Document LKYj02NrdgYNpove1DYkvVpaX

Sublethal Effects of Structurally Related Tetrachloro-, Pentachloro-, and Hexachloroblphenyl on Juvenile Coho Salmon "'SSSwow* ZJax return ^ /o-7 Edward H. Gruger, Jr.*, Thomas Hruby, and Nava L. Karrlck Northwest Fisheries Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration. 2725 Montiake Boulovard East, Seattle, Wash. 08112 Four groups of cohorts of juvenile coho salmon {Oncorhynchu* kieutch) were fed 2,6,2,,5'-tetrflchlorobipheriyl, 2,4,5,2',6'-pcnuchIorobipheny), and 2,4,6,2',4/,6'-hexachlorobiphcnyl together at combined total concentrations of 1,2, and 12 parts per million in food pellets. Salmon fed 12 ppm chlorobiphcnyla gained weight significantly (P < 0.05) faster during a one-month treatment than aalmon in a control group. Whole body concentrations of the three chlorobiphenyle in the fish during a 72-day period indicated a possible steadystate condition for the chlorobiphenyle when administered at the concentration of 1 ppm, but not at 2 ppm or greater in the food. Biphenyls chlorineted in the 2,6- and 2,4,6-positions on the aromatic rings were accumulated to e similar extent in the fish. Rearrangement of chlorine atoms from the 2,6-po- silions to the 3,4-positions in isomeric tetrachlorobiphenyls resulted in an apparent decrease in the accumulation of the compound in young salmon. For the first time a pentochlo- robiphcnyl was found to cause enhanced activity ofthe hepatic aryl hydrocarbon hydroxylase system in treated fish. Polychlorinated biphenyls are well recognized as persistent contaminants in the environment Chlorobiphenyle are quite resistant to chemical and enzymatic degradations (7). Chlo robiphenyle possess a wide range of molecular structures, which can be classified according to the degrees of chlorina tion. The structural arrangement of chlorine atoms on the biphenyl molecule arc expected to have considerable influence on the resistance of the molecules to chemical and enzymatic alterations, especially in biotransformations. Fen' experiment* have been reported on the metabolism of chlcrobiphenyls in flsh. A review by Hutzinger etal. (1) fur nishes information on the chemistry of these compounds in flsh and other animals, in addition to providing references to studies on the uptake, accumulations, and metabolism of chlorinated biphenyls. Biochemical and physiological effects, such as enlargement of liver, altered cholesterol metabolism, blood anaemia, and hyperglycemia, in brown trout treated with polychlorinated biphenyls after 43 days were observed by Johansson et a). (2). Analyses ofchlorobiphenyls In Atlantic aalmon after 243 days of treatment with a polychlorinated biphenyl (Aroclor 1254) in food showed a reduction or dis appearance of the chlorobiphenyls having low degrees of chlorination (I). However, Hutzinger at al. (3) found no evi dence for the excretion of hydroxylated chlorobiphenyls from brown trout which had consumed 4-chloro-, 4,4'-dichloro-, 2,6,2',5'-tetrachloro-, and 2,4,6,2',4',5'-hexachlorobiphenyi In food. Chlorobiphenyls, like other foreign aromatic compounds, ara believed to be metabolized via an snzymatic system that involvos aryl hydrocarbon hydroxylation. Evidence supporting this view is the observation that hydroxy chlorobiphenyls are excreted from animals fed chlorobiphenyls (3). Although .certain chlorinated biphenyls are depurated from treated fish in spite of the lack of detectable hydroxylated metabolites, furt her work is needed to understand the role of mplecular struct ure of chlorobiphenyls in the accumulations In flsh and the effect* that these compounds have on biochemical systems such as the aryl hydroxylases. - Chlorobiphenyls in organisms from various trophic levels in the marine food web are ready sources of these contend, nants in higher levels of marine fish (4). An understanding of the fate of chlorobiphenyls after they are transported through food to fish will enable predictions to be made about food web interactions and persistence of different chlorobiphenyls in fish. The present work is a continuation of a related study (5) ofspecific structurally pure chlorobiphenyls fed to young coho (Oncorhynchus kisutch) salmon. In examining growth rates of young salmon fed chiorobiphenyls in diets, we found that small populations (50-100 fish) of cohorts establish divergent ranges of body weights during the course of experimentation. For example, 106 salmon fry weighed 0.3-0.4 g each at the start of an experiment, while in one year in captivity they grew to have a weight range of 3.6-19.6 g, according to a certain feeding schedule (5). The resulting wids range may be related to a natural aggressive behavior of some fish to establish territorial dominance, which would allow dominant fish to get more food than subordinate flsh (6-4). In the present research, we designed our experiments to take into account the problem of a wide weight range due to the presence of dominant and subordinate fish that result* in individual differences among fish to feed and consume chlo robiphenyls in food. The purpose of the research was to ex amine the relationship of dietary concentrations of the chlo robiphenyls to growth of individual young salmon of uniform weight distribution, to examine fish of uniform weight dis tribution for accumulations of three structurally related chlorobiphenyls of different degrees of chlorination, and to make a preliminary determination of the effect of a pentachlorobiphcnyl on enzymatic activities of an aryl hydrocarbon hydroxylase. Experimentol Juvenile coho aalmon were reared in a manner previously described (5). The fish were cohorts held in reserve from the initial study, and they were nearly otto year older when uaed. The fish were fed diets at a rate of 2% of body weights each day, five days per week. At several intervals, the fish were individually identified and weighed, and the amount of food fed was adjusted to the weight of fish remaining after the re moval,of fish for analyses. Experiments were designed to determine the influence on fish growth of and accumulations in fish of 1,2, and 12 ppm of structurally related chlorobiphenyls which wsre added to the source of food, Oregon moist pellets (9). The additives were 2>5,2/,6'-tetrachlorobiphenyl, 2.4,5,2',6'-pentachlorobipheny), and 2,4,5,2',4',5'-hexachlorobiphenyl, which are among the principal components of Aroclor 1242,1248, or 1254 (10). We included 12 ppm chlorobiphenyis in the diets to allow us to relate the present results to the previous work (5). Treatment of Fish. To examine fish fed the three con centrations of chlorobiphenyls vs. a control group, we selected four groups of fish weighing as nearly alike as possible, with similar standard deviations of mean weights. This was done by selecting 64 fish having the narrowest weight range from among 106 cohorts, dividing them into two groups of 32, and measuring the individual fish weights according to each group. Next, the groups were divided in half, and groups of 16 fish MGNS 083327 Volume 10, Number 10. October 1976 1033 were sorted to five a distribution of about the same number of low, medium, and high weight Huh in each group. Each fish was branded with an identifying code mark, which was made using rapid cold-brands with branding tools at temperatures near that of liquid nitrogen (11). At the start of feeding food-impregnated chlorobiphenyls, the groups ranged in weight from II to 20 g; their mean weighte A standard de viation were 15.6 A 2.0 g. There were 400 ml of water/g of fish in arparate rearing tanks for each group at the etarl of the feoding. Such water space was believed to be sufficient to preclude problems from overcrowding (6). When the Hah wero taken from the rearing tanks for anal yses, thoy were' identified by the cold-brand markings and selected in such a way that the lowest weight, the highest weight, and woighta closest to the mean weight of the group were used. This method of selection was designed to negate the effects of Ute individual differences of Hah to consume food sujscrimposcd on the growth effects associated with the di-' etary chlorobiphenyls. At different times during the feeding period, the fish were identified, their weights recorded, and selections made for analytes. The fish were killed by placing them in water con taining 200 ppm (w/v) of MS-222 (ethyl m -aminobenzoate methnneaulfonate) anesthetic until their movements ceased, then wrapping them in aluminum foil, quick freezing them at -60 *C In a freezer, and holding them at -60 *C until ana lysed. Generally, four fish were taken for analysis each time, and individual measurements were made for the concentra tions of the three chlorobiphenyls in homogenized samples of each whole fish. Chloroblphenyl Analyses. The methods of analyses were essentially the same as rejwrted previously (5). A Virtis Model 45 homogenizer (the Vertis Co., Gardiner, N.Y.) was employed to thoroughly grind and mix the tissues. Weighed aliquots of homogenized tissue samples were extracted with chloro form-methanol. water mixtures, and aulwnqucntconcentrated extracts were analyzed by electron capture gas-liquid chro matography. Standards of the three chlorobiphenyls (Analabs, Inc., North Haven, Conn.) were analyzed prior to each ex perimental sample. Statistical analyses of covariance F test (12) and multiple oom]Mrieons of ail pairs of regression coefficients (13,14) were performod on growth data. Student's t test was performed on the data for the accumulations of chlorobiphenyl in fish tissues. Aryl Hydroxylalion. The effect of a pcntachlorobiphenyl on aryl hydrocarbon hydroxylalion was determined by com paring hepatic enzymatic activities for two treated fish with those for two control fish. All four Hah were coho salmon which had been maintained in laboratory freshwater tanks for 3 years. During this experiment, the water temperature for the fish was 11-12 *C. The treated fiah were injected intraperitoneally with 0.2 ml of corn oil containing 10 mg of 2,4,B,2',5'-pentachk>robiphenyl, while the control fish received only coni oil. The Inal feeding of the fish was 3.6 h after the injections. Pairs of treated and control fish were sacrificed at 46 and 69 h after the injections. Immediately after sacrificing, the livera were excised end quickly chilled in ice-cold 0.26 M-sucrose. Assays for aryl hydrocarbon hydroxylase, as bcnzo|aJpyrenc hydroxylase, were performed on 16000 X g (20 min) supernatant fractions from homogenates of 1 part liver in 14 volumes of cold 0.26 M-sucrose. The enzyme assay method of Nebcrl and Gclbpm (75), but using radioactive labeling techniques, was applied to incubation mixtures at 25 C, which is approximately the optimum temperature of re lated enzymes in fish (76). Assay blanks were performed using heat-denatured portions of the 15 000 X g supernatant The quantitation of the products of the enzymic reaction was made by performing parallel assay reactions using ,4C-labeled- benzojajpyrene (Amereham/Searle Corp.) as the substrate and determining from scintillation counts and specific radioac tivity (21 mCi/mmol) of the substrate the picomoles of phe nolic and other hydroxylated products produced by the re action. Protein analyses were performed on the 15 000 X g supernatant fractions by the Lowry method as modified by Miller (17). Reaultt and Ditcussion Studies of biological and biochemical effects of chlorobi phenyls often utilize complex mixtures of PCH's, and for this reason it is difficult to ascribe some biochemical effects of PCB's to specific or joint action of such undefined groups of compounds. The present study with individual chlorobi phenyls Is an effort to alleviate some of the uncertainties as sociated with complex mixtures and learn how specific structural entities in PCB's relate to certain observed effect* on fiah. Effects on Growth. Body weights of the fish were deter mined at each time interval when fish were selected for anal yses. The data are summarized in Table I. The rale of growth of each fish which remained until 17 or 36 days was deter mined from the slope of s linear regression plot of wet body weight and time, using a 700 Series Wang calculator pro grammed for point plotting and regression analysis. The data according to each group are given in Table II. An analysis of covariance F test showed that the regression coefficients were Table I. Body Weights of Salmon Fed 1, 2, and 12 ppm Chloroblphenyl! in Diet! No. Time. of days fiah Concentration of ehloroblphenyli In dists, ppm 0 1 2 12 Mean body weights, g* 0 64 15.1 t 1.8 15.7 l 2.7 15.8 t 1.8 15.6 i 2.3 3 64 16.3 i 3.5 16 4 i 3.? 16.8 t 3.0 16.3 s 3.3 (5.8%) (3.8%) (7.0%) (6 5%) 7 48 16.7 * 1.4 17.1 * 3.1 18.0 * 2.6 17.3 t 2.7 (5.0%) (8.2%) (11%) (12%) 17 31 19.1 *1.5 20.5 s 3.2 21.0 * 3.7 21.3 s 3.2 (25%) (38%) (33%) (37%) 35 16 23.4 t 1.1 25.) *3.9 25.3 i 1.6 28.1 t 2.3 (55%) (59%) (60%) (68%) 72 4 (20.7)5 (31.1)5 (23.5)5 (36.4)5 *Means t standard deviation. Percent weight gains relative to rare time ere given In parentheses. P Weight of single fish, which it not necessarily representative of e group. Table II. Growth Rales of 31 Individual Experimental Coho Salmon Fed 1,2, and 12 ppm Chlorobiphenyl! ppm treated Control 1 2 12 0.242 0.154 0.198 0.228 0.203 0.233 0.204 Gram per day0 . 0.272 . 0.211 0.229 0.478 0.336 0.309 0.267 0.220 0.453 0.271 0.215 0.172 0.255 0.274 0.343 0.334 0.331 0.390 0.3/6 0.367 0.264 0.236 0.284 0.449 0.208 Mean 0.296 0.284 0.337 SD 0.030 0.078 0.095 0.072 Slope of linear regression curve for body weightt to 17 or 35 av. 1034 Environmental Science & Technology MONS 083328 significantly different (F m 3.164, df 3 and 88, F < 0.05) (12). A multiple comparison of oil point of regression coefficients showed that the cunt rol group was significantly different from the 12 ppm group (/* < 0.00) (13,14). All other comparisons did not show a significant difference. In this way, we found thnl the fish fed 12 ppm of chlorobiphcnyls gained weight significantly faster than those in the control group. Thus, calculations for mean growth rates between groups of treated fish showed no significant difference-- not even for a com parison of the 1 ppm treated group to the 12 ppm treated group- The treatment of fish, therefore, with these particular chlorobiphcnyls resulted in a greater gain in body weights during one month of feeding than did fish receiving the basal control diet. The physiological events that result in the ob served weight gain arc not readily explained at this time but constitute on attractive area for future research. When the results of tiro present work are compared to those of the previous work (5), both the ages of the fish and the solubilit ies of the chlorobiphcnyls may have been factors in the observed differences. The molecular structures of the chlorohiphenyls were not the same between present and previous experiments. Tclrachlorobiphenyl isomers have different solubilities in water (/). Chlnrobiphcnyl Accumulations In Salmon Bodies. Prior to feeding chlorohiphcnyl-trcnted food pellets to the experi mental fish, the foods were extracted as in the case of fish tissues, and analyses were performed by gas-liquid chroma tography as previously reported (5). The results of the food analyses arc shown in Table 111. The Gl-C of the basal control diet ext ract gave numerous peaks at high sensitivity of de tection, hut only the three peaks corresponding to the reten tion times of the reference standards were quantitated for comparisons to the chlorubiphenyl-treated diets. The GLC pattern of the control fish is presumed to be a reflection of the ubiquity of lipophilic contaminants in the basal food pel lets. Our results indicate that a need exists in research for fish foods that arc free of organic contaminants. In the present study, the concent rations of chlorobiphcnyls were one to two orders of magnitude greater than ubiquitous foreign sub stances in the food, but these substances become important factors when one wishes to use concent rations much less than 1 ppm of chlorohiphenyls or other compounds in animal food. However, not enough is known to permit the preparation of a synthetic clean balanced diet for salmon that is free of the interfering contaminants. In fart, it is likely to take years to develop a clean diet that is without nutritional deficiencies. Jiy plotting the total combined concentration of the three chlorohiphenyls found in tire cxjrerimcntal fish against time, it appeared that nearly constant values were reached for fish fed at t lie 1 ppm level (Figure 1), beginning as early as about 2 weeks; however, this was not true for fish fed 2 ppm chlo* robiphenyis or more. Kach point in Figure 1 is based on three or four fish except for the points at 72 days, which are for single fish. The plateau line for the lowest concentration of chlorohiphenyls between 17 and 35 days indicates a possible tendy-state equilibrium condition, whereby the rates of up take and storage of chlorobiphcnyls in the fish are equal to the rates of metabolism and depuration, or residues are parti tioned Into water to meeting equilibrium requirements. The total food ingested in 2 weeks amounts to 20% of the total weight of fish. Thus, if there were no excretions and ig noring weight gains, t he total body burden of chlorobiphcnyls theoretically would be 0.2 ppm (wt/wl) for the 1 ppm con centration in the food. This 0.2 ppm is in good agreement wit h the data in Figure 1. Hesidues do not increase further with additional feeding; thus, an equilibrium has been reached. At 2 weeks, the group receiving 2 ppm has equivalent body con centrations; one-half of the amount ingested must have been eliminated or otherwise excreted or lost. The 12 ppm group had less than six or 12 limes the concentrations in the whole bodies of the 2 ppm or 1 ppm groups, respectively. If the chlorobiphcnyls are taken up equally from the food, then the results suggest an ability of the fish to melnbolbe or excrete chlorobiphcnyls at low concentrations. There is no reason to believe that less food was ingested by the 12 ppm group than by the other groups; because the fish generally consumed all food pellets that were given to them. The fish were healthy and they ate vigorously. Data for the individual chlorobiphcnyls are expected to show the effect of molecular structure on accumulations oc curring in fish. Karlier work showed that 3,4,3',4'-tctrachlorobiphenyl accumulated in coho parr at about one-half of the rate for the 2,4,5,2',4',6'- and 2,4,6,2',4',6'-hcxachlorobiphrnyI isomers (5). The concentrations of 216.2',5'-tetrachloro-, 2,4,5,2',5'-pentachloro*, and 2,4,5,2',4',5'-hexachlorobiphenyl found in the present experimental fish are shown in Tables IV-VI, respectively. In Table IV we show a progressive increase of 2,5,2',5'tetrachlorobiphenyl with time at concentrations above 1 ppm Table III. Analysis of Chlorobiphenyls in Prepared Food Pellets Found concentration, pg/g food Theoret ooneni ppm ` 2,5,2' ,5'Tetraehloro- biphenyl 2,4.5,2' ,5`. Pcntachlorobiphenyl 24 5.2,4 5- Hexaehlorobipltenyl 0 2 2 12 ^Control. 0.027 0.38 0.69 4.07 0.063 0.30 0.73 3.82 0.018 0.31 0.65 3.82 Totel 0.11 0 99 2.07 11.7 Figure 1. Total concentrations oi accumulated 2,5.2'.5'-tctrachlorobiphenyi, 2,4.5.2',5'-penlachlorobiphonylt and 2,4,5,2'.4'.5'-hcxachkxoblphenyl In salmon throughout feeding period Fleh red chlorobipltenyls at () 12 ppm. !) ? ppm, (A) 1 ppm. (Ot etmiioi lovel SONS 0 8 3 3 2 9 Volume 10. Numbo 10, October 1976 1035 chloiobiphcnyltf in the f<*l. The tetrnchlorobiphenyl in creased from one and one-half to twofold in the fish as the feeding Intervnl was doubled during 72 days. A significant accumulation of tctrachlorobiphenyl in the treated fish was found as early as 3 days. A concentration of 1.2 ppm (wi/wl) in fish is reached in 72 days when feeding the highest level in food. Analytical result* for the pcnlachlorobiphcnyl (Table V) and the hcxachlorobiphenyl (Table VI) were nearly iden tical and at*o nearly the same as for the tctrachlorobiphenyl. Student's t test showed no significant differences among paired data for the chlorobiphrnyl accumulations. It appears that a chlorine atom occupying the 4(4')-position of the chlorobiphcnyls has no effect on bioaccumulations in these fish when the 2,6,2',6'-po8itionfc are also occupied by chlorine. The comparative result* for the tctrachlorobiphenyl conflict with what was found previously (5). Data in Table VII are given for comparison of two tctrachlorobiphenyl isomers. Iterance 2,416,2',4/,5'-bexachlorobiphrn.yJ is common to the prrvinua and present experiments, it serves as a marker to allow the comparison to be made. The chlorine atoms in the 2,5-positions of the tctrachlorobiphenyl seem to allow the molecules to accumulate much more, perhaps twice as fast, Table IV. Concentrations of 2,5,2',5'-Tetrchlorobiphenyl In Juvenile Coho Salmon Fed 1, 2, and 12 ppm Combined Chlorobiphenyls in Diels Concentration in diets, ppm Osy 0" 1 7 12 Concentration in wet tissue, ng/g/> 3 29 i A 42 i 7 58 i 5 142 i 24 7 28 i 6 66 t 8 93 i 9 252 116 17 24 i 1 144 i 24 122 i 23 450 t 87 35 45 i 17 139 i 35 198 i 22 775 * 8? 72 45 180 300 1230 Control diet, tnto ppm theoretical, 0.1 ppm round. ^Mean values, 1 ttantferd deviation*, for tltivc or lour fish analyzed at 3 35 dayi <itie tisti eneiyred at 72 days. Table V, Concentrations of 2,4,6,2',6-Pantechlorobiphgnyl in Juvenile Coho Salmon Fed 1, 2, and 12 ppm Combined Chlorobiphenyls in Diots Concentration in diets, ppm Days O'1 * 2 12 Concentration in wet tissues, ng/g& 3 51 i 10 53 t 6 65 i b 133 l 19 7 47 i 9 78 i 9 93 i 9 218i 17 17 46 i 2 156i 17 140 i 20 444 i 76 35 69 i 16 162 i 42 214 i 21 762 i 66 72 82 235 355 1380 In labt* IV. at In labia iv. Table VI. Concentrations of 2,4,6,2',4',5'Hcxachlorobiphenyl in Juvenile Coho Salmon Fed 1, 2, and 12 ppm Combined Chlorobiphenyls in Diets Concentration in diets, ppm D*y* 0" 1 2 17 Concentration iin wot tissue, ng/gt* 3 3? i 10 7 26 i 6 46 i 10 57 * 1? 59 ii 16 72 i 11 130 i 18 214i 13 17 38 i 9 36 58 i 1? 146 i 28 151 t 2D 121 i- 26 197 ii 16 444 i 106 775 i 87 7? 62 190 32? 1380 *Same at in 1 able I v. ^Sime a* In 1 able IV. than the molecules with chlorine nlmils in tin- ion* This is readily illustrated by comparing the ratios in Table VII of the concentrations of tetrachiorobiphenyls lo that of the bexachJorobiphenyl. It appears that the placement of chlorine atoms at the 2,5-positions on the aromatic nucleus hinders the reactions involved in biotransformations. One explanation of bioaccu mulations is that the stereo configuration of the molecule hinders the fixation of the substrate to an aryl hydrocarbon hydroxylase, which may be involved in the first step of me tabolism of chlorobiphenyls; thereby, the molecule with a hindered structure may accumulate in an organism. On the other hand, the greater water solubility of 3,4,.T,4'-tetrach- lorobiphenyl compared to other chlorobiphenyls, as noted by Hutzinger eta). (/), is a factor in reducing the quantity ac cumulated. Hut according to what is presently understood, the difference in accumulations of the 2,5,2',5'- and 3,4.3',4'- tetrachiorobiphenyls is more plausibly related lo a preferential loss of the d^.S'^'-isoiner.RS a result of higher water solubility and a lower partition coefficient between fish and water. A similar explanation can be made for the relative accumulal ions of other component* of J`CH's in fish. Zinek and Addison (18) inlmvctiowly administered 2-, 3-, and 4-chlorobiphcny! to two species of skate (Hajo radiotn and It. ocellata) and found that the 3-isomer was excreted to a greater extent than the other two isomers. Here, too, the 3-position for chlorination of biphenyl is pcrhai>s involved so as to enhance the hydrophilic character of the comjtound. A determination of a complete material balance for chlorohiphenyls entering and leaving fish could possibly lend to an understanding of the role of solubility, gastrointestinal ab sorption, and other physiological processes related to uptake and bioaccumulations of chlorobiphenyls. It is interesting to find in Table VII that the concent rations of 2,4,5,2\4',5'*hexachlorobiphenyl in cohorts of salmon one year different in age were nearly the same. This is likely in port a reflection of the closeness of the feeding regimens. Over a period of time, the regimens allowed nearly identical absolute quantities per unit body weight of the compounds to be fed to the fish at the levels of 10 and 12 ppm. Chiorobiphcnyl Induction of Aryl Hydrocarbon Ily- droxylation. Certain chlorobiphenyls ere metairolized in birds and mammals (3), and there arc indicat ions from studies with industrial PCB's (Aroclor 1254) that some chlorohi- phenyls arc metabolized extremely slowly in fish (/). If the metabolism involves the biochemical transformations that are associated with other aromatic hydifrcarlmns, then a first step could be hydroxylation, perhaps via an arene oxide interme diate (19). Such oxide intermediates are more likely to be formed from chlorobiphcnyls of low chlorinnt ion as opjx>scd lo highly chlorinated biphenyls, because two adjacent non- chlorinated positions on the biphenyl nucleus are a possible prerequisite to the reaction (notwithstanding dechlorination). Fish are generally recognized to possess enzymic systems ca pable of xenobiolir metabolism (16,20, 21). Mole and female trout (Salmo fario) were shown'by Crenven ct al. (22) to possess hepatic microsomal hydroxylase activity toward bi phenyl. However, there is no evidence reported for an asso ciation between aryl hydrocarbon hydroxylase and chlorobi phenyls in fish. Data in Table VIII show the enzyme inductive effects of single doses of the pcntachlorobiphenyl in two mate coho salmon. At 45 and 69 h after the treatment, hepatic enzymatic activities of test fish were 2.6 and 2.4 times greater, respec tively, than those of contra1 fish. Although these data are obtained from only four salmon livers, we routinely measure bcnzo|a]pyrene hydroxylase in our laboratory and find that replications among assays for a like group of fish have stan dard deviations which are d37% of the mean assay values. 1036 Environmental Science & Technology MOMS 063330 Table VII. Concentrations of Chlorobiphenyli in Juvenile Coho Salmon Fed 10-12 ppm Chlorobiphanyls pg chlorobiphenyl/g wet tissue Exp) Day* 3,4,3 Tetrechioro- biphenyl 2,6,2\6'letrechloro- biphenyl 2.4.5.2',4,.6'Hexachloro- biphenyl Ratio4 |5 53 0.59 t 0,23 0.93 l 0.21 0.6 1 108 0.65 *0.32 1.41 * 0.35 0.5 IK 35 0.78 1 0.08 0.78 i 0.09 1.0 11 77 1.23 1.38 0.9 *Cntt<fn|raMon o totracWorut'lptionyl divided by concent(St)on of hexachiorohlphcnyl. /'let* pnni of equal portions of 3,4,3 .4 teltid.loiU', enci ?,4,6,?*,4`.6 -hexactiloiobiphtnyi, feeding 3,!>% body welglii/dsy, 3 deys/wccK (5). ("twelve oom of equal motions of ?,&,? ,B'-lclreciiioro-, 2,4,5.?' 5 -pentachtoro-, end 2,4.4',t,' hOMclilo>oblphonyl, feeding 2.0% body weight/ day, & deys/woeh. Table VIII. Aryl Hydrocarbon (Benzo[a] pyrene) Hydroxylaio Activity in Hepatic Tissue* of 3-Year Old Coho Salmon Treated with 10 ms of 2,4,5,2',5'- Pontachlorobiphcnyf Group Fish Sex Wt, Time after Hydroxylase treat activity, length, ment,4 pmol/mg cm h protoin/30 min lost A 240 33 45 Control 9 225 30 45 1e*l 4 1C7 26 69 Control d 164 26 69 180 * 18 68 i 3 49 2 20 t 8 *$ee 6 xperlmentsl section, Thus, i the present case, the effect is significant for in vivo induction of the enzymic system by a single dose of pcnlarhloroblphcnyl in snlimm. The induction of n aryl hydrocarbon hydroxylase in fish by a chlorobiphenyl has been demonstrated for t he first time, using 2,4,&,2',6'-pentchlorobiphenyl. It is tempting to spec ulate on the basis of our research with a single biphenyl that certain other aromatic substrates, including certain other chlorinated biphenyls, may allrr the activities of the aryl hydrocarbon hydroxylases. Sanborn et al. (23) exposed green sunfish (Lepomis cy- oncllui*) to aqueous solutions of pure HC-labeied chlorobi* phenyls to st udy mctalxdiam. They found percentage distri butions of 18.39, 98.84, and 99.41%, respectively, for 2,5,2'trichlorobiphcnyl, 2,5,2',6'-tetrachlorobiphenyl, and 2,4,fi,2',f>'-|H>nUtch)orobiphenyl in the fish as parent materials, and the remainder of radioactivity occurred as polar residues. Thus, sunfish arc apparently side to metabolise the chloro* biphenyls, CHjKTinUv the trichlorobiphcnyl. Zinck and Addison (]R), on Iho other hand, showed that a large fraction of monochlorobiphenyls was excreted from skate when the compounds were administered by intravenous injection. Al though they did not indicate the excretion of polar derivatives of the chlnrobiphcnyls, that possibility remains an open question. Aside from the above specie* differences, different routes of metabolism could be involved in fish depending on the mode of ent ry of t he compounds into the organisms. I'CTt's induct' niicmsoinul in/vinrs m higher mmhiihIs, notably in rats (24-26). 'I'hc priiclicnJ iuiplianiion vt Mich induced enzymatic activities is that the animals may be ex pected to metabolize many foreign compound* al elevated rates. The rates of metabolism of I'CH's arc very slow in fish. That a chlorobiphenyl induces the microsomal enzyme system for aryl hydrocarbon hydroxylntion suggests that aromatic hydrocarbons from the environment, such a* those occurring in petroleum, are more readily metabolized in fish which have been preexposed to chlorobiphcnyls. The present evidence suggesting that the chlorobiphcnyls alter the activities of the aryl hydrocarbon hydroxylase in fish is an im|K>rtant area for future research. Acknowledgment The authors thank James L. Mighell for cold-bi'anding of our fish, Philip Nurnoto for technical assistance, and Frank J. Ossiander for statiaiical analyses. 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(25) l.ilterat, (\ I... van I .non, K. J, Frw. Sue F.rp Hint Mod. 141, 765-68 (1972); Hull Environ. Contain. Toxicol, II, 206 12 0 974). (26) Turner, J. C.,Green, It. S., Hull Environ Contam Toxicol, 12, 669-71 (1974). Received (or review November 13,1975. Afccpted May 3, 1976 Fnprr presented at the 30th Annual Northwest Regional Meeting </ the American Chemical Society, Honolulu, Hawaii, June 13- I t, 1975 Mention of commercial products is for identification only and doc s not constitute endorsement by the US Department of Com merce. M0NS 083331 Volume 10, Numbor 10, October 1976 1037