull. Eiwirnn. Conlavi, 7'n.v/rn/. 4 ( I ). 211-21. 30. Bum mi\. M. C.. ami Hhio/.a. M. (1005). Journal of the Assoeiutioii of Official Auri- eidluial (.7irm/\(.\ 4S (5), 013-052. Waj.m.m, k. C\, AMI Hi-.ko/.a, M. (J003). Journal of (he Association of Official Auii ruhund Chemists 40 (2), 253-201. 32. Hiamii.ds. I.. M. ( J000). Hull. Eucirou. Contain. Toxicol. (3), 28-12 33. III \ t.M J . A., \M) ( Ic.VTA, J. \ ( 1070). J. Cfuomato. 50. I 12-1 I. 34. AllMlM II. j.. AM) IlUUKJi, J. (1070). .Journal of the Association of Official Analytical Chemists 53 (4), 701 -08. 35u. JUu.s, W. J.. Jh.. ( 1050). "DOT,'* S, W. Simmons, Hirkhausci Vcrlau, IVisrl. 1). Hymn. W. J.. Jh. ( 1005). Aim. Iltv. Phannol. 5, 27-52. D.si.e. . \r i-; * ) ). 1'I. pi'; 1". IV, AMI ( 1002), 37. Lam. . i :. i'.. AM) l*`i i / III ( ii. o. i; (1010). J. Phann. 87( I ), 18-23. 3K. Dvi.i . w. i: , ( Iainks, 3* . 11.. VSl) II WIN, \\', ( 1903). Sr ieiirc 3508). 70. 311. Luv, 1.. M. , YM) (aiKIU.ir/., n. r. ( 1070). Jouimd of the Awnriofiou of Official .Aiui- lytieul Chemist. 53 (0 ). I27(i-K(). 10. kiMimot (.m, H. 1)., 1M) (.AIMS, T. A/ch. i'.minm. Health (in press).. OSW 026045 STLCOPCB4010007