Document jgkNBLaNE4Ep4y7Dnzp5zEgz5

KVVIIHIVMI.M Polychlorinated Biphenyls: Distribution and Storage in Body Fluids and Tissues of Sherman Rats' Ai't.ttvi (!umi,i:y, Vihi.yn \V. Ihmsi:, Maiiy 1. tuuM. lUi.m \V. Jknnincs, a\o Hai.i-ii K. !,i\i)ki< CtMliulihv i.iihoHilon/, l.iit ininiiit nlal Pivtcftiaii Afteniy, J770 lUifont llitil.wtitj, Cltuiuhtcc, i'.t'nruiti .10341 An iiiiiilvlirtil nirtliin! Ini determination nl PCH (Aroilnr 1251) in blood, mini-, bees. and (issues nl Slin until strain rots niter prulnuued dietary intake (100, 500, .inti l(KI() |>|iin) is presented. 'Hie method involves lioiiHM'ciii/iilioii ntul extraction will) hex.me, cleanup ou u silica ttel uitetucoluum unit analysis by elcctmu-cupUnc nxlapod clirumntour.ipby. Ihcovery data arc slims n at 0 1. 5. I I, uod 100 ppm. Mass spi'i liosc ipic characteristics nl PCH in tissues are yi'en. Dtslribntiim, storage, nod cMieOon rales dittinu lecdinu and allei discontinuance ol PCH io tile diet an pieseiited. Pulyihlurinateil biphenyls (PCBs) were first descrilx'd in 1881, by Schmidt ami Shultz. ( I ). Commercial production was begun in 1930 (2). These compounds lime been ami still are widely used as plasticizers and resins and in chlorinated rubber (3. 4). They are known In enbanee the insecticidal properties of lindane (5), DOT, ami dieldrin (ft). A bes- may be used as stationary phases in gas-liquid vhmniiilugrnphy (7). Their widespread use may have icMtlhd in contamination of the eco-systcm as suggested by many recent reports. Aioelor' and DDT were both used during World War II. In 11)18, total organic chlorine analysis provided data for the first determination of DDT storage in man (ft). Since this method has no specificity lor DDT. the lotal organic chlorine content would not preclude the presence nl oilier organic chlorine-containing moieties. In terms of halide .specificity, the Dnlirmami miet'ocoulomelric gas chrnmalogruphic method for detecting pesticides was well established by I960 (9), and eleelum-eaptmo was in use by 1961 (10). Iloburn (11) first observed extraneous peaks in 1965; lie used n potcntiomrtiic mode ol analysis lor total chlorine and found disagreement between this method ami total chlorine content calculated from gas chromatographic results in which \rgnn ionization and electron-capture were used for tin- analysis of tissues and 'Tlu- Mumuntu Company mamifaetmes I'd)* chiefly io the United States under the trade imine of Aioelor. They are manufactured in |;ram'(` by Prodelee, and in Urtninny by Hayer, ivilli trade names, I'liemicbloi and Colphi-n, respectis'cly. These compounds me designated by numbers. Tbe first two diuits represent tbe molecular type; 12, vblminnled hipbemix; 25 and II, blends of chlorinated bi|dieoyls and cldoiiuatcd teiplienyls (75V biphenyl and ftOV biphenyl, iespc< lively); 51, cblminated terphenyls. The last I"a illitils ul\e tile Weight percent ol cblnrine. Tims, Aioelor 1251 ami ]2(f0, with whieli this is concerned, aie chlorinated biphenyls containing 51 and (i()V chlorine, respecUvcly. 5) IO by Academic Press, Inc. 481 oa^oas 482 CUtU.KY t~r Al,, eggs of wild birds. The samples showed significant amounts of unknown electruu-eapturing compounds. Kobmn postulated that these were metabolic intermediates of one or mure of the ovgannchlorinr pesticides. The diiFerrmrs could also have been indicative of PCIl contamination. It was not until i960 (hat S. Jen sen (J2) using electron-capture and mass spectrometry was able to identity as lX'Bs unknown peaks found in the analysis of fish and a bird carcass. Undonbtedly, tbc time lag between initiation use and detcetion of PC,H in (he en\'iroitment is a matter of accumulative concentration and/or mslunm utatiim development. The presence of PC Iks in environmental samples has been well established ( Id, If). 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 hunts ol marine life ( 19. 21, 22). radiological changes in the liver mid other organs have been shown in guinea pigs, rats, and rabbits at high concentrations (22). Ilydropericaidium ami growth depression proportional to dietary levels of the toxicant have Im-cm pro duced in the chicken (23). Polychlorinated biphenyls are known to affect estrogen levels in birds which in turn reduces calcium reserves and results in thin eggshells. Chemical porphyria from oral adnmestration has also been shown in chicks ( IS). This paper presents distribution, storage, and e xcretion rates of Aroelor in en tail) tissues, body fluids, and excreta of rats following (1) a single oral dose. (2l repeated dietary intake, and (3) after discontinuance of PCB in the diet. j . 1 ! < 1 METHODS Aroelor 1254 and 1260 were supplied by the Monsanto Chemical Company with respective lot numbers, AK-38 and AK-3. (dietary formulations wen- prepared according to jhr methods described by Games ami Kimbrough (24). ISxi'KMIMKNT A Aroelor 1254 and Aroelor 1260 were administered at 16(X) mg/kg and 3200 mg/kg, respectively, as a .single oral dose, by stomach tula* to three female. 173to lS3*day-o!d. 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 (oxalatcd), fat, muscle (abdominal), liver, kidney, him*, and brain were taken for analysis. j 1 FxctCUlMKNT It Arnelnr 1254 at KMX) ppm was tod for OS 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 or death. The rats consumed an aver age of 72 7 mg/kg/Aroelor/day. blood (oxalatcd), la I, muscle (abdominal), liver, kiduev. lung, and brain were taken from each rat at time of sacrifice. Tissues wen preserved in 10V formalin and held lor annlys s. M0NS 0B.ZQ90 Slur chow < days. Fort icensers p of S day s. \ is-ing rat rats su re collection* kidnev, an feces wen plasma n maii/cd a (iced on I diet, two . Shenna dietary le initially a measured and 36.4 pies were FofvcM highly lij tins prop Plasma in the s|t No sigml with the Sumph lloinugt m (ham tracts svi Urine to a voh pies svrr I lie lu x. with 1.5 Feces pnls-eri/. on an at liXlTaOMCN'T C Sherman strain male uvunling rats (61- to 66-dav.s-old) were frd a diet of plain chow or chow fortified with Aroclor 125-1 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 ot S days, alter 42 davs food consumption was ineasored daily for 4 days. Sur viving lilts were weighed once a week. At various times during the experiment rats were ind vidnally homed in metalro) sin cages. Urine and feces were collected over a Ifidiuur period during which time plain chow was fell. 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 Iro/en 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, 31, 42, and 58. After discontinuance of PCB in the diet, two animals were sacrificed on l>a\ S 18. 24. and 71. Fxi'kiiimi nts D and 1 Sherman strain male weanlings (36- to 41-days-old) were fed Aroclor 1254 at dietary lewis of 100 ppm m 5(X) 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 Aroclor 1254 were 6.8 mg/kg/day and mg/kg/day for 100-ppm and 500-ppm dietary levels, respectively. Sam ples wen* collected in the same manner as Experiment C. Sami'lk Phiwamatiox Polychlorinated biphenyls like DDT, are not water soluble hut 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 et l. (25). Iaitcr in the study, tin* Bryaiit-Thompsun modification of this method was used (26). No significant dilfcrence has Ix-rn found between these methods in this laboratory with the exception that convenience favors the Bryant-Thompson modification. Samples of fat (230 mg), liver, muscle, kidney, mul lungs (500 mg each) were homogenized two times in 4 ml of hexane, and the extracts were combined. Brain (500 mg) was homogenized two times in 4 ml of acetone, and the ex tracts were comlrnvd (27). Urine (mean volume of 10 ml) at pll <7 (using pllydrion paper) was diluted tu a volume nl 25 ml hy the addition of water am) 1 ml isopropanol. These sam ples were extracted in 125-ml separatory funnels three times with 10 ml hexane. The la xaue extracts were combined, washed once with 3 ml of water, and dried with 1.5 g of anhydrous sodium sulfate. Peers were desiccated out 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 and placed in a 50-inl 4S4 CUMJCY KT AL. centrifuge tube. Particulate matter in the extract was removed hv centrifuging and/or decanting. Tlie extracts were evaporated with a stream of dry nitrogen to a volume of 0.5 ml in a water 1>atli (40). The acetone extracts were evaporated just to dryness and made to u volume of 0.5 ml in hexane. The samples were dried with anhydrous sodium sulfate ami held for cleanup and analysis liy election capture gas-liquid chromatography. j Phkuauation' of Su.ica (ha. and Mk:i>-C<u,ums Silica gel (27) from Woelro (Activity grade i) was activated at 130 for 4 hours ami desiccated over calcium chloride for 16 hours before deactivation at (v/w) lv the adtlition 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 alter activation, it was found to be activity grade 1 as specified by the manu facturer and further activation was unnecessary; however, each new batch was routinely activated. One gram of die deactivated silica gel was placed in a disposable pipet. winch had been loosely plugged "'ith silnnr/ed glass wool, and gently seated. Columns were prewashed with 10 ml of hexane. Kaeh sample was placed on the column in a volume of 0.5 ml. At least 1 ml of hexane was used to quantitatively transfer die sample' to the head of tlx.1 column. The column was eluted with 10 od ol a 1:1 benzene:hexane mixture (27). Samples were chromatographed in sets of eight with duplicate column blanks. Aroclor 1254 standards were eluted' simul taneously in order to ascertain tin- variance in silica gel activity and to detect any possible contamination from solvents, glassware, or silica gel. ' | i i ` ^ ('.as C.'itHOMAroenvenv Sample? from experiments, A, C, D, and K as treated above wen- 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 / sic-, pulse rale, 120 /see; detector, 'll, parallel plate design. No. 1 203. No. 2 10S0, column. (V X H" o.d. U-shapcd pyrex glass packed with 5'J OV 210 on SO/100 mesh ('hromosorb W, high performance, 170; inlet 2.4S"; earlier gas, argon-nietliam (95-5V), 50 psi; How-through Detector No. 1. 50 cc/ininnlc; Detector No 2. Wt ee/niinute. The DC mode was as follows: cell voltage, 13 volts; detector, II. parallef plate design, 210; Column, 0' X )V o.d. U-shaped pyrex glass packed villi 1.5V ()\M7/1.95`/ QF-1 on 100/200 mesh Chmmosorh \V. IIP, 200''; inlet, 2,10 carrier gas, udrogen, 40 psi; Hmv-through detector, 75 ee/minute. Samples from Kxperiment Jl were injected directly as hexane extracts and analyzed by electron-capture gas-liquid chromatograph)- under the following conditions using a MicroTek MT 220 gas clnomiitogi.iph; cell voltage, 40 vein DC; detector, ```Ni, 250; cuhuun, 6' X Ji" o.d. U-shaped glass column packed with J.5V OV-17/1.95'/ <V>F-1 on 100/200 mesh (Tromosorl, \\\ IIP 200C; inh-t 210; carrier gas, nitrogen, 40 psi; flow-through detector, SO ce/mnmte, 20 iv/imn ntc scavenger phis 60 ee/minnt(` carrier gas How. j j j j l I t 3 * P d t! tr g. h. mi th. th. m (h. st.l h.u HONS 0820 t I'Ol.VCill.OHINATI I) mi'lli:\YLS 485 | Hkcovkiiy Stuoics Hecmcry studies were complelrd using the following methods lor control ti.smicn, plasma, ami IS-hour mine a11< 1 trees collections. The fort ilical ion level lor radi type of sample was tin* mean concentration, with tin- exception of fat, of Aroclor 12-54 funnel for that sample in Jixperiment C. Tat samples were fortified with 100 ppm. Tissues (lat, liver. kidney, brain, and muscle): Aroclor was added directly to | the hexane extract prior to liquid chromatography. Plasma: Aroclor was addeil directly to fresh plasma, thoroughly mixed using I a mini-mixer, and allowed to equilibrate overnight under refrigeration. Samples i were then I reap'd as metilioneil in methods. | lares: Aroclor was added directly to pulverized dry feet's ami treated as men tioned in methods. 1 Urine (three slightly different approaches were list'd): t \o. 1--Amcloi was added to I ml of isopropanol, then mixed with a solution of twine and water ( Id ml + lo ml) and analyzed as mentioned above; No. 2-- I Aroclor was added to a hexane extract of 10 ml of urine which had been prepared ,k mentioned above; No. .1--Urine (5 ml) was added to a known quantity of ' \mrlnr in a 15-nil centrifuge tube, mixed on a minipiixcr, and allowed to equili brate at room temperature for 1(1 hours. Urine was extracted three limes with . 1 ml of hexane; the hexane extracts were combined ami evaporated to 0.5 ml. HKSl'l.TS AM) DISCUSSION | Hontnely. this laboratory operates dual channel solid state electrometers at a uiisitivity of 4 X It) amps hill scale which permits the detection of 10 to 30 Ijiicograins of the more' common chlorinated hydrocarbons. There was a .significant ililfcrcmv between the response characteristics of the Aroclors as compared to the chlorinated hydrocarbons. To achieve a representative gas chromatographic I traei*, at least 330 and 400 pg of Aroclor 1254 and Aroclor 1260 were required. A | gas chromatogram illustrating the ddlcvenee between the electron-capture rej vpimse characteristics ol these compounds as compared to chlorinated hydrocar bons is shown in Fig. I. bxi`:niM!-;\T A Data resulting from electron-capture analysis of plasma and tissues in Fxperiinmt A are presented in Table I. Quantitation is an approximation. In all eases, , the peak heights (in mm) of all IK'll comjMmentx were summed for the respec* live standard at a given pieogram quantity injected. A response factor was deter mined for each standard in pg/tmn. For each sample, a summation was made of j the total response (in mm), and the product of this times the response factor cal minted to give pieograms of I'Cll or PCH-derived material in the sample. When I the gas chromatograms for the samples were compared with those of the Aroclor slaixlanl, there were no recurrences of the general Aroclor pattern. Some peaks had completely disappeared. This was also true lor each of the succeeding studies. Although flic dosage level ol Aroclor 1260 was twice that of Aroclor 1254, the ! HONS 081093 480 CUHLliY KT A!.. Ah fr. in mi tlv 1'k;. 1. (..'its i-hrunintngraim for Aroclor 125-1 itml eliloiiniitcd hydrocarbon standard*. (A plot of detector response vs tone.) TA11I.K l DirentimmoN OK Ahoci-ohs 24-iioukh akti.u Ojiai. Imostion in Stomach Tuiik Avovlor 1254 (<ioMKt! level-- HHW HiR'kK) Aioelur 1200 /iliwatfe level- 4200 jiir Wr) I'lnsma Mean sK Bruin Menu SIC Km Mean SIC Liver Menu SK Kiln*y Mean SIC 1 Mean SIC Muscle Mean sic 24 <U s as i:ts on :m 41 1140 0 571 r> 141 . JJO 47 SO 27405 M) 17 05 111 2!l 4S Sll 20 07 ;>s 15 71> 0 00 145 17 <:'> :>u u;im n 420 0 250 I i i iii :I2S 5 114 o 105 17 21 ]:; .17 IHJ I di. tin -V> Ai 1 tvt mi f<-> T pr /V MUNS 082094 l rt>i.Yi:m.ont\Ari:i> m/vijk.vs i.s 4<S/ mean storage levels after 21 hours were essentially (lie same. Considerable vari ation iu (hr slor;ijif levels was fount! in each group. Dillrring absorption rates from (hr gut might aerounl for the variation in levels tumid; variation was highest in (hr hit and lowest in (lie plasma. According to Haves ('15), acute dosages, such as tlir one given here, do not result in storage, hut rather in a Hooding of |Ih* organism. ExeiJUMKNT B Only surviving rats wore 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. ICight 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 Aroelor 1251 ami reported lethal effects to none of 6 by 14 days, 1 of 5 by 28 days, 3 of 4 bv 43 days and 4 of 4 In 53 days including a 21% reduction in food con sumption. All survivors in Experiment B appeared to he normal; however no in crease in weight was observed in surviving males after 84 days of dietary intake mid after 77 days for surviving I.....airs. Food consumption for dosed males and females was 6ft and 047, respectively, of consumption hv controls. Data from electron-capture analysis of tissues and plasma are presented in Table II. The variation in die concentrations of PCB in this experiment, as in Ex periment A. is least in tile serum and l'ain and highest in the muscle and fat. A significant difference between males and females was nut found for storage of TABI.K II I liKTiiiw tiii.n iik l'( 'li-l liauvv.i Men mu. Iuh.uim imi tis-Do Kxivihchi; rn \ lJu.TUO i.i.Ml. m HMHl I'I'M Aihicuhi Mull- i|i|nl ; > 0 jn 08^095 IKS CUItLEY ET AL. Aroclor 1254. Hayes (35) and Dale at ol. (30) report that storage of DDT in Iniulr rats exceeds that <>l male rats when both are in.lint.lined on (lie same diet and that the enhancement in storage exceeds die greater loot) intake by females. The absence of certain peaks in tlu- Aroclor standard when compared to PCHderived material in the samples became more dramatic in tins experiment as compared to Experiment A. Cenorally, the percentage of matching Aroclor peaks found in each tissue was as follows: plasma 81V, fat 74V, brain 66V, liver 61V, long 637, muscle 62V, and kidney 5-SV, These differences could be attributed to: ( 1) selectivity in storage for each tissue; (2) enzyme activity resulting in some lonn of metabolism; (3) excretion favoring some isomeric lorim as opposed to others. liXl'KIUMl'NT t- The data front die 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 sleatly buildup of Aroclor in all tissues, while the excretion trend is quite erratic. I I I t U4U HUNS 0d-'096 l'0l.Yau.0lUX4TH)> mrilENYLS 489 1? I (I ft 'J: In l it., (ImuvtiliiUion nt I'CII mul l*(.'H-U'nvitl nmtvriul In frees ami urine fitmi nils mi u tllrl.if) level nl 10!) ppm .\mclur 1231. The excretion trriul may in- due in purl to the discontinuous collections; there fore, lliese results reflect only the excretion pattern at specific times. The residue levels ohserveii in ICxperimeut B created some doubt that levels observed alter the oS-ilay dietary intake were representative oi steady slate values for Aroclor 1251. Haves ( 55) states that DDT fed to rats at a toast.ml rale is increasingly stored in their iat until it reaches a plateau, ami on a diet containing 200 ppm or less this plateau is readied within 90 to 140 days. Dale et al. (38) foam! mean concentrations of DDT and DDL in the fat of malt* rats led 200 ppm in the diet ior 90 days to he 525 and 51 ppm, respectively, white male rats on the same diet fur 140 days had mean concentrations of 506 anil 42 ppm. A maximum concentra tion in liver, kidney, and hraiu is achieved within a lew days for a dietary level of 1000 ppm (35). lixmuMe.vr D and i Hesults of lxpcrhm*nt D showed that rats on the same dietary levels stnn d more Arodor in their tissues alter 240 days than at the end of 58 days. The 240day group showed no significant difference in the (piantity of the FOD-derived material loimd in the urine hut showed significantly more FOB excreted in the feces. Analysis of tissue, plasma, ami excreta from li.xprrmrnt E indicates that if; .a, h\ MONS U82097 490 CUMLEY ET AL. Aroclor storage, like DDT, is directly related to tin- daily dosage However, tlw * puiiils al which equilibrium storage is achieved arc yet to he determined. AI dietary levels of 100, -100, and S(K) ppm lor 2 years (37), tin- mean values >l DDT stored in the fat ol rats at equilibrium were reported as 93, 1028, am) 42tX) ppm. r< spectivelv. The concentrations of Aroclov 1254 found in the fat after 240 days at dietary levels of 100 and 500 ppm were 1101 and 10,021, rcsprctivrlv Ciemrally, the levels of DDT stored in the fat of male lats alter ISO days are equivalent to levels after 2 years (37), although there is some- reduction in DDT storage hryond 2 years. These levels in the fat illustrate the vast differences in storage at the steady states for these two compounds, and the vast differences in tlieir relative toxicides and/or lipid solubilities. (The brain concentration, rather than fat concentration is a better imlicat.'un of toxicity) (38). DDT storage cun lx* reduced if exposure is reduced or discontinued. Lang ami 1IV I.IVI .Mil lint KhI bin Kitzhugh (37) found refent on of 50-75* and 25a of DDT in the fat. '10 amlfW days after discontinuing diets containing 5 to 50 ppm. After 58 days on a dietarv level of 100 ppm Arcelor and a recovery time of 71 days, StW ol the concentration in the fat remained. Elimination seems to parallel that of DDT. These data indicated the concentration of Aroclor found at turn' of sacrifice to be in the following order: fat > liver > feces > kidney > brain > plasma > mine. The order of storage for tissues and plasma .urin.s to parallel lipid content. Petal excretion exceeds urine excretion with Aroclois as it does with DDT ami other chlorinated hydrocarbons, * t ' I | I Evaluation ok Mictijoo .4.) stitWilree levmo am per (L. The efficiency of the silica gel mieroeolumn and tin* extraction techniques em ployed were evaluated by determining the recovery of Aroclor 1254 from in oilm fortification. Recovery data for all samples elated from a silica gel mieroeolumn arc presented In Table 3. All in vitro recoveries, whether from liquid chromatog raphy or a combination of extraction ami liquid chromatography, were at an acceptable level with the exception of urine. Recoveries of DDT from human fn. cm ert wit col urine using liquid-liquid extraction techniques have been reported by Cwtu ami Riros (28) and Cranmcr et al (29) with recoveries of 72 and 96'i, respectively. i he' In both instances, their extracting solvent or combination of solvents was more ren polar than hexane. The recovery in approach No. 1 ami possibly 3 might he * 12: attributed to the partition coefficient of hexane in an aqueous system. In order to | 12` evaluate this, reciprocal p-vuhies were determined using the technique of Row- i sai man and Rernza (30). The average of triplicate analyses gave a reciprocal g-viilmof 0.761, or 76.4^ of the Aroclor was partitioned into the hexane layer from the aqueous system. This value agrees favorably with approach No. 3 but docs not amount for the 17V difference in approach No. 1. Zitko (20) states that he found fractionation taking place when emulsions resulting from mixing Aroclor 12.51 with water were broken by centrifugation. (Jus chromatographic analysis l ,i hexnuc extract of the supernatant showed it to he richer in the lower ehloiinated biphenyls than the original preparation. This varied with batches ot Aiocluv 1 . l J 1 aI of an Tl When a peak-for-pcak comparison was mad*1, whether in vitro fortification ni p-value studies were being considered, recovery was generally low for all cun- ) MONS 08^09 i'(>i.van.oiii\AT:j) diphenyls 491 Ih.ruvuit m In Vitimi Fme Ti-m- TAItl.K 111 Wciiilii Sami'I.ks w ith Auoi.wm IJAI Fi>rlifii><l (ppm) Alt l'2A4 Pm mil mi-overy" I'iUNllUl Km Livvr Miwrlc lluiili Kiilin-v Fi*ri> I iiim' (Appnmeh l,J,4) 2 <1 ,2.j() .'>011 .Ann .Ann Aim 1 IHHI o r too <> 1) in :s .o 20 Hi) o. 1 0l 0I !IN < S2.2 `14 S int i 100 4 100 (i `HI N ttt 1) 114.(1 77.0 H I'liiMtmi, fill, liver, nniM'lr, liniin, kiiiney, fr>v*--metin of 4 (li'tmniimlioim, mill miiic A menu of iliiplMiiie ileifiiiimiiiiiiiii*. Ktiluenls. ami the average recovery was reported. Low recovery from the urine was probably due to tile extracting solvent. The values reported were not correele;l for in oilro recoveries. Although p.//DDT and p,//-DI)L 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 tissue or exerctu extracts cannot In* determined at this time, Before and alter elution from silica gel there usually was a marked dificrcncc in the appruruncc 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 sample's.) Muscle mid plasma were the least affected by li<jnid-solid chromatography and could have been analyzed very easily without this treatment. In fact, for the con centrations of Arodor found, most of the samples could have been analyzed without mn' cleanup, taking into consideration the problems associated with column and detector contamination. Alter elution patterns of Arodor 1254 Mere established, 10 nil ol benzine: hex.me were not retpiired for i|uantitutivc elution of PCB, in fact, only 4 ml were required. This saved time in subsequent analysis. The elution pattern of Arodor 12-54 was unchanged at the fortification levels mentioned in 'Fable III. The Arodor 1251 standard that was chromatographed simultaneously with each hatch of samples analyzed had a mean recovety of I0W with a SIC of 3.1 for 23 samples. Ixtkiokulncl ok DD T with PGB Analysis The presence of PCBs, DOT, and DDT-likc moieties in samples presents n pruhlem hot)) qualitatively ami quantitatively during analysis. Initially, samples of liver and fat from control rats for each sampling period were analyzed for DDT am) its metabolites; samples were taken from the oldest control rat in the study. These tissues usually showed measurable amounts cif DDT, DDD, ami DDL. The values for liver and fat. respectively, were as follows: ;:.;/-DDT, 0,046 and 1.306 ppm; p.p'*DDD, 0.036 and 0.546 ppm; p.p'-DDK, 0.016 and 1.09'S ppm. Since preliminary evaluations showed liver ami fat storage of PCBs to be a m cuiu.ly rrr al. minimum ol JO to JOO times the above mentioned values, interference was urn- sidered to lu* nonexistent. However, three possible approaches to separation <>1 F('R aiul l)l)T-like materials without any ehemieal degradation of the pesticides eoneerned were evaluated. Thin-layer chromatography was considered first. Using siliea gel (J and essentially the techniques as outlined by Walker and tlero/.a {ill), both Aroelor 1251 and Aroelor 1260 in the presence of the chlorinated hydro carbons, p.p'-DDIC, p,p'-DDT, <>,p'-DDF, o.p'-DDT, diehlrin, and heptachlor epoxide, moved with the solvent front. Using polar and nonpolar mobile phases, die Hf values for FCRs ami p,p'-DDF were identical and not resolved. This can possibly be used in confirmation and identification nf Arndors. The two other approaches utilized column chromatography. Horisil was used. as proposed by Reynolds (32). PCiRs are eluted from the column with hexane, while other chlorinated hydrocarbons are eluted with a dn-diyhthci hexane mixture. Reynolds evaluated Florisil with a mixed standard of Aroelor 1254 ami a chlorinated hydrocarbon mixture containing lindane, licplacliloi. aldrin, heptsichlor epoxide, p,p'-DDF, diehlrin, p.p'-DDD, and p.p'-DDT. Alehin. p.p'-DDIC, and heptaeblor are eluted in (he Will fraction. Although these pev ticides were not separated, this approach might still be acceptable since aldrin ami heptaeblor are seldom found in environmental samples in their uncpoxyduled tonus. However, whin an attempt was made to reproduce the work of Reynolds using PH grade Florisil, a mixture of Aroelor 1254 plus the chlorinated hydrocarbons, lindane, hcplachlor epoxide, diehlrin, p.p'-DDD, p,p'-DDli and p.p DDT, measurable quantities of p.p'-DDT, p.p'-DDD and lindane were found in Fraction 1, i.e., 66, 16, and .'12/ of the total, respeelivelv. Reynolds diK*s not mem lion the quality of the iTorisil although he docs mention how it was handled. The problem, however, seems not to be related to the grade of Horisil. Revenue and Ogata (33) have presented data using both qualities of Horisil ami were unable to reproduct* tile work of Reynolds. There is some disagreement between the fuu-tinning pattern of Revenue and Ogata (33) and the one observed in this work. This eimld probably be due to tin* quality and treatment of the Florisil These problems suggest that the elution characteristics of Florist] should he evaluated before use for separating PC'R and DDT. . Armour ami Rurke (34) used a column of silicic acid (treated) and Celite 545. The column is eluted first with petroleum ether lor PCBs and then with a mixed solvent system of dichlorometham'-acrtoniliile-liexane (80:I;16) for the chinrinalcd 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 /^-isomer of RJIC, heptaeblor epoxide. o,p' DDF, p.p'-DDF, diehlrin, p.p'-DDD, and p.p'-DDT. Separation has Ihcsv a problem with the p.p'-ODF; Fraction 1 contained 20'* p,p'-DDF with then-maming NOtt in Fraction 2. Although some recoveries were less than 85*/, all other peslcides evaluated were only found in Fraction 2. According to Armour and Ibuke (34) the elution pattern can be altered by the water content of the silica acid. The first pesticide to be a/leeted in the group of compounds that wen* used to show tin* changes was p.p'-DDF. The lact that there was not a complete peak for peak match between stored and l J | I | I > * ) j i j 1 1 , * I i 1 i I 1 MUSS 082100 TOT?PO SNOW 194 cuiiley i:r ai.. excreted PC^II and the dietary standard is not new (Pig. I). Whether tins is evidence tf metabolism is vet to be diter'mined. These dilli remcs were successlolly demonstrated hy eleetron capture gas-liquid chromatography ami gas chro matography-mass spectrometry in (hat there1 we re changes in the relative abun dances of some of the components of Aroclor 125-4 in fat ami the mine samples. This has not yet her n demonstrated in any other tissue. Tin- mass spectra showed molecular ions at m/c 2S.S, .123. 33.8, ami 192 eonlaming Cl,, (3.., C'l.,, and 01- isotopic clusters in the fat and 234, 288,124, and 358 in (lie urine. Two additional ions which were observed in the urine nnlv, were m/e 304 and 405. These ions contained the (3, isotopic cluster The presence of unaltered Aroclor in the feces may he indicative of lack of absoipt on; this was the only sample that showed no alteration w hen compared to standard Aroclor 1254. The neutral urinary excretion pattern was quite the contrary to feces. This mav in part he due to a solubility phenomena which Zilko (20) rucunntercd when working with IT.'B and water. The situation ol Zitku with either urine or alcoholwater mixtures under in vitro conditions was not reproduced. Under iii vivo ton* dilions, there may be selective solvation of certain constituents of Aroclor Selected s imples of urine were analyzed lv gas chromatography us'iig the electrolvtiv conductivity detector in the reductive mode. The pic.senee of chloride ion was further substantiated. The retention times of these components compared fax nrablv will) peaks in the Aroclor standard. * I L . ! ( I ^ I ' ' I \ [ COXCJ.t'SlOXS it is hoped that these data xx'ill elucidate some oi the problems inherent in studying the polychlorinated biphenyls; and that some correlations can be found between the residue levels reported and the LIT,, detemviiations including the pathological findings to be reported iu a later public at.on by Kimbrough ef <il (40). There are some basic generalizations that can be drawn Inmi the data presented: (1 ) I'ollowiiig dosage with Aroclor 1234 or 1200, whether acute or chronic, residue amounts ean be detected in all body tissues, fluids, and excrement. (2) At the same dosage- level rats store more ITUs than DOT. (3) No sigirlicant d llerence is apparent in I be storage o| l*CBs bv male or !i male rats when led the same dietary levels. (-1) Bolvchlorinatcd biphenyls are stored primarily in adipose tissue. (5) S uer there was no rmimiur of the general Aroclor pattern in the gas chromatograms or total ion current traces of the mass spectrometer betxvcen the Aroclor standards and components observed iu the fat and urine, pos sible metabolism nr differential absorption is suggested. * , I i ! ' .1. ' I. > 5. I (i. I 7. t n. i y. t 10 it. 12. I.) II. 15. HI 17. 18. 10. 20. 21. 22 21. 21 20. 27 2H 20 :VJ Jl 32 Ti 31 35 V a(;k\o\vi.kix;mi:nts \\V l*iiuik Mrs. KstelU1 Cav for statistical analysis. Mi. Tliim.is It. t.auws amt l)t U D kiinlnniiuli lor assistance vvitli 11 it- animal studies. 1. N* iiwinr, 11 2. I'l.VKU.l.. I ) V 1 hki'i:mi:.\ci:.s I) Snei.i v, i;. ( IBH1 ). A/m. C/mm. 207. 13K-II. It., asm !.i\u a, J. t,, (1U70). lint. Sri. 20 ( 17). l MoiimiiiIii Trrl.Mli;,I H,,11,1ms 0/f|,ill\, O/I'l ,-KHj. O- IT/ 1 l. Mnii\uiilo IVrliiiti.il lltillrliiiN O/l'l.- lI I A, 20. 5. (ltm\SIKI\, |., AMI SUI.UVAN, W. \. (1 JJ.VJ >. J. I'.tint. I'ntoauil. 4(1, 037. (. I.ICIIIIMIHN', K. I*. (1000). J. linm. I.nlnutol. 02, 701. 7. Stiprlm, Inc. CiitnlnK (1070). . Iltiuil.i,, |). l\. ( 1048), htH\ Okfo. Aeuil. Sei. 21), 31. 0. (.'m i.miv, I). M,, Civwawi, L. A., Di.Vhii-.s, J. K., a\ii Wot.intai, IF (1900), ). Auric. Fihh( Cftrm. H, 300. )(). (atnitvvi\, K. S., Cnvi.noN, IF. am> UivY.voi its, J, (J. ( 1901 ). Auahjst (London) 81, 007. 11. IlniiuiN, J, < 1005). Analyst ( I.iiikIihi ) 00, ( 1073) 4 13. 11 Atillmr imUmvii (1000). .Yen S.i. 32, 012. 13. Mimhimih.ii, U. \V., Kiimim, I'., I'iakii.i., D, II., IJi:imv\, S. and kinvi.s, M. N. I 10001. VfiMnr 220, I00.N. J FJismn, S. (1000). Salute 224. 217. 15. Tii'kni. M. k.. (Mi Chvmtioa:, I). <. (1070). "IkmiltiiMik of Tovicily of IVsik-kles to WilillHr" I'. S. Dcpl. oi hilriior, Ilinriiti ol Sp irt Fi>,lw-rirs imd Wildlife. HrMtmvrs I'til,- liiiihnii Nn, HI. 10, Fio.am. I). 1/ ll. (1008). "IVsiicidr llumllio.ik Knloiim." ('ullrur Science 1'iiMisliris, Skill- Cull, ur. 10801. 17. I'm m i . I., Jkimuks, 11. J.. and Mooiii:, ,\. W. Kmimn. I'hIIhF 1(1), 3-20. 1H. )): Vns. J. ()., ami ki:vi\\, J. II. ( 1070). Toxicol. A)/</.I'hariiun-ol. 17, 050-08. 10. Dim. T. \V , Lnvu. J. I., ami W'ii.min. \. j., |n. (|070).Hull. Kmiron. Coutamin. Toxicol. 5. (2). m. /.iimi. V. ( 1070). Hull. limiiiMi. Ctinitim. Toxicol. 3 (3), 270-285. 21. Wildish. I). J. (1070). Hull. T.iiviton Contain. Toxicol. 5 (3), 202-201. 22. Mii.u t, J. W. ( 1011). full. Ilnilih Heft. 50 ( 33), 1085-03. 21. Fl.H K. D I-'. ( 100)). foultiy Sri. 44. I 100. 21. Cvimis. T. IF, am, kiMHnniH.li, M. |). ( 1001), Hull. W. II. O. 31, 737-715. 25. Dm.i.. Wm K.. Chiu a. A., ami Cn.io, C. ( 1000). Life Sei. 5, -15-51. 20. I'm (Vi, A. C.. ami TiioMfsov. |. (to lie pidilislied). 27. jiNMM.s, K. W. ( 1008). llinlnuUill Sample Clr-.mnp lor CMiiviimU-d IlytUm-.irLon mul OitfiuiupImspltDiuv AihiIamh Cmiiu KleeUnii-CVptiiie (kiN-LJipjid Cltruumkiuraplty. Pupil pn-M-iilnl Nul l. ACS Slitting, Allunlie < Tl>, \j, JH. Cu.m, C.. ami limns, F. (1007). Toxicol. .\)/>), Phannol. 10, 201-200, 2(1. Chas'M m. M. Cahi ni.i.. J. |.f and Ooi'h.avii, M. F. ( 1000). Hull, Knvlron. Contain. Toxicol. 4 I I). 211-2). 30. MnVVMW. M, <!.. AND lll-lin/.A. M. (UHi5). Journal of the A.wocinlion of Official Auri- euiluittl Chemist* 4S (5), 01.3-052. 31. W.u.ki.h, K. C.. and Dmio/.a, M, ( 11813). Journal of the Amiciation of Official Afiii- cuhnnil Chemists 40 (2), 250-201, 32. HtiiNni.Ds, I.. \l. ( 1000), Hull. F.nciron. Conlam. Toxicol. 4 (3), 28-12. 31. lim.Nt i , A., ami Oi.aia. J. \. (1070). J. Ctnamulo. 50. 112-11. 3-1. Aii'ioin, Jami liiuiKii, J. (1070). Journal of the Association of Official AnahjtUul Chenuxis 53 (4). 701-0K. 35u. IIavi.s, W, J.. |h., ( 1050). "I)I)T," S. \V. SiDimims, lliikliimsn Verlay. Husrl. It. IJ vw s, W. J.. |h. ( 1005), Aim. Hit. fhanaol. 5, 27-52. 30. Dau-:. W. Caim-.s, T. IF. ami Hams, W. |.. In. (1002). Toxicol. Afi//. Pharm. 4 i l ). `"i.t'W. 37. Lam,. F. I'., and Fii/iik.ii. O. (;. ( 1010). /. fhann. 87( I ), tK-23. 3H. Dvl.i, \V. J!.. Cainks, T. I)., (Mi Uvu.s, \\. Jn. ( 1003). SWrnrr 142, (3508), ) 171. .10. l.vvv. ).. M.. ,mi Cuihuiv., I). Ihjiicul Chemist. 53 (0), 1270-80. 10 kiMimniui, It. I) , vMt Caims, T ( 1070). Jnninul of the Axxticialinn of Official Anai. Health (in pn-s: MUNS 0dZ103