Document 22YXROBL1qX3ObeEdV2ndbrR

In lout; i)i a j liy keeping \vnl(i. TL he ir measuring u nt o d/s- ii.ilysis. The TllC Ilufl(T O. ml, taking ill'll)'. lug All!Am dated from g of rrian- li odes ivas h occur in dcU-iminnmcll loci is 'dwnj. Chief <*)nrnl. BsmilONXIENTAL HESEARCII 4, 481--195 (197i) Polychlorinated Biphenyls: Distribution and Storage in Body Fluids and Tissues of Sherman Rats1 August Cuuley, Viiu.yn W. Buhse, Mauy E. Ghim, Rai.pii W. Jennincs, and Rai.i'ii E. Livdeu Cbamhlcc Toxicology Laboratory, V.nvironmental protection Agency, 4110 Buford Hie' unj, Cbamhlcc, Georgia 303-11 Hccciocd July 1C, 1071 An analytical method for determination of I'CU (Aroclor 12S-I) in Mood. urine, feces, and tissues of Sherman strain rats after prolonged dietary intake (100, 500. and 1000 ppm) is presented. The method involves homogenization and extraction with hexane, cleanup on a silica gel microcotuinn and analysis hy electron-capture gasliquid chromatography. Recovery data arc shown at 0.1, 5. 14, and 100 ppm. Mass spectroscopic charactciistics of PCB in tissues arc given. Distribution, storage, and excretion rates during feeding and after discontinuance of PCB in the diet arc presented. Polychlorinated biphenyls (PCBs) were fitsl described in 1SS1, by Schmidt and Slmll/ (1). Commercial production was begun in 1930 (2). These compounds hare been anti still are widely used as plasticizers and resins and in chlorinated rubber (3, 4). They are known to enhance the insecticidal properties of lindane (5), DDT, and dicldrin (6). They may be used as stationary phases in gas-liquid chromatography (7). Their widespread use may have resulted in contamination of the cco-system as suggested by many recent reports. Aroclor' and DDT were both used during World War 11. In 1943, total urganie chlorine analysis provided data for the first detenoination of DDT storage in man (8). Since this method has no specificity for DDT, the total organic- thimine content would not preclude the presence of other organic chlorine-containing moieties. In terms of halide specificity, the Dohrmann microcuulomctric gas chromatographic method for detecting pesticides was well established by 1960 ( 9), and electron-capture was in use by 1961 (10). Robum (11) first observed extraneous peaks in 1965; he used a pntenlionietiie mode of analysis for total chlorine and found disagreement between this method and total chlorine content calculated from gas chromatographic results in which Argon ionization and electron-capture were used for the analysis of (issues and `The Moi isBnto Company manufactures PCBs chiefly in the United States under the trade naiiK- of Aroelor. The) are manufactured in Prance by Prodelee, and in Germany h) Bayer, with tiade names, PLenoehlm and Colphcn, respectively. These compounds aie designated by mmikci.s The first two digits represent the molecular type: 12, chlorinated biphenyls; 25 and 44, Meeds of chlorinated biphenyls and chlniiiiaUd tciphcuyls (757 biphenyl and 007 biphenyl, rt-- pcclis-cly); 51, chlorinated terphcnyls. '1 In' ia-l two digits give the weight percent of chlorine. T ims. Aroclor 1254 and 1200, with which this paper is concerned, are chlorinated biphenyls containing 54 and 007 chlorine, rcspccliscly. 481 1972 hy Academic Piers, Inc. * OSW 031109 STLCOPCB4015071 482 CURLEY ET AL. r eggs of wild birds. The samples showed significant amounts of unknown elec tron-capturing compounds. Robura postulated that these were metabolic inter mediates of one or more of the organochlorino pesticides. The differences could also have been indicative of PCB contamination. It was not until 1966 that S. Jen sen (12) using electron-capture and mass spectrometry was able to identify as PCBs unknown peaks found in the analysis of fish and a bird carcass. Un doubtedly, the time lag between initiation use and detection of I'CB in the en vironment is a matter of accumulative concentration and/or instrumentation development. The presence of PCBs in environmental samples lias been well established (13, 14). 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 of marine life (19, 2), 22). Pathological changes in the liver and other organs have been shown in guinea pigs, rats, and rabbits at high concentrations (22). Ilydropcricnrdium and growth depression proportional to dietary levels of the toxicant have been pro duced in the chicken (23). Polychlorinated biphenyls arc known to afTecl estrogen levels in birds which in turn reduces calcium reserves and results in thin eggshells. Chemical porphyria from oral administration has also been shown in chicks (18). This paper presents distribution, storage, and excretion rates of Aroclor in cer tain tissues, body fluids, and excreta of rats following (1) a single orai dose, (2) repeated dietary intake, and (3) after discontinuance of PCB in the diet. METHODS Aroclor 125-1 and 1260 were supplied by the Monsanto Chemical Company with' respective lot numbers, AK-3S and AK-3. Dietary formulations were prepared according to the methods descriiicd by Caines and Kimbrough (24). ' Expf.iumf.nt A . Aroclor 1254 and Aroclor 1260 were administered at 1600 mg/kg and 3200 mg/kg, respectively, ns a single oral dose, by stomach tube to three female, 173- to 183-dav-old, white Sherman rats weighing 260-318 g. Four control rats, two in each group, were given peanut oil only. Twenty-four hours later the animals were killed, and blood (oxidated), frit, muscle (abdominal), liver, kidney, lung, and brain were taken for analysis. - . Experiment B ' Aroclor 1254 at 1000 ppm was fed for 9S days to 29- to 34-day-old Sherman strain male and female weanling rats, in groups of 10. Controls were fed only plain chow. Food consumption was measured at intervals as described previously by Kimbrough and Caines (4). All rats were weighed on a weekly basis from experiment initiation until time of sacrifice oi death. The rats consumed an aver age of 72.7 mg/kg/Amelor/day, Blood (oxidated), fat, muscle (abdominal), liver, kidney, lung, and brain were taken from each rat at time of sacrifice. Tissues were preserved in 107 formalin and held for analysis. DSW 031110 STLCOPCB4015072 K>wn elcc>olie inter nees could hat S. Jonidenlify ns. rcass. Un in the cnumentnlion established nnd some 2?). Tol- ifr (19, 21. i shown in udiuin nnd bee n pn>cl estrogen n eggshells, hicks (IS), pckn in ecrd dose. (2) lirl. inp;ut>' with c prepared g nncl 3200 'cinnle, 173- o) rats, two the animals idney, lung, >ld Slier nun re fed only d previously . basis from nod an nverninal), liver, Tissues were POLVCHL03IXATSI) BIPII2NYLS I 483 Experiment C Sherman strain male weanling rats (61- to 66-days-old) were fed n diet of plain chow or chow fortified with Aroclor 1251 at a concentration of 100 ppm for 58 days. Fortified chow was discontinued, and samples were collected during the recovery period. Food consumption was measured at 2-day intervals for a period of 8 days; after 42 days food consumption was measured daily for 4 days. .Sur viving rats were weighed once a week. At various times during the experiment rats were ind.vidually housed in metabolism cages. Urine nnd feces were collected over a IC-liour period during which time plain chow was fed. Following excreta collections, rats were sacrificed. Blood (oxalated), fat, muscle (abdominal), liver, kidney, and brain were taken from each rat at time of sacrifice. Tissues, urine, and feces were frozen at time of collection. Whole blood was centrifuged and the plasma refrigerated. The outline of the experiment as aforementioned is sum marized as follows: These animals (two on the diet and one control) were sacri ficed on Day 4, 9, 13, 17, 25, 34, 42, nnd 58. After discontinuance of 1'CB in the diet, two animals were sacrificed on Day 8, 16, 24, nnd 71. Experiments D and E Sherman stiain male weanlings (36- to 41-dnys-old) were fed Aroclor 1251 at dietary levels of 100 ppm or 500 ppm for 252 days. Body weights were determined initially and weekly thereafter for 12] days. On the basis of food consumption measurements, the average rates for intake-of Aroclor 1254 wcie 6.8 mg/kg/day and 36.4 mg/kg/day for 100-ppm and 500-ppm dietary levels, respectively. Sam ples were collected in the same manner as Experiment C. Sampi.e Preparation Polychlorinated biphenyls like DDT, are not water soluble but are, in fact, highly lipid soluble. Solvents for tissues and plasma extractions woe selected for this properly. . Plasma (at least 2 ml) was extracted by the method of Dale et til. (25). Later in the stud)', the Bryant-Thompson modification of this method was used (26). No significant difference has been found between these methods in this laboratory with the exception that convenience favors the Bryant-Thompson modification. Samples of fat (250 mg), liver, muscle, kidney, and lungs (500 mg each) were homogenized two times in 4 ml of hexane, nnd the extracts were combined. Brain (500 ing) was homogenized two times in 4 ml of acetone, and the ex tracts were comlrned (27). . Urine (mean volume of 10 ml) at pi I < 7 (using pllydrion paper) was diluted to a volume of 25 m! by the addition of water and 1 ml isopropanol. These sam ples were extracted in 125-ml separatory funnels three times will, 10 ml hexane. The hexane extracts were combined, washed once with 3 ml of water, nnd 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 ml of acetone on an automatic shaker. The acetone extract was decanted nnd placed in a 50 ini DSW 031111 STLCOPCB4015073 484 CUW.EV ET AL. centrifuge tube. Particulate matter in the extract was removed by centrifuging and/or decanting. The extracts were evaporated with 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 sulfate and held for cleanup and analysis by electron capture gas-liquid chromatography. PnF.i'AHATiON or Silica Cel and Miciio-Coi.umn Silica gel (27) from Woclm (Activity grade 1) was activated at 130 foi 4 hours and desiccated over calcium chloride for ]6 hours before deactivation at 3% (v/w) by the addition of water. The silica gel was allowed to equilibrate at least 2 hours before use. After comparing the weight of the silica gel before and after activation, it was found to be activity grade I as specified by the manu facturer and fqrlhcr activation was unnecessary; however, each new batch was routinely activated. One gram of 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 JO ml of hexane. Each sample was placed on the column in a volume of 0.5 ml. At least J ml of hexane was used to quantitatively transfer the sample to the head of the column. The column was eluted with 10 ml of a 1:1 benzene:hexane mixture (27). Samples were chromatographed in sets of eight with duplicate column blanks. Aroclor 1251 standards were eluted simul taneously in order to ascertain the variance in silica gel activity and to detect an)' possible contamination from solvents, glassware, or silica gel. CAS ClinOMATOCHArilV Samples from experiments, A, C, D, and E as treated above were analyzed by election-capture gas-liquid chromatography under one of the following condi tions using a MicroTek MT-220 gas chromatograph: pulse Mode (using dual columns and dual detectors); cell voltage, 36.5 volts; pulse width, 5.5 i-scc; pulse rate, 120 /tsec; detector, 'll, parallel plate design, No. 1 205, No. 2 198: column, 6' X K" o.d. U-shaped pyrex glass packed with 57 OY 210 on SO/100 mesh Chromosoil) W, high performance, 170; inlet 23S; carrier gas, argon -methane (95-57), 50 psi; flow-through Detector No. 1, 50 cc/minute; Detector No. 2, 60 cc/minutc. 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 OY-17/1.957 QF-1 on 100/200 mesh Chromosorb \V, IIP, 200; inlet, 230''; carrier gas, nitrogen, 40 psi; flow-tinough detector, 75 cc/minute. Samples from Experiment 11 were injected directly as hexane extracts and analyzed by electron-capture gas-liquid chromatography under the following conditions using a MieioTek MT 220 gas chromatograph: cell voltage, 40 volts DC; detector, ,;~h'i, 250; column, 6' X Yd" o.d. U-shaped glass column packed with 1.57 OY-J7/J.957 QE-l on 100/200 mesh Chromosorb \V, IIP 200C; inlet, 210; earlier gas. nitrogen, 40 psi; flow-through detector, SO cc/minutc, 20 cc/ininutc scavenger plus 60 cc/minutc carrier gas flow. DSW 031112 STLCOPCB4015074 vntrifuging nitrogen to evaporated w ere dried by election J30 for 4 ctivntion at oilibralc at before nnd tin- manubatcb was ipel, which ;!. Columns (be column ely transfer 10 ml of n in sets of uted simuldetect any nnlyzed by \ big condiiising dual see; pulse eoliimi), >/ ](!() mesh m metliane i No. 2, 00 or, "H, par aded with inlet, 230; stiacts and following r,e, 40 volts mo packed inlet, 20 ee/min- > ' , , POLYCHLORINATED BIPHENYLS Recoveky Studies I r 485 Recovery studies were completed using the following methods for control tis sues, plasma, and 4S-hour urine and fcccs collections. The fortification level for each type of sample was the mean concentration, with the exception of fat, of Aroclor 1254 found for that sample in Experiment C. Fat samples were fortified with 100 ppm. Tissues (fat, liver, kidney, brain, and muscle): Aroclor was added directly to the hexane extract prior to liquid chromatography, Plasma: Aroclor was added directly to fresh plasma, thoroughly mixed using a mini-mixer, and allowed to equilibrate overnight under refrigeration. Samples were then treated as mentioned in methods. Feces: Aroclor was added directly to pulverized dry feces and treated as men tioned in methods. Urine (three slightly different approaches were used); No. 1--Aroclor was added to 1 ml of isopropanol, then mixed with a solution of urine and water (10 ml 4 15 ml) nnd analyzed as mentioned above; N'o. 2-- Aroclor was added to a hexane extract of 10 ml of urine which had been prepared as mentioned above; No. 3--Urine (5 ml) was added to a known quantity of Aroclor in a 15-ml centrifuge tube, mixed on a minimixer, and allowed to equili brate at room temperature for 16 hours. Urine was extracted three times with 3 ml of hexane; the hexane extracts were combined and evaporated to 0.5 ml. RESULTS ANI) DISCUSSION' Routinely, this laboratory operates dual channel solid slate electrometers at a sensitivity of 4 X 10 amps full scale which permits the detection of 10 to 30 picograms of the more common chlorinated hydrocarbons. There was a significant difference between the response characteristics of the Aroclors as compared to the chlorinated hydrocarbons. To achieve a representative gas chromatographic trace, at least 3-50 and 490 pg of Aroclor 1254 and Aroclor 1260 were required A gas chromatogram illustrating the difference between the electron-capture re sponse characteristics of these compounds as compared to chlorinated hydrocar bons is shown in Fig. 1. . Experiment A Data resulting from electron-capture analysis of plasma and tissues in Experi ment A are presented in Table I. Quantitation is an approximation. In all cases, the peak heights (in mm) of all PCB components were summed for the respec tive standard at a given picogram quantity injected. A response factor was deter mined for each standard in pg/mm. For each sample, a summation was made of the total response (in mm), and the product of this times the response factor cal culated to give picograms of PCB or PCB-derived material in the sample. When the gas chiomalograms for the samples were compared with those of the Aroclor standard, there were no reeuriences of (he general Aroclor pattern. Some peaks had completely disappeared. This was also true for each of the succeeding stvidics. Although the dosage level of Aroclor 12G0 was twice that of Aroclor 1254, the DSW 031113 STLCOPCB4015075 486 CURLEY ET AL. 4* I'KIUKINaTM* KVpH M'AHM*H Fie. 1. Gas chromatograms for Aroclor 1254 and chloiinntcd hydrocarbon standards. (A plot of detector response vs time.) TABLE I IllRTRIMUTlON OK AhOCI-OKS 24-HOURS ATTKlt OltAI. INGESTION BY STOMACH I'D Ilf. Aroclor 1254 (dosage level--1C00 mg/kg) Aroclor 12C0 (dosngc level--3200 mg/kg) Plasma 'Mean SE Brain Mean SE Fat Mean RE Liver Mean SE Kidney' Mean PE Lung Mean SE Muscle Menu RE 24.03 R.OS 138.00 30.41 . 1146.9 i 674 5 141.20 47. SO 274.0.3 30.47 65.01 29.48 80 29 1 67.58 ' . 15,79 0.99 145.17 i 23.50 930.0 420.6 236.1 116 3 328.5 114 9 105.17 21.13 37.00 i 22.37 OSW 031114 STLCOPCB4015076 e 5 i:--"'nl -- tnndards. (A i Tunr: i POLYCHLOMNATEI) BIPHENYLS f 487 mean storage levels after SA hours were essentially the same. Considerable vari ation in the storage levels was found in each group. Differing absorption rates from the gut might account for the variation in levels found; variation was highest in the fat arid lowest in the plasma. According to Hayes (35), acute dosages, such as the one given here, do not result in storage, but rather in a flooding of the organism. Experiment B Only surviving rats were analyzed in Experiment B. Five of the ten male rats died--one on the last day (day 98), and one each on days 85 and 56 and two on day 50. Eight of the ten female rats died.--one each on days 89, 80, 47, 41, and 35, and three on day 55. Tucker and Crabtree (15) fed male albino rats 1000 ppm Aroclor 1251 and reported lethal effects to none of 6 by 14 days, 1 of 5 by 28 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 B appeared to be normal; however no in crease in weight was observed in surviving males after 84 days of dietary intake and after 77 days for surviving females. Food consumption for dosed males and females was 66 and 64%, respectively, of consumption by controls. Data from electron-capture analysis of tissues and plasma arc presented in Table II. The variation in the concentrations of 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 difference between males and females was not found for storage of tabu; it Disthihvtion or PCU-Di:itm:i> Material Foliowing OS-IJ.w Exposure to a Dietary ]Level or 1000 ri'M Aunctou 1254 . , Males (ppm) Females (ppm) Males vs Females Plasm** Menu BE Fat Mean SE Muscle Menu SF. lame Mean SE Brain Menu BE Kidney SE Liver Menu SE 17 6 1127$ 5742 155 117 7$ 22 111 14 5G 9 155 30 ._ 1R 5 8431 570 753 721 52 20 94 21 54 24 210 13 ' p > 0.50 p > 0.50 p > 0.20 . p > 0.50 ' p >0.50 p > 0.50 p > 0.20 . DSW 031115 STLCOPCB4015077 iU U 488 CURLEY rr AL. Aroclor 125-1. Hayes (35) and Dale et al. (36) report that storage of DDT in female rats exceeds that of male rats when both arc maintained on the same diet and that the enhancement in storage exceeds the greater food intake by females. The absence of certain peaks in the Aroclor standard when compared to PCBderived 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 ns follows: plasma 812, fat 742, brain 662, liver 612, lung 632, muscle 622, and kidney 582. These differences could be 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. Experiment C The data from the analysis of tissue, plasma, and excrement obtained according to the schedule outlined in Experiment C are shown in Figs. 2 and 3. There is a steady buildup of Aroclor in all tissues, while the excretion trend is quite erratic. o rt liver O Kidney Mute le A Ptiln A Plain Fic. 2. Distribution of PCIl-dcrived material in tissues and plasma from rats on a dietary level of 100 ppm Aroclor 125J. DSw 031116 STLCOPCB4015078 f DDT in same did >v females, d to TCBTimrnl ns cJor peaks liver 617, ibutvd to: g in some pposed to according Tlicic is a itc erratic. - z=i * if, M- \ <m ft dietary POI,YCHI/>RINATEI> BIPHENYLS c 489 Fie. 3. Concciitinlion of PCB and I'CB-derivcd material in feces and urine from rats on a dietary level of 100 pjmi Aroclor 1251. ' ' The cxcietion trend may be due in part to the discontinuous collections; there fore,these results reflect only the excretion pattern at specific times. The residue levels observed in Experiment B created some doubt that levels observed after the 58-day dietary intake were representative of steady stale values for Aroclor 1254, Hayes (35) stales that DDT fed to rats at a constant rate is increasingly stored in their fat until it reaches a plateau, and on a diet containing 200 ppm or less this plateau is readied within 90 to 140 days. Dale ct al. (36) found mean concentrations of DDT and DDE in the fat of male rats fed 200 ppm in the diet for 90 days to be 525 and 51 ppm, respectively, while male rats cm the same diet for 140 days had mean concentrations of 506 nnd 42 ppm. A maximum conc entra tion in liver, kidney, and brain is achieved within a fen' days for a dietary level of 1000 ppm (35). Exit.himent D ant> E, Results of EjqxTiment D showed that rats on the same dietary lev els Ston d more Aroclor in their tissues after 240 days than at the end of 58 days. The 240day group showed no significant difference in the quantity of the PCR-dcrived material found in the urine but showed significantly more PCH excreted in the feces. Annlysis of tissue, plasma, and excreta from Experiment E indicates that DSW 031117 STLCOPCB4015079 490 CURLEY ET AL. Aroclor storage, like DDT, is directly related to the daily dosage. However, the points at which equilibrium storage is achieved are yet to be determined. At dietary levels of 100, 400. and 800 ppm for 2 years (37), the mean values of DDT stored in the fat of rats at equilibrium were reported as 95, 1028, and 4200 ppm, respectively. The concentrations of Aroclor 1254 found in the fat after 240 days at dietary' levels of 100 and 500 ppm were 1101 and 10,021, respectively. Generally, the levels of DDT stored in the fat of male lats after 180 days arc equivalent to levels after 2 years (37), although there is some reduction in DDT storage beyond 2 years. These levels in the fat illustrate the vast differences in storage at the steady slates for these two compounds, and the vast differences in their relative toxicities and/or lipid solubilities. (The brain concentration, rather than fat concentration is a better indication of toxicity) (38). DDT storage can be reduced if exposure is reduced or discontinued. Tang and Fit/hugh (37) found retention of 50-757 and 257 of DDT in the fat, 30 and 90 days after discontinuing diets containing 5 to 50 ppm. After 58 days on a dietary level of 100 ppm Aroclor and a recovery time of 71 days, 807 of the concentration in the fat remained. Elimination seems to parallel that of DDT. These data indicated the concentration of Aroclor found at time of sacrifice to be in the following order: fat > liver > feces > kidney > brain > plasma > urine. The order of storage for tissues and plasma seems to parallel lipid content. Fecal excretion exceeds urine excretion with Aroclors ns it docs with DDT and other chlorinated hydrocarbons. Evaluation of Methoo The efficiency of the silica gel microcolunm and the extraction techniques em ployed were evaluated by determining the recovery of Aroclor 1254 from in vitro fortification. Recovery data For all samples eluted from a silica gel microcolumn arc presented 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 hv Cucfo and Biros (28) and Cramncr el al. (29) with recoveries of 72 and 967. respectively. In both 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 of hexane in an aqueous system. In order to evaluate this, reciprocal p-valucs were determined using the technique of Bow man and Bcroza (30). The average of triplicate analyses gave a reciprocal ;>-value of 0.761, or 70.47 of the Aroclor was partitioned into the hexane layer from the aqueous system. This value agrees favorably with approach No. 3 but docs not account for the 177 difference in approach No. 1. Zitko (20) states that he found fractionation taking place when emulsions resulting from mixing Aroclor 1254 with water were broken by centrifugation. Gas chromatographic analysis of a hexane extract of the supernatant showed it to be richer in the lower chlorinated biphenyls than the original preparation. This varied with batches of Aroclor. Wien a prnk-for-pcak comparison was made, whether in vitro fortification or p-value studies were being considered, recovery was generally low for all con- DSW 031118 STLCOPCB4015080 urvor, the cd. i values of , and !f?00 l after 240 spcclivcly. ) days arc n in DDT i-rentes in ieiencos in ion, rather I .any and 30 and 90 ii a dietary ccnlration ;ac'i ificc to plasma > d content. DDT and nques cinm in vitro rocohnni! hromatogcic at an -in Innnan .'urto and pectivcly. seas more might be ii Older to o of lk>wal p-value fioin tlic does not he found cloi 1:734 dysis c>f a idoi inated f Arcelor. cation or r all con polychlorinated biphenyls 491 TABLE III IRecovery or n Vitro Fortu-ication or Control Samples with Aroclor 1254 Tissue Weight Fortified (ppm) AR 1254 Fflrcent recovery* Plasma Fat Liver Muscle Hmin Kidney Feces Urine (Approach 1,2,3) 20 .250 .500 .500 .500 .500 1.000 . 0.5 100 6.0 4.0 3.0 2.0 14.0 0.1 0.1 0.1 08.6 82.2 94.8 103.1 100.4 100.0 90.8 62.0 114.0 77.0 I'lnsinn, f*(, liver, muscle, brain, kidney, feces--mean or 4 determinations, and urine X mean of duplicate determinations. . stituents, and the average recovery was reported. Low recovery from the urine was probably due to the extracting solvent. The values reported were not cor rected for in vitro recoveries. ' Although p,p'-DDT and p,/>'-DDE were determined at the 0.01 ppm sensitivity level in liver from a control rat, the degree to which silica gel removes interfering moieties from tissne or cr.crcla extracts cannot be determined al this time, before and after elution from silica gel there usually was a marked difference in the ap pearance of the extracts depending on the weights of tissue and excreta used. (Law and Gocrlitz (39) report the success of pigment removal with silica gel from water samples.) . Muscle and plasma were the least affected by liquid-solid chromatography and could have been analyzed very easily without this treatment. In fact, for the con centrations of Aroclor found, most of the samples could have been analyzed without any cleanup, taking into consideration the problems associated with column and detector contamination. After elution patterns of Aroclor 125-1 were established, 10 ml of benzene: hexane were not required for quantitative elution of PCb, in fact, only 4 ml were required. This saved lime in subsequent analysis. The elution pattern of Aroeloi 1254 was unchanged at the fortification levels mentioned in Tabic III. The Aroclor 1254 standard that was chromatographed simultaneously with each batch of samples analyzed had a mean recovery of 1018 with a SE of 3.1 for 23 samples. Inteiu-euenck of DDT with PCb Analysis The presence of PCbs, DDT, and DDT-likc moieties in samples presents a problem both qualitatively and quantitatively during analysis. Initially, samples of liver and fat from control rats for each sampling period were analyzed for DDT and its metabolites; samples were taken from the oldest control rat in the stud)-. These tissues usually showed measurable amounts of DDT, DDD, and DDE. The values for liver and fat, respectively, were as follows: p.p'-DDT, 0.045 and 1.360 ppm; p,;'-DDD, 0.036 and 0.516 ppm; jyp'-DDE, 0.016 and 1.09S ppm. Since preliminary evaluations showed liver and fat storage of PCbs to be a DSW 031119 STLCOPCB4015081 492 CUHLEY ET AL. minimum of 10 to 100 times tlic above mentioned values, interference was con sidered to be nonexistent. However, three possible approaches to separation of PCB and DDT-like materials without any chemical degradation of the pesticides concerned were evaluated. Thin-layer chromatography was considered first. Using silica gel G and essentially the techniques as outlined by Walker and Beroza (31), both Aroclor 125-1 nnd Aroclor 12G0 in the presence of the chlorinated hydro carbons, p,p'-DDE, p,p'-DDT, o,p'-DDE, o,p'-DDT, dieldrin, and hcptaehlor epoxide, moved with the solvent fionl. Using polar and nonpolar mobile phases, the Rf values for PCBs and p,p'-DI)E were identical and not resolved. This can possibly be used in confirmation and identification of Aroclors. The two other approaches utilized column chromatography. Florisil was used, as proposed by ltcynokls (32). PCBs are eluted from the column with hexane, while other chlorinated hydrocarbons arc eluted with a diethylethei: hexane mixture, ltcynokls evaluated Florisil with a mixed standard of Aroclor 1251 and a chlorinated hydrocarbon mixture containing lindane, hcptaehlor, aldrin, hcptaehlor qpoxide, p,p'-DDE, dieldrin, p.p'-DDD, and p,p'-DDT. Aldrin, p,p'-DDE, and hcptaehlor are eluted in the PCB fraction. Although these pes ticides were not separated, tin's approach might still he acceptable since aldrin and hcptaehlor are seldom found in environmental samples in their unepoxydaled forms, lion ever, when an attempt was made to reproduce the* work of Reynolds using PR grade Florisil, a mixture of Aroclor 1254 plus the chlorinated hydro carbons, lindane, hcptnctilor epoxide, dieldrin, p.p'-DDD, p,p'-DDE and p,p'- DDT, measurable quantities of p,p'-DDT, p.p'-DDD and lindane were found in Fraction 1, i.c., 66, 10, and 32J of the total, respectively. Reynolds docs not men tion the quality of the Florisil although he does mention how it was handled. The problem, however, seems not to be related to the grade of Florisil. Revenue and Ogata (33) have presented data using both qualities of Florisil and were unable to reproduce the work of Reynolds. There is some disagreement between the fractioning pattern of Revenue and Ogata (33) and 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 be evaluated Irefore use for separating PCB nnd DDT. Armour and Burke (34) used a column of silicic acid (treated) and Cclilc 515. Tire column is eluted first with petroleum ether for PCBs and then with a mixed solvent system of dichloromethane-acctonitrile-hcxanc (80:1:19) for the chlo rinated hydrocarbons. This system was evaluated using standards at levels below those dial would he expected in environmental samples, i.e., 0.5 ppm Aroclor 125-1 and Aroclor 1260 and 0.1 ppm y- and /S-isomer of B1IC, hcptaehlor 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 1 contained 20Y p,p'-DDE with the* remain ing SOY in Fraction 2. Although some recoveries were less than 85Y, all other pesEcides evaluated were only found in Fraction 2. According to Armour and Burke (34) the elution pattern can be altered by the water content of the silicic acid. The first pesticide to be adected in the group of compounds that were used to show the changes was p,p'-DDE. . Tire fact that there was not a complete peak for peak match between stored and DSW 031120 STLCOPCB4015082 I'm. ). Ciis eltroinatonmm for rut feces, fut nod mine (n plot of detector response vs time). OSW 031121 STLCOPCB4015083 494 CURLEY ET AL. r excreted PCB and tlie dietary standard is not new (Fig. 4). Whether this is evidence of metabolism is yet to be determined. These differences were success fully demonstrated by electron capture gas-liquid chromatography and gas chroniatography-mass spectrometry in that there were changes in the relative abun dances of some of the components of Aroelor 1254 in fat and the urine samples. This lias not yet been demonstrated in any other tissue. The mass spectra showed molecular ions at u/e 28S, 323, 35S, and 392 con taining Cl(, ClSl Clc, nnd C1T isotopic clusters in the fat and 25-1, 2SS, 324, and 35S in the urine. Two additional ions which were observed in the urine only, were mfe 304 and 405. These ions contained the Ch isotopic cluster The presence of unaltered Aroelor in the feces may be indicative of lack of absoipfon; this was the only sample that showed no alteration when compared to standard Aroelor 1254. The neutral urinary excretion pattern was quite the contrary to feces. This may in part bo due to a solubility phenomena-which Zitko (20) encountered when working with PCB and water. The situation of Zitko with either urine or alcoholwater mixtures under in vitro conditions was not reproduced. Under in oiuo con ditions, there may be selective solvation of certain constituents of Aroelor. Selected samples of urine were analyzed by gas chromatography using the elec trolytic conductivity detector-in the reductive mode. The presence of chloride ion was further substantiated. Tire retention times of these components compared favorably with peaks in the Aroelor standard. .CONCLUSIONS It is hoped that these data will elucidate some of the problems inherent in Studying tire polychlorinated biphenyls: and that some correlations can be found between the residue levels reported and the LDi0 determinations including the pathological findings to he reported in a later publication by Kimbrough et al. (40). There are some basic generalizations that can he drawn from the data presented: (1) Following dosage with Aroelor 1254 or 1200, whether acute or chronic, residue amounts can be detected in all hod)' tissues, fluids, and excrement. (2) At the same dosage level rats store more PCBs than DDT. (3) No significant difference is apparent in the storage of PCBs by male or female rats when fed the same dietary levels. (4) l'ol)chlorinated biphenyls are stored primarily in adipose tissue. (5) Since there was no recurrence of the general Aroelor pattern in the gas chromatograms or total ion current (races of the mass spectrometer between the Aroelor standards and components observed in the fat nnd urine, pos sible metabolism or differential absorption is suggested. ACKNOWLEDGMENTS ' We thank Mrs. Estelle Cray for statistical analysis. Mr. Thomas It. Caines anti Dr. K. D. Kimbrough for assistance itli the animal studies. REEKUKNCES 1. Sciimipt, Jl., ash Sioxta, C. (18SI). Ann. Chrm. 207, 138--11. 2. rt'.vKAi.i., David It., ano Eiscm, J. L. (1070). Hio. Sc/. 20 (17). 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