The distribution and elimination of 2,5,2′,5′-[14C]tetrachlorobiphenyl in rainbow trout (Salmo gairdneri)

TOXICOLOGY

The

AND

APPLIED

PHARMACOLOGY

Distribution biphenyl

(1977)

39,329-338

and Elimination in Rainbow Trout

of 2,5,2’,5’-[i4C]Tetrachloro(Salmo gclirdneri)’

PATRICK D. GUINEY, RICHARD E. PETERSON, MARK J. MELANCON,JR., AND JOHN J. LECH’-~ Department

School oj’pharmacy, of Pharmacology,

University of Wisconsin, Madison, Wisconsin 53706 and Medical College of Wisconsin, Milwaukee, Wisconsin 53233

Received

July 9,1976;

The Distribution and Elimination in Rainbow Trout (Salmo guirdneri).

accepted

October

7,1976

of 2,5,2’,5’-[14C]Tetrachlorobiphenyl

GUINEY, P. D., PETERSON, R. E., M. J., JR., AND LECH, J. J. (1977). Toxicol. Appl. Pharmacol. 39, 329-338. The distribution and persistence of 2,5,2’,5’-[r4C]tetraMELANCON,

chlorobiphenyl were studied for 140 days in rainbow trout which were exposedto [14C]TCB for 36 hr and transferredto hatchery raceways.The distribution of 14Cwas determined immediately following the exposure and at 1, 2, 7, 15, 28, and 56 days later. Carcass, muscle, skin, lower

gastrointestinaltract, and fat containedmost of the 14Cand a total of 88% of the radioactivity

was located in these tissues. During

the first 14 days,

14Cin adiposetissue,carcass,and eyesincreased,possiblyat the expenseof other tissues.The disappearance of r4Cfrom most tissuesstudiedappeared to be a biphasicprocess.Approximately 30% of the compoundwaseliminated after 2 weeks,but only a 6% losswasobservedin the following 126 days. The initial rapid lossof the [14C]TCBhad a half-life of 1.55 days. Subsequent,slowereliminationhad a half-life of 2.66 years. The bile and blood initially contained relatively high concentrations of 14C, but disappearancefrom thesesiteswasrapid and nearly complete. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants. Because of their chemical stability, inability to conduct electricity, and fire-resistant properties, they have been used in a variety of industrial processessince the 1930s but were not recognized as environmental pollutants until 1966(Jensen, 1966). PCBs are persistent, and they accumulate in the food chain in much the samemanner asthe organochlorine pesticides(Lieb et al., 1974). Veith and Lee (1971) have reported the presenceof polychlorinated biphenyls in a wide variety of fish. Fish taken from Lake Michigan often contain higher concentrations of PCBs than those caught in other Great Lakes (Panel on Hazardous Trace Substances, 1972). Lake Michigan lake trout, rainbow trout, and coho salmon have been found to contain PCBs in concentrations of lo-20 ppm (U. S. Fish and Wildlife Service, 1974). 1 Supported by Grants EPA R803971010 and NIEHS ES01080, University of Wisconsin Sea Grant College Program, and University of Wisconsin Graduate School. * USPHS Career Development Awardee, ES-00002. 3 To whom reprint requests should be addressed. 329 copyright 0 1977 by Academic Press, Inc. All rights of reproduction Printed in Great Britain

in any form

reserved.

ISSN

004-008X

330

GUINEY

ET AL.

It has been shown that goldfish (Hattula and Karlog, 1973), spot and pinfish (Hansen 1974) take up and accumulate PCB even at very low water concentrations. After being absorbed by the fish, the PCBs did not appear to be eliminated very readily. Lieb et al. (1974) studied the elimination of dietary PCBs from rainbow trout by using a PCB-free diet or fasting. The trout which received the PCB-free diet grew and the PCB concentration decreased, however, the total amount of PCB per fish remained constant. The fasted fish lost weight and their PCB concentrations increased, but the total amount of PCB per fish remained constant. This study was initiated to examine the distribution and elimination of [14C]TCB in rainbow trout over an extended period of time. Commercially produced PCBs usually contain a mixture of about 50 different isomers. Since the complexity of these mixtures makes most studies difficult, a single 2,5,2’,5’-[14C]tetrachlorobiphenyl (TCB) isomer was used to circumvent some of the problems with interpretation of data encountered in other studies which used industrial PCB mixtures. This particular PCB isomer was selected because Veith (1974) showed that it was one of the major PCB isomers found in Lake Michigan fish. et al., 1971), and yellow perch and rainbow trout (Melancon,

METHODS Chemicals and Animals

2,5,2’,5’-[14C]Tetrachlorobiphenyl (TCB), with a specific activity of 4.2 mCi/mmol, was purchased from California Bionuclear, Sun Valley, California. The labeled TCB was diluted with unlabeled 2,5,2’,5’,-TCB obtained from Analabs, New Haven, Connecticut. The final specific activity was 0.004 &i/,ag. The labeled compound was purified by thin layer chromatography on silica gel using hexane, after which the purity by TLC was found to be greater than 99 %. Two-year-old rainbow trout, reared at the Wisconsin Department of National Resources (DNR) Class A Nevin Fish Hatchery in Madison, Wisconsin, were obtained as a gift from the Wisconsin DNR. Analytical Procedure

The initial background contents of PCB in five unexposed fish were determined before exposing the test fish. The Fforisil column extraction technique for removal of PCBs in fish, as described by Hesselberg and Johnson (1972), was used with the following changes. The Florisil, 60-80 mesh, was activated for 4 hr at 450°C instead of at 600°C for 2 hr. Chromatography columns used were 2 x 34 cm without integral reservoirs. The PCB extracts were eluted at a column flow rate of about 5-6 ml/min. The final column eluate, 170-180 ml, was concentrated to 6 ml on a hot plate and injected into a Hewlett-Packard 402 gas chromatograph equipped with a 63N electron capture detector. The column was packed with 80-100 mesh Corning GLC-110 glass beads coated with 1.5 % OVI 7 and 1.95 % QF- 1. The column was operated at 170°C the detector was operated at 250°C and the nitrogen flow rate was maintained at 31 ml/min. The characteristic PCB isomers contained in each of the samples were compared by relative retention time to Aroclor 1248 reference standards run under identical conditions. The areas were compared quantitatively and the concentrations were calculated by the procedure described in the National Pollutant Discharge Elimination System, Appendix A (1973). A similar prodecure was followed to determine PCB background

TETRACHLOROBIPHENYL

IN

RAINBOW

TROUT

331

contained in the fish meal used to feed the fish. Extractions of PCBs were all greater than 95 % by this technique and there was less than 4 % variation between duplicate samples. Exposure

Three hundred microcuries of [14C]TCB in a small volume of acetone was added with vigorous stirring to 150 liters of spring water (0.5 ppm). One hundred 2-year-old rainbow trout weighing about 120-130 g were then exposed to the water-borne 2,5,2’,5’[r4C]TCB. Water samples were taken throughout the exposure and analyzed for 14C content. The water temperature was kept constant at 11°C and was aerated with pump and air stones. After 36 hr of exposure, the trout had taken 71% of the [14C]TCB from the water. At this time, the fish were transferred to a 60 x 3-ft outdoor concrete raceway with PCB-free flowing spring water, where they remained throughout the rest of the study. Analysis of 14C Radioactivity

Complete necropsies were performed on five fish, sacrificed by a blow to the head, at the time of transfer to the raceway (T,) and at 1,2,7, 14,28, and 56 days. In addition, total radioactivity in five fish was measured at 140 days. The weight of each fish was recorded, as were the weights of each total tissue mass analyzed. Radioactivity was determined in adipose tissue (obtained from the mesenteric walls of the GI tract), muscle, liver, gonads, all skin except from the head and caudal fin areas, brain, gills, heart, kidneys, upper GI tract, lower GI tract, eyes with periorbital fat, bile, and gallbladder. The carcass, including fins, bones, and cartilagenous material, which remained after removing all other tissue, was homogenized with an equal amount of dry ice which was allowed to sublime before analyzing for 14C. To estimate the total amount of 14C in each of these tissues, 4-125 mg of material was oxidized in a sample oxidizer (Model 306, Packard Instrument Company, Downers Grove, Illinois) and collected in a mixture of 7 ml of Carbosorb II/12 ml of Permafluor V (Packard Instrument Company), and the radioactivity was estimated in a liquid scintillation counter (Isocap 300, Searle Analytic Inc., Des Plaines, Illinois). Duplicate samples were oxidized and 14C was counted for each tissue. The amount of radioactivity found in the first group of five fish (To) was arbitrarily designated as 100% and 14C analyses thereafter were expressed either as a percentage of this initial loading dose for each tissue mass or as the actual concentration in each tissue (parts per million). RESULTS

Analysis of the fish performed before exposure showed that there was background contamination with PCBs of 0.13 f 0.02 ppm (approximately 16 pg/fish). The fish meal was found to contain 0.16 + 0.03 ppm and, based on the amount fed, the maximum dietary PCB intake by the fish studied for 140 days was less than 70 pg. Apart from one spontaneous death in the group, shortly after the exposure to 300 ,Ki of [14C]TCB for 36 hr, there were no observable effects on the fish and no other mortalities occurred. The initial concentration on a whole fish basis was 4.2 5 0.9 ppm. The mean total amount of TCB in the first five fish analyzed after the exposure was 530.9 + 14.7 pg, which was assigned the value of 100 ‘A 14C residue.

332

GUINEY ET AL.

I

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T t = 2.66 ‘12

50

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yrs.

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2.0

t = 1.55 days

‘ts

$2

s

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20

40

60

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100

120

140

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to TCB-free

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1. Whole body elimination of [‘%]TCB residuesfrom rainbow trout. The values used to calculate the initial rapid phase were obtained by extrapolating the slow phase to zero time and then subtracting the values of the extrapolated slow phase from the uncorrected rapid phase portion of the curve. The t$ for the slow phase was calculated from data points from 14,28,56, and 140 days. Each point represents the mean + SE of five fish. FIG.

The elimination of radioactivity from the whole fish is shown in Fig. 1. Initially, there was a rapid loss of the radioactivity with a t1/2 of 1.55 days. This was followed by a slower elimination with a t$ of 2.66 years. Approximately 30 ‘A of the compound was eliminated after 2 weeks, but only a 6 % loss was observed in the following 18 weeks. Figure 2 shows that, immediately after exposure (T,), 94% of the radioactivity in the fish was located in the carcass, muscle, skin, lower and upper GIJracts, visceral fat, eyes

Muscle Skin Visceral fat Rricfbital fat Lower G.I.T.

UPW

G.I.T.

0.5

*’0

28 time after

tranferto

35 K&free

42

49 water

56 (days)

2. Elimination of 14C from major tissues of distribution. Values shown are the percentage of the initial total fish residue contained in the indicated tissues. Each point represents the mean + SE obtained from five fish at each sampling time. FIG.

2.6 f 0.29 1.6_+0.39 0.97 ?c0.09

0.36 f 0.03 0.33 0.11 f* 0.13 0.01

Gills Gonads Liver

Kidney Brain Heart

0.36 + 0.21 0.09 0.07 k* 0.03 0.01

1.8+ 0.13 0.09 * 0.04 0.66 + 0.04

Day 1

0.33 f 0.01 0.06 0.06 + &-0.02 0.01

2.2 f 0.69 0.04 f 0.14 0.61 f 0.08

Day 2

3.0 IL 0.2 8.8 + 2.8 2.7 f 0.1 2.1 + 0.1 3.5 f 1.0 2.0 IL 0.1

Gills Gonads Liver Kidney Brain Heart

1.4-tO.l

1.9kO.5

2.8 + 0.1 3.6 f 0.8 2.0 f 0.2 2.0 + 0.1

Day 1

[14C]TCBIN

2.8 & 0.6 3.2 f. 0.9 2.0 f 0.1 2.0 t- 0.2 1.4 f 0.3 1.2 + 0.1

Day 2

a Data represent mean f SE of values from five fish at each sampling time.

Day 0

Tissue

OF

MINOR

TISSUES OF DISTRIBUTION

0.23 + 0.02 0.04 0.05 + f 0.01 0.01

1.9 + 0.30 0.03 + 0.01 0.54 f 0.14

Day 14

0.17 f 0.02 0.03 0.03 + & 0.01 0.01

3.2 + 0.46 0.08 + 0.01 0.50 + 0.12

Day 28

2.6 + 0.4 3.2 + 0.9 1.7 * 0.1 1.5 & 0.1 2.0 + 0.3 1.2 * 0.2

Day 7

0.7 * 0.1 1.1 4 0.1

1.0 + 0.1

2.2 & 0.2 1.2kO.2 1.2 + 0.2

Day 14

2.9 + 0.1 1.1 + 0.2 0.9 Yf0.2 0.7 * 0.1 0.7 5 0.1 0.6 -t 0.2

Day 28

Concentration (parts per million) after transfer to TCB-free water”

CONCENTRATION

0.26 f 0.08 0.08 0.06 ff 0.02 0.01

2.0 + 0.31 0.02 f 0.01 0.55 f 0.05

Day 7

TABLE 2

’ Data represent mean + SE of values from five fish at each sampling time.

Day 0

Tissue

OF [14C]TCB FROM MINOR TISSUES OF DISTRIBUTION

1

Percentageof initial total 14Cafter transfer to TCB-free watera

ELIMINATION

TABLE

1.2& 0.3 0.6 -t 0.1 0.8 + 0.1 0.6 + 0.1 0.6 & 0.2 0.5 + 0.1

Day 56

0.19 + 0.03 0.03 0.03 *+ 0.01 0.01

1.4i- 0.39 0.04& 0.02 0.41 & 0.06

Day 56

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$ 5

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334

GUINEY ET AL.

with surrounding periorbital fat, bile, and blood. Within the first 14 days, there was a redistribution of the radioactivity to visceral fat and periorbital fat (eyes), as seen in Fig. 2. An examination of these elimination curves for the major tissues reveals that the percentage initial total residue contained in most of these organs reached a plateau by the end of the 2%day period. Carcass and muscle contained the highest percentage of the total 14C residue, probably due to their large tissue mass. These were followed by skin, visceral fat, eyes, lower GI tract, and upper GI tract in decreasing order. Bile initially contained 3.12 f 1.88 ‘A of the total 14C residue, but this decreased to the low value of 0.03 + 0.01 y0 after 14 days. Blood showed an even more rapid decrease from

-‘I----------+

Periotbltal fat

0.6

0.1 I 0

7 14 time after

21 28 35 42 transfer toTCB-free

49 56 water (days)

FIG. 3. Concentrations of 14C in major tissues during washout period. Values shown are calculated as parts per million of TCB in the major tissues of distribution. Each point representsthe mean + SE obtained from five fish at each sampling time.

1.12 + 0.01% at To to 0.04 4 0.01% after 2 days (Fig. 2). The data compiled for all of the other tissues analyzed, which were involved to a lesser extent in the distribution of TCB, are presented in Table 1. In general, each of these organs also showed a decrease in the percentage residue with time. After 56 days, these organs accounted for only 2 % of the initial 14C residue. Figure 3 shows the change in concentration (parts per million) of TCB-derived radioactivity during 56 days. Visceral fat and eyes with surrounding periorbital fat contained the highest concentrations. Adipose fat reached a maximum of 54.14 + 9.15 ppm after 7 days. Skin, carcass, lower GI tract, and upper GI tract contained lower concentrations in decreasing order, respectively. Muscle contained the lowest concentrations of TCB, despite incorporating a large percentage of the total body 14C residue in its mass, Bile actually contained the second highest concentration (19.65 f 3.09 ppm) initially, but decreased rapidly (0.85 it 0.11 ppm) after 28 days. Blood showed a similar pattern

126 & lib 126 + 20 149 * 15 151 + 14 153 + 19 163 & 14 193 + 11 304 + 22

0

[“‘C]TCB

t 0.4 2.4 + 0.6 2.0 + 0.8 1.2 k 0.7

26

4.2 & 0.9 3.9 + 0.9 3.0 f. 0.7 2.8 + 0.5

TCB (Ppm)

3

100.0 ?c 6.4 93.8 * 9.4 82.2 * 13.1 76.3 + 10.6 69.2 + 5.4 68.6 + 12.3 67.6 + 7.1 63.3 fi 4.4

+6

62

+6

55 * 13 51 + 14 53 + 11 56 f 18 56 + 14 61

Weight (53 4.8 * 0.4 3.1 +0.1 3.4 + 0.2 3.4 * 0.4 3.3 +0.3 2.7 * 0.2 2.7 kO.3

TCB (mm)

Carcass

30 + 1.74 38.9 f 1.78 35.7 + 5.73 30.1 + 4.89 25.9 + 4.23 28.7 + 3.86 26.9 + 3.27

Percentage of total residue 37 z!z 3 36 f 7 40 * 5 39 IL- 9 48 + 12 72 + 18 86 * 11

Weight k) 2.7 + 0.6 2.0 + 0.4 1.4 + 0.3 1.3 * 0.4 0.9 kO.1 0.6 + 0.1 0.5 * 0.1

TCB (wm)

Muscle

f.

+

+

+

+

+

*

22.4 5.15 16.0 2.36 9.7 2.39 10.6 3.04 10.1 1.52 9.9 1.97 11.2 0.98

Percentage of total residue

FROM WHOLE FISH, CARCASS, MUSCLE, AND VISCERAL FAT WHEN [Y]TCB TRATION (ppm) AND AS PERCENTAGE OF INITIAL TOTAL BODY BURDEN

Percentage of total residue

Whole fish

OF

a Time after transfer to TCB-free water. b Data represent mean + SE of values from five fish at each sampling time.

140

56

28

14

7

2

1

Weight (8)

OF ELIMINATION

Time” (days)

COMPARISON

TABLE

0.33 * 0.5 0.41 f 0.13 0.38 * 0.04 0.36 * 0.07 0.59 * 0.18 0.87 + 0.18 0.83 + 0.14

Weight (24

16.9 2.9 39.1 & 5.3 39.7 + 3.8 54.1 * 9.1 44.9 f 5.8 32.5 rf. 1.9 25.9 + 1.1 f

TCB Cmm)

1.2 + 0.15 3.9 + 0.71 3.4 + 0.36 5.5 &- 1.70 6.2 + 1.78 6.3 + 0.68 5.3 2 1.28

Per centage of total residue

Visceral fat

LEVELS ARE PRESENTED AS CONCEN-

336

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AL.

with a more rapid loss of radioactivity. The concentrations contained in the organs which were involved to a lesser extent in the distribution is shown in Table 2. Except for adipose tissue and eyes with surrounding periorbital fat, the concentrations in each group of tissues analyzed decreased with time. This was due, in part, to the removal of radioactivity from the fish, but was largely a function of the increased tissue mass caused by the gain in whole body weight for each fish. Table 3 summarized these effects for whole fish and for three of the tissues. For each tissue, the concentration decreased steadily with time, while the decrease in the percentage initial total 14C residue contained in each tissue was much slower, indicating that the decrease in TCB concentration with time was largely due to dilution by growth rather than by elimination per se. DISCUSSION Some general comments concerning the distribution and elimination of [i4C]TCB can be made. Shortly after the exposure, the 14C was rapidly eliminated from the blood and most of the radioactivity was found in the muscle, carcass, and skin. The large percentage of the total 14C residue stored in these tissues may be due to their large mass (80 % relative to the whole body). Redistribution of TCB to visceral fat and periorbital fat (eyes) occurred during the first week after exposure. The redistribution was basically to tissues of high lipid content. Skin initially contained about 20% of the total 14C residue and the rate of TCB removal from this depot was slow. The liver was found to contain a lower concentration and a smaller percentage of the 14C residue than expected. Hansen et al. (1971) found that, in spot, an estuarine fish which was exposed to PCBs in water, the liver contained a greater concentration of PCB when compared to visceral fat. In rats, the liver also contains high concentrations of PCB (Peterson et al., 1976; Matthews and Anderson, 1975). However, Lieb et al. (1974) showed that, in rainbow trout fed 15 ppm (dry weight basis) of Aroclor 1254 for 32 weeks, the lowest concentration of PCBs occurred in the liver. This is in agreement with our results. The elimination of [14C]TCB was very slow on a whole body basis and in all tissues except blood and bile (Fig. 2). Hattula and Karlog (1973) have found that goldfish containing 70 ppm of PCB eliminated 50 ‘A of that dose after 3 weeks and almost 80 % after 70 days. Results obtained by Hansen et al. (1971) in experiments with spot show that 61 ‘A of PCBs taken up were excreted over the course of an 84-day experimental period. Our results show a 30% elimination after 14 days, but then only a 6 % loss in the following 126 days. Excretion in bile was one of the major routes of eliminating the radioactivity initially. From 0 to 14 days, the percentage of 14C residue in bile dropped from about 4 to 0.4%. In contrast, Peterson et al. (1976) found that male rats which were given 2,5,2’,5’-TCB into the stomach excreted approximately 42 y0 of the dose in bile after only 24 hr. Allen (1975) reported that the same TCB isomer had a t+ of 2-3 months in rhesus monkeys. This compares with the t+ of 2.66 years found for rainbow trout in this study. It has been reported by Gage and Holm (1976) that the retention of different PCB isomers in mice may be related to the number and position of chlorine atoms on the molecule, but it is difficult to compare the mammalian data with those obtained for TCB in this report at the present time.

TETRACHLOROBIPHENYL

IN RAINBOW

TROUT

331

In a study by Lieb et al. (1974), chromatographic profiles of PCBs isolated from exposed rainbow trout were identical to standards of Aroclor 1254 and this was interpreted to indicate that rainbow trout do not metabolize PCBs. Hutzinger et al. (1972) found that rats and pigeons formed hydroxyl metabolites of pure TCB, but they could not detect any hydroxyl metabolites in the water in which TCB-fed brook trout were maintained for 4 days. These studies suggest that trout do not have a hydroxylating mechanism for metabolizing PCBs. Reports on the biliary excretion of metabolites of 3trifluoromethyl-4-nitrophenol (Lech, 1973), sulfobromophthalein (Schmidt and Weber, 1973), carbaryl and DDT (Lech et al., 1973), and Bayer 73 (Statham and Lech, 1975) by rainbow trout suggest that trout bile may be a potential route of elimination of PCB metabolites. Recently, Melancon and Lech (1976) have isolated a glucuronide conjugate of TCB from bile of rainbow trout exposed to 2,5,2’,5’-TCB. This conjugate, however, represented less than 1% of the accumulated dose. The data in this report are consistent with the idea that rainbow trout have the ability to metabolize and excrete TCB in bile to a limited extent. The high concentrations of 14C in bile during the initial rapid elimination phase indicate that biliary excretion of TCB may be greatest at the time of elimination and redistribution of a “mobile” compartment of TCB and, when this compartment is exhausted, both overall elimination and biliary excretion are reduced considerably. ACKNOWLEDGMENTS

The authors wish to acknowledge the advice and assistance of Mr. Paul Degurse and Mr. Wes Warwick of the Wisconsin Department of Natural Resources Nevin Fish Hatchery and Mr. Tom Gibson of the Wisconsin State Hygiene Laboratory. REFERENCES J. R. (1975). Response of the nonhuman primate to polychlorinated biphenyl exposure. Proc. Fed. Amer. Sot. Exp. Biol. 34, 1675-1769. GAGE, J. C., AND HOLM, S. (1976). The influence of molecular structure on the retention and excretion of polychlorinated biphenyls by the mouse. Toxicol. Appl. Pharmacol. 24,555-560. HANSEN, D. J., PARRISH, P. R., LOWE, J. I., WILSON, A. J., JR., AND WILSON, P. D. (1971). Chronic toxicity, uptake and retention of Aroclor 1254 in two estuarine fishes. Bull. Environ. Contam. Toxicol. 6, 113-l 19. HATTULA, M. L., AND KARLOG, 0. (1973). Absorption and elimination of polychlorinated biphenyls (PCB) in goldfish. Acta Pharmacol. Toxicol. 32,237-245. HESSELBERG, R. J., AND JOHNSON, J. I. (1972). Column extraction of pesticides from fish, fish food and mud. Bull. Environ. Contam. Toxicol. 7, 115-120. HUTZINGER, O., NASH, D. M., SAFE, S., DEFREITAS, A. S. W., NORSTROM, R. J., WILDISH, D. J., AND ZITKO, V. (1972). Polychlorinated biphenyls: Metabolic behavior of pure isomers in pigeons, rats, and brook trout. Science 178,312-314. JENSEN, S. (1966). Report of a new chemical hazard. New Scientist 32, 612. LECH, J. J. (1973). Isolation and identification of 3-trifluoromethyl-4-nitrophenyl glucuronide from bile of rainbow trout exposed to 3-trifluoromethyl-4-nitrophenol. Toxicol. Appl. Pharmacol. 24, 114123. LECH, J. J., PEPPLE, S. K., AND STATHAM, C. N. (1973). Fish bile analysis: A possible aid in monitoring water quality. Toxicol. Appl. Pharmacol. 25,43&434. LIEB, A. J., BILLS, D. D., AND SINNHUBER, R. 0. (1974). Accumulation of dietary polychlorinated biphenyls (Aroclor 1254) by rainbow trout (Salmo gairdneri). J. Agr. Food Chem. 22,638-642. ALLEN,

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ET AL.

H., AND ANDERSON, M. (1975). The distribution and excretion of 2,4,5,2’,5’pentachlorobiphenyl in the rat. Drug Metab. Disp. 3,211-219. MELANCON, M. J., JR. (1974). The uptake and metabolism of some organic pollutants in fish. Eighth Great Lakes Regional Meeting, American Chemical Society, West Lafayette, Indiana. MELANCON, M. J., JR., AND LECH, J. J. (1976). Isolation and identification of a polar metabolite of tetrachlorobiphenyl from bile of rainbow trout exposed to [14C]tetrachlorobiphenyl. Bull. Environ. Contam. Toxicol. 15, 181-188. National Pollutant DischargeElimination System,Appendix A. (1973).Fed. Reg.38, No. 75, Pt. II. Panelon HazardousTrace Substances.(1972).PCB’senvironmentalimpact. Environ. Res.5, 249. PETERSON, R., SEYMOUR, J., AND ALLEN, J. (1976).Distribution and biliary excretion of polychlorinated biphenylsin rats. Toxicol. Apl. Pharmacol.38, 609-619. SCHMIDT, D. C., AND WEBER, L. J. (1973).Metabolism and biliary excretion of sulfobromophthalein by rainbow trout (Salmogairdneri).J. Fish Res.Board Canad.30, 1301-1308. STATHAM, C. N., AND LECH, J. J. (1975). Metabolism of 2’,5-dichloro-4’-nitro-salicylanilide (Bayer 73) in rainbow trout (Salmogairdneri). J. Fish. Res.Board Canad.32, 515-522. U. S. Fish and Wildlife Service.(1974). DDT-PCB Level in Lake Michigan fish. Great Lakes Fishery Laboratory, Ann Arbor, Michigan. VEITH, G. D. (1974).1971 BaselineConcentrations of PCB’s andDDT in Lake Michigan Fish. U. S. EPA, National Water Quality Laboratory, Duluth. VEITH, G. D., AND LEE, G. F. (1971).PCB’sin fish from the Milwaukee region. In Proceedings of the 14th Conferenceof the Great LakesFisheryLaboratory, pp. 157-l 69. MATTHEWS,