Chronic Oral Exposure of Rainbow Trout (Salmo gairdneri) to a Polychlorinated Biphenyl (Aroclor 1254): Pathological Effects H. Nestel and Joan Budd*
ABSTRACT
RESUME
Groups of eight rainbow trout (Salmo gairdneri) were fed rations containing 0 ppm, 1 ppm, 10 ppm and 100 ppm of the polychlorinated biphenyl, Aroclor 1254, over a period of up to 330 days. Growth rates were unaffected by these levels. Pathological changes were found in the kidneys of 13 fish on the polychlorinated biphenyl ration. Foci of nephrosis with cellular or granular cast formation were seen. The greatest number of cases occurred in fish at the 10 ppm concentration. An increase in the number of hepatocytes per unit area was related to the greater concentration of polychlorinated biphenyls in the diet. A reduction in the amount of white pulp in the spleen was observed in 23 of 27 fish on the 10 ppm and 100 ppm diets. The fish with nephrosis had reduced splenic white pulp and lowered white cell counts. Fish fed the 1 ppm and 10 ppm levels had mean tissue residues of 1.4 and 2.3 ppm polychlorinated biphenyl respectively. Fish on the 100 ppm ration had a mean value of 80.1 ppm polychlorinated biphenyl content.
L'alimentation de quatre groupes de huit truites arc-en-ciel (Salmo gairdneri) avec des rations contenant respectivement 0, 1, 10 et 100 ppm de diphenyle -1 polychlorure, Aroclor 1254, au cours d'une periode allant jusqu'a 330 jours, n'affecta pas le taux de croissance de ces poissons. On decela des changements pathologiques dans les reins de 13 truites experimentales. Ces changements consistaient en des foyers de nephrose avec formation de cylindres cellulaires ou granuleux. Le plus grand nombre de cas de nephrose survint chez les truites dont la ration contenait 10 ppm d'Aroclor. L'augmentation du nombre d'hepatocytes par unite d'aire s'avera proportionnelle a la teneur de la ration en Aroclor. On nota une diminution de la quantite de pulpe blanche splenique chez 23 des 27 truites dent la ration contenait 10 et 100 ppm d'Aroclor. La quantite de pulpe blanche splenique et le nombre de leucocytes s'avererent aussi moins eleves chez les truites qui presentaient des lesions de nephrose. La moyenne des residus tissulaires des truites dont la ration contenait 1, 10 et 100 ppm d'Aroclor s'eleva respectivement 'a 1.4, 2.3 et 80.1 ppm de cette substance.
*Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. From a thesis submitted by the senior author to the Graduate Faculty, University of Guelph, in partial fulfilment of the requirements for the degree of Master
INTRODUCTION
of Science.
Supported in part by the Ontario Department of Health Grant #PR257 and by funds from the Ontario Ministry of Agriculture and Food. Present address of senior author: I.D.R.C., Apartado Aereo, 53106 Bogota, D.E. Columbia. Address reprint requests to Dr. Joan Budd.
Submitted July 29, 1974.
208
Polychlorinated biphenyls (PCBs) have been identified as widespread contaminants in aquatic and terrestrial ecosystems (16, 23, 27) and high levels of PCBs have been found in fish from European (16, 18) and Ncrth American waters (26, 31, 32, 35). In the aquatic environment these compounds enter the biota by way of the water and food (10). Invertebrates associated
Can. J. comp. Med.
with contaminated sediments contain high levels of PCBs (21). Biological magnification can occur as these contaminants move up the food chain (11, 28, 36). Acute toxicity studies in several species of fish have established lethal levels for some of the more common chlorobiphenyls (31) but sublethal exposure to these compounds over a long period of time may present the greatest hazard. Fish and fish products are utilised as a source of protein by man and his domestic animals, thus man becomes the end point on the food chain. Pathological effects of PCBs on fish have received little attention. Degeneration of the liver and lowered reproduction in salmon (Salmo salar) were noted by Jensen et al (17). Retarded growth and stimulation of thyroid activity (12, 30) were seen in lake trout (Salvelinus namaycush) fed PCBs and it was suggested that this might affect viability and reproductive success. This is a report of the effects in fish of oral exposure to sublethal levels of a polychlorinated biphenyl for up to 330 days. Rainbow trout (Salmo gairdneri) were selected for study because of their increasing use as a food fish. Aroclor 1254 was used because it appears to be one of the common contaminants in tissues of aquatic species (10, 32).
MATERIALS AND METHODS Four groups, each of eight fish, were placed in 15 gallon capacity glass tanks. A charcoal filtered water supply provided a flow of 12 changes every 24 hours and artificial light was provided for 12 hours daily. The environment was monitored and controlled to maintain close to optimal conditions for maintenance of fish. A replicate experiment was set up 60 days after the first. The basic ration was composed of two parts raw frozen pig liver and one part dry commercial trout food'. Prior to use an aliquot from each batch of food was analysed and found to be free of detectable levels of PCBs. A stock solution of the PCB was prepared by the addition of 10 g of commercial Aroclor 1254' to 90 g of olive oil. This
PCB stock solution was further diluted with olive oil (w/w) and added to the basic ration to give concentrations of 100 parts per million (ppm), 10 ppm and 1 ppm. The control ration contained olive oil but no PCB. To each 500 g of mixed food 15.5 g of gelatin in 100 ml hot water was added. Rations were stored in air tight aluminum containers at -25°C until required. Fish were fed once daily on a calculated intake of 4% of body weight initially but later were fed according to demand. Each group of fish was weighed as a unit at monthly intervals. Fork lengths were measured at the time of killing. Satisfactory rate of gain in the experimental groups was judged by comparison with the control groups. Food intake per day expressed as a percentage of body weight was plotted against the average weight of each group. Haematological examinations were performed every two weeks on one fish randomly selected from each test group and on all fish at the time of killing. Individual fish were anaesthetised with methane tricaine sulfonate3 and 0.2 ml of blood was withdrawn from the dorsal aorta into a heparinised syringe. Total erythrocyte and leukocyte counts were made using the dilution method of Yokoyama (34) and an improved Neubauer haemocytometer. Two fish from each group were killed in carbon dioxide saturated water at 21 day intervals from day 229 to day 330. The following tissues were examined for histological lesions: gill, spleen, gonad, liver, heart, pancreas, head and midkidney and brain. Tissues for routine examination were placed immediately in Susa fixative and after 24 hours were transferred to 65% alcohol. All sections were stained with haematoxylin and eosin (H & E). Liver specimens to be examined for fat were fixed in 10% neutral buffered formalin and frozen sections were stained with oil red 0. The liver glycogen content was assessed using fixation by the Lisson Vokear technique and staining with periodic acid Schiff (PAS) reaction. The brains were fixed in formalin as above or formalin/ammonium bromide and frozen sections were stained by Penfield's, Ortega's and Ramon y Cajal's
2Aroclor 1254 (a chlorinated biphenyl containing 54% chlorine) was generously supplied by Monsanto Canada Ltd., Toronto, Ontario.
'Martin Feed Mills, Elmira, Ontario.
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April, 1975
3MS222, Sandoz (Canada) Ltd., Dorval, Quebec.
209
methods for cell identification (6). A stage micrometer and a 0.8 x 0.2 mm field was used to document the number of hepatocytes per unit area. PCBs were extracted from homogenized whole fish by the method of Grant et al (13) and analysed by the method of Saschenbrecker and Ecobichon (29) using a Micro-Tek model MT-2204 gas chromatograph equipped with a 63Ni high temperature electron capture detector. The quantitation and separation of PCBs was obtained by the method of Armour and Burke (3). Total fat content of three specimens from each group was extracted by a 24 hr ether extraction process in a soxhlet apparatus followed by a gravimetric method using Bloor's reagent (2).
the 10 ppm group and two from the 100 ppm group). Most of the fish affected were killed after day 258 (Table I). There was no nephrosis in kidneys from 13 control fish. The earliest recognized change was an increase in the size of epithelial lining cells of the tubule and vacuolation of the nucleus in some cells. The cytoplasm of affected cells frequently contained vacuoles some of which had eosinophilic proteinaceous inclusions. In what appeared to be a later stage there was a loss of cytoplasmic volume and a flattening of the cells. Pycnotic nuclei were present in many of the flattened cells. In affected tubules many of the epithelial cells were detached from the basement membrane and were visible in the lumen. Adjacent cells seemed to have covered the exposed but intact basement membrane so RESU LTS that severely affected tubules appeared as structures filled with proteinaceAt the termination of both trials in the ousdilated by a single layer of low casts and experiment there were 13 fish in each of epithelial cellslined 1). (Fig. the control and 1 ppm groups and 14 fish to identify It was not always in each of the IO ppm and 100 ppm groups. the exact location of thepossible in lesion the renal the exThe fish died during the course of a which The distal segments tubule. periment from causes unrelated to the diet. larger lumen lined by low columnar have epitheTen fish died during the course of the exseemed to be mainly involved. Howweight throughout the entire experimental lium in ever the more extensive lesions other segperiod and there were no significant dif- ments of the tubules were affected also. ferences among the growth rates of control The severity of the nephrosis varied but and principal groups. Occasional behavioureven in the most severely affected fish some al changes were noted in the fish fed the of the kidney tubules were unaffected. 10 ppm and 100 ppm PCB diets. Sporadic Data obtained from the 13 fish which of two duration days periods of one to lesions were examined for had nephrotic increased aggression and hyperexcitability from normal values. The deviations other were evident in these groups. There was an were all within the normal counts red cell increase in the degree of pigmentation in the the mean values obto close being range fish on all rations containing PCB after 50 days. Initially the entire body was darker but later the pigmentation became concentrated in the fins and caudal peduncle. Mean blood cell counts of principals did not differ from control fish. Renal tubular degeneration (nephrosis) and changes in the liver and spleen were found in some fish fed PCBs compared with tissues from fish on the control ration. All other tissues appeared to be normal. KIDNEY
Renal lesions were present in 13 fish (four from the 1 ppm group, seven from 4Tracor Inc., Augustin, Texas, U.S.A.
210
Fig. 1. Renal tubule with granular cast (C). Flattened epithelial cells (arrow) line the affected tubule. Normal haematopoictic tissue (H) and tubules (N) on the periphery. X545.
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TABLE I. Number of Cases of Nephrosis Related to Time and the Level of PCB in the Diet of Rainbow Trout
Days on Experiment 229 ...................................... 243 250 ....................................
258 ................................... 264 ................................... 285 ................... 307 ................................... 330 .................................. Total number of affected fish per group
...
0 ppm 0/2&
0/2 0/2 0/1 0/3 0/2 0/1 0/13
Level of PCB in the Ration 1 ppm 100 ppm 10 ppm b 1/2 0/2 _ 0/2
0/2 0/2 1/2 2/4 0/1 1/1 4/14
0/2 0/2 0/2 1/4 0/1 0/1 2/14
1/4 2/3 3/4 1/1 7/14
aNumber of fish affected/total number of fish bNo fish were killed
TABLE II. A Comparison of White Blood Cell (WBC) Counts at Time of Killing From Rainbow Trout With and Without Nephrosis
Mean WBC of all (56) Fish................. Standard Error............................ Mean WBC of 13 Fish with Nephrosis....... Standard Error............................
tained. The total white cell counts at the time of killing were below the mean values obtained from fish on the same treatment which had been bled previously (Table II). However those differences were not statistically significant at the 5% level. LIVER There was considerable variation evident in the degree of vacuolation and the apparent density of the cytoplasm of hepatocytes. In some sections from both controls and principals most cells appeared vacuolated, in others the cytoplasma was dense in appearance, occasionally more basophilic and there was an increase in the number of cells per unit area. No other changes were noted in the livers. Hepatocytes were counted in ten randomly selected fields from 4 micron sections of livers from each group. A significant increase in the number of cells per unit area was related to the higher levels of PCB in the diet (Table III). There was no association between the hepatocyte cell count and the prevalence of nephrosis. Mean cell density values were 36.91 for controls and 47.40, 51.00 and 54.50 for the 1 ppm, 10 ppm and 100 ppm PCB fed fish respectively.
Vol. 39-April, 1975
Level of PCB in the Ration l ppm 0 ppm 10 ppm 34.3 33.5 34.3 -+d 5.6 :1 6.5 0 20.5 23.8 6.5 + 8.7
100 ppm 42.4
9.1 27.3 + 9.8 +
In the PAS stained sections of liver from fish in the control groups the hepatocytes contained abundant glycogen-positive material. In the livers from the groups on 1 ppm and 10 ppm PCB containing rations there were variable quantities of glycogen. This appeared to correlate with the degree of vacuolation present in H&E stained sections. The hepatocytes with dense cytoplasm had only small amounts of glycogen near the sinusoidal margins of some cells and around the blood vessels. These liver sections had a high hepatocyte cell count per unit area (Table III). Fat, identified with the oil red 0 stain, was present only in variable but relatively small quantity in the livers of 53 fish examined. A comparison of the liver weight to body weight ratio was made from the data collected at the time of killing. The liver weight was expressed as a percentage of the total body weight and a mean value was obtained for each PCB fed group killed between days 229 and 330. The mean percent liver weight to body weight for fish on the control ration was 1.27. The means were 1.24, 1.44 and 1.53% respectively for the 1, 10 and 100 ppm PCB fed fish. These differences were not significant at the P > 0.5
level.
211
TABLE III. Hepatocyte Cell Counts per 0.8 mm x 0.2 mm Unit Area of Rainbow Trout Livers (Sections 41 thick) 0 ppm
Number of Livers .......................... Mean Cell Count ........... Standard Error ............................ .
11 36.91 i. . 1.84
Level of PCB in the Ration 10 ppm 100 ppm 1 ppm 10 10 10 54.501 51.001 47.40a i 1.29 i 4.02 4 3.50
'Significant at P > 0.05
SPLEEN
Changes in the spleen included variation in degree of pigmentation, the number of corpuscles and the amount of white pulp (lymphoid elements) present. To evaluate these observations spleen sections were examined without an identification i.e. "blind evaluation". As a base for comparison the white pulp was considered "normal" when it was present in amounts approximately equal to that of the red pulp and "reduced" when the white pulp was less than the red. The amount of pigment and number of corpuscles visible were classified into three overlapping categories, abundant, moderate or sparse. These criteria were used to compare the effects of the various levels of PCB in the diet. Spleens from fish on the higher levels of PCB in the diet had less pigment, less corpuscles per unit area and reduced amounts of white pulp compared with spleens from control fish (Table IV). Of the 13 fish with nephrosis 11 had low total white cell counts and reduced splenic corpuscles and white pulp. In fish which had low white cell counts but no nephrosis there was no association with
splenic change. Whole fish were analysed for total PCB concentration. The PCB concentrations found in the tissues of the fish on both the 1 ppm and 10 ppm PCB containing diets were much lower (mean values 1.3 and 2.3 ppm respectively) than those from the fish fed at the 100 ppm level of PCB (mean value 81.1 ppm) (Table V). Analysis of whole blood for PCB content indicated a similar trend with mean values from 0.7, 0.5 to 3.7 ppm respectively (Table V). There was no observable difference in the PCB concentrations found in fish killed on day 229 compared with those killed on day 330. The analysis of total fat content showed
212
no significant variation between the groups. The mean for all groups was 57.8% with a range from 39.8 to 65.9% wet weight of whole fish.
DISCUSSION Renal physiological processes in freshwater fishes differ from that of mammals in that only about 5% of the total glomerular filtrate is reabsorbed (15). Renal tubules of healthy fish are almost impervious to fluid in both the proximal and distal regions. One of the early signs of the PCB lesion in the kidney was a swelling, apparently due to fluid intake, of the epithelial lining cells of the affected tubules. One might speculate that the PCB or its break down products in the glomerular filtrate exert a deleterious effect on cell membranes resulting in increased permeability and thus allowing fluid to enter the cells. At the cellular level PCBs are reported to reduce enzyme activity (7, 9, 18, 22, 33) especially that concerned with energy production. The cells of the proximal renal tubule are rich in mitochondria as would be expected from the amount of energy required for the positive absorption of materials from the glomerular filtrate against the osmotic gradient. Diminution of the activity of these mitochondria would lessen their effectiveness, adversely affecting the permeability of the cell membrane. Damage to the upper nephron allowing absorption of fluid would result in a concentration of PCB in the filtrate. This may explain why the distal segment of the tubules appeared to be more severely affected than the proximal ones. The concentrations of PCBs in tissue from the fish on the 1 ppm PCB containing ration were low in relation to the blood PCB levels and considering the duration
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TABLE IV. Relationship Between Splenic Change and PCB Intake Levels in Rainbow Trout Levels of PCB in Ration O ppm 1 ppm 10 ppm 13 13 14
Number of Fish per Group .................. Pigment: Abundant............................. Moderate .............................
lOppm 13
Sparse ................................
7 3 3
4 6 3
1 3 10
2 4 7
Corpuscles: Abundant............................. Moderate ............................. Sparse ............................... Reduced White Pulp .......................
8 3 2 5
6 7 0 8
1 4 9 12
2 10 1 11
TABLE V. PCB Concentrations in Whole Fish and Whole Blood of Rainbow Trout
Level of PCB in Ration 1 ppm 10 ppm
O ppm
Number of Whole
Fish Sampled....
Mean PCB Value...
Range .............
Number of Blood Samples. . . Mean PCB Value ..
Range .............
3 N.D.N.D. 2
N.D. (0.0 - 0.0)
4 1.3 (1.1
-
1.4)
4 2.3 (1.9
-
3
4
0.7 (0.24 - 0.89)
0.5 (0.28
3.1)
-
0.77)
100 ppm
4 81.1
(70.1
-
112.0)
3 3.7
(2.37 - 5.27)
&N.D. = Nondetectable
of feeding (Table V). The concentrations of PCBs in whole fish on the 10 ppm intake were less than double those of fish on the 1 ppm PCB diet but the concentrations in the fish on the 100 ppm PCB diet were
much higher (Table V). Detoxifying enzymes are present at a low rate of efficiency in fish (4, 5) and it would seem that these rainbow trout were able to detoxify or eliminate the PCB to a degree and that it was the process of elimination which resulted in a high prevalence of nephrosis but at 100 ppm the system was overloaded and a greater proportion of the PCB was absorbed to be deposited in the tissues. Rehfeld et al (25) found that dosage was critical in the amount of PCB (Aroclor 1248) retained in the tissues of chicks. The ability of the surviving nephrons in mammalian kidneys to compensate in the event of loss of function of many of their number has been well documented. It seems probable that the kidneys of fishes may be able to do the same as they function in-
Vol. 39 -April, 1975
termittently in the normal control of urine production (15). Extensive damage would have to occur therefore before the fish would suffer from physiological incapacity. The significance of the kidney lesions alone in the health of these fish was probably limited since all had some unaffected tubules. The increased numbers of cells per unit area seen in the livers of fish on the 100 ppm ration with a concomitant depletion of glycogen may indicate that some physiological processes were becoming diminished. Other workers have demonstrated that faecal excretion of PCBs is minimal in mammals and birds which suggests that the biliary system is not a factor in its elimination (2, 8, 24). In this investigation the chromatographic peaks obtained from the food samples containing Aroclor 1254 were almost identical with those obtained from the whole fish tissue residues which suggests that little degradation of the compound occurred in the body. 213
The changes in the spleen, more marked Platonow was invaluable. Mrs. K. Hodson's with the higher PCB intakes and commonly technical assistance is gratefully acknowlpresent in those fish with nephrosis, as well edged. as the lowered white cell counts in these cases, suggest another potential threat to the health of the fish. It appeared that REFERENCES many of the affected spleens might have 1. ALBRO, P. W. and L. FISHBEIN. Intestinal abreduced ability to respond to antigenic sorption of polychlorinated biphenyls in rats. Bull. stimulation and that this might be an imenvir. Contam. Toxic. 8: 26-31. 1972. portant factor in considering the effect of 2. ANNINO, J. S. Clinical Chemistry. p. 270. Boston: Brown & Co. 1964. PCBs. Further investigations are required 3. Little, ARMOUR, J. A. and J. A. BURKE. Method for to evaluate the role of PCBs in conjunction separating polychlorinated biphenyls from DDT and its analogs. J. Ass. off Analyt. Chem. 53: 761-768. with physiological stresses and infectious 1970. agents before the importance of PCBs in 4. BUHLER, D. Hepatic drug metabolism in fishes. Fedn. Proc. 25: 343. 1966. the environment can be fully understood. P. J., D. V. PARKE and R. T. WILThe relatively low tissue residues from 5. CREAVEN, LIAMS. A fluorometric study of the hydroxylation of biphenyl in vitro by liver preparations of various the fish fed the diets containing 1 ppm or Biochem. J. 96: 879-885. 1965. 10 ppm PCB suggest that the trout were 6. species. CULLING, C. F. A. Handbook of Histopathological capable of eliminating the PCB at those Techniques. London: Butterworths. 1963. CUTKOMP, L. K., H. H. YAP, E. V. VEA and levels in the diet even when it was adminis- 7. R. B. KOCH. Inhibition of oligomycin sensitive (mitotered over a long period (Table V). Mayer chondrial) Mg2 ATPase by DDT and selected analogues in fish and insect tissues. Life Sci. 10: 1201. ('20) has stated that rainbow trout re1971. quire an oral dose of PCB in excess of 8. DAHLGREN, R. B., Y. A. GREICHUS and R. L. LINDER. Storage and excretion of polychlorinated 300 mg per kg of body weight for five days biphenyls in the pheasant. J. Wildl. Mgmt. 35: 823that states he 828. However, 1971. lethal. before it is D., L. K. CUTKOMP, H. H. YAP and deaths occur after exposure to low levels 9. DESAIAH, R. B. KOCH. Inhibition of oligomycin-sensitive and of PCBs in the water. The rainbow trout insensitive magnesium adenosine triphosphatase activity in fish by polychlorinated biphenyls. Biochem. in this study survived an oral intake of Pharmac. 21: 857-866. 1972. 4 PCB at the rates of 0.04 mg, 0.4 mg and 10. DUKE, T. W., J. I. LOWE and A. J. WILSON, JR. A polychlorinated biphenyl (Aroclor 1254) in the mg per kg body weight for 330 days. water, sediment and biota of Escambia Bay, Florida. Bull. envir. Contam. Toxic. 5: 171-180. 1970. The importance of fish as a link in the E. H., L. F. STICKEL, L. J. BLUS, transfer of PCBs from an aqueous to a ter- 11. DUSTMAN, W. L. REICHEL and S. N. WEIMEYER. The occurrestrial environment was stressed by Hamrence and significance of polychlorinated biphenyls in the environment. Trans. N. Am. Wildl. and Nat. mond (14). The tissue residues in fish on Resources Conf. 36: 118-131. 1971. the two lower levels of PCB in this study 12. GRANT, B. and D. SWEDBERG. Physiological effects of PCBs on fish. In Progress in Sport Fishery indicate that, although the danger of Research. p. 36. Washington, D.C.: United States Dept. of the Interior. 1970. magnification exists, rainbow trout feedGRANT, D. L., W. E. J. PHILLIPS and B. C. ing on the biota with a concentration of 13. VILLENEUVE. Metabolism of a PCB (Arochlor not more than 10 ppm of Aroclor 1254 1254) mixture in the rat. Bull. envir. Contam. Toxic. 6: 102-112. 1971. would be unlikely to contain concentrations 14. HAMMOND, A. Research topics, chemical pollution: of the PCB acutely toxic to man or other polychlorinated biphenyls. Science 175: 155-156. 1972. HICKMAN, C. P. and B. F. TRUMP. The kidney. In predators but these trout may be unable 15. Fish Physiology, Vol. I. W. S. Hoar and D. J. Ranto withstand the rigours of a natural envidall, Eds. New York and London; Academic Press. 1969. ronment.
ACKNOWLEDGMENTS The authors wish to express appreciation to the faculty and staff of the Departments of Pathology and Biomedical Science
(Toxicology), Ontario Veterinary College and to the personnel of the Veterinary Services Branch, (Poultry Pathology), Ontario Ministry of Agriculture and Food for their support and assistance. Throughout the course of this study the advice of Dr. N.
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16. JENSEN, S., N. JOHANSSON and M. OLSSON. PCB-indications of effects on salmon. PCB Conference. Stockholm. Sept. 29, 1970. Swedish Salmon Research Institute. Report LFI MEDD7. 1970. 17. JENSEN, S., A. G. JOHNELS, M. OLSSON and G. OTTERLIND. DDT and PCB in marine animals from Swedish waters. Nature, Lond. 224 (5216): 247-250. 1969. 18. KOCH, R. B., D. DESAIAH, H. H. YAP and L. K. CUTKOMP. Polychlorinatd biphenyls: effect of long-term exposure on ATPase activity in fish, Pimephales promelas. Bull. envir. Contam. Toxic. 7: 87-92. 1972. 19. KOEMAN, J. H., M. C. TEN NOEVER DE BRAUW and R. H. DE VOS. Chlorinated biphenyls in fish, mussels and birds from the River Rhine and the Netherlands coastal area. Nature, Lond. 221: 11261128. 1969. 20. MAYER, F. L. Oral toxicity of Arochlor 1242 and 1260 to rainbow trout. In Progress in Sport Fishery Research. p. 33. Washington, D.C.: United States Department of the Interior. 1970.
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21. NIMMO, D. R., P. D. WILSON, R. R. BLACKMAN and A. J. WILSON, JR. Polychlorinated biphenyl absorbed from sediments by fiddler crabs and pink shrimp. Nature, Lond. 231: 50-52. 1971. 22. PARDINI, R. S. Polychlorinated biphenyls (PCB); effect on mitochondrial enzyme systems. Bull. envir. Contam. Toxic. 6: 539. 1971. 23. PEAKALL, D. B. and J. L. LINCER. Polychlorinated biphenyls - another long-life widespread chemical in the environment. BioScience 20: 958-964. 1970. 24. PLATONOW, N. S., R. M. LIPTRAP and H. D. GEISSINGER. The distribution and excretion of polychlorinated biphenyls (aroclor 1254) and their effect on urinary gonadal steroid levels in the boar. Bull. envir. Contam. Toxic. 7: 358-365. 1972. 25. REHFELD, B. M., R. L. BRADLEY and M. L. SUNDE. Toxicity studies on polychlorinated biphenyls in the chick. Poult. Sci. 51: 488-493. 1972. 26. RISEBROUGH, R. W. and B. DE LAPPE. Accumulation of PCBs in ecosystems. Environmental Health Perspectives. Expl Issue 1: 39-45. 1972. 27. RISEBROUGH, R. W., P. RIECHE, D. B. PEAKALL, S. G. HERMAN and M. N. KIRVEN. Polychlorinated biphenyls in the global ecosystem. Nature, Lond. 220: 1098-1102. 1968. 28. SANDERS, H. 0. and J. H. CHANDLER. Biological magnification of a polychlorinated biphenyl (Aroclor 1254) from water by aquatic invertebrates. Bull. envir. Contam. Toxic. 7: 257-263. 1972.
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29. SASCHENBRECKER, P. W. and D. J. ECOBICHON. Extraction and gas chromatographic analysis of chlorinated insecticides from animal tissues. Agric. Food Chem. 15: 168-170. 1967. 30. SCHOETTGER, R. A. In Progress in Sport Fishery Research. Resource Publication 121. p. 2. Washington, D.C.: Bureau of Sport Fisheries and Wildlife. 1973. 31. STALLING, D. L. and F. L. MAYER, JR. Toxicities of POBs to fish and environmental residues. Environmental Health Perspectives. Expl. Issue 1: 159-164. 1972. 32. VEITH, G. D. and G. F. LEE. Chlorobiphenyls PCBs) in fish from the Milwaukee region. Proc. 14th Conf. Great Lakes Res. Internat. Ass. Great Lakes Res. pp. 157-169. 1971. 33. YAP, H. H., D. DESAIAH, L. K. CUTKOMP and R. B. KOCH. Sensitivity of fish ATPases to polychlorinated byphenyls. Nature, Lond. 233: 61-62. 1971. 34. YOKOYAMA, H. 0. Studies on the origin, development and seasonal variations in the blood cells of the perch (P. flavescens). Bull. Jap. Soc. Sc. Fish. 34: 1066. 1960. 35. ZITKO, V. PCB's and organochlorine pesticides in some freshwater and marine fishes. Bull. envir. Contam. Toxic. 6; 464-471. 1971. 36. ZITKO, V. and P. M. CHOI. PCB and pp-DDE in eggs of cormorants, gulls and ducks from the Bay of Fundy, Canada. Bull. envir. Contam. Toxic. 7: 63-67. 1972.
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ABSTRACT
RESUME
Groups of eight rainbow trout (Salmo gairdneri) were fed rations containing 0 ppm, 1 ppm, 10 ppm and 100 ppm of the polychlorinated biphenyl, Aroclor 1254, over a period of up to 330 days. Growth rates were unaffected by these levels. Pathological changes were found in the kidneys of 13 fish on the polychlorinated biphenyl ration. Foci of nephrosis with cellular or granular cast formation were seen. The greatest number of cases occurred in fish at the 10 ppm concentration. An increase in the number of hepatocytes per unit area was related to the greater concentration of polychlorinated biphenyls in the diet. A reduction in the amount of white pulp in the spleen was observed in 23 of 27 fish on the 10 ppm and 100 ppm diets. The fish with nephrosis had reduced splenic white pulp and lowered white cell counts. Fish fed the 1 ppm and 10 ppm levels had mean tissue residues of 1.4 and 2.3 ppm polychlorinated biphenyl respectively. Fish on the 100 ppm ration had a mean value of 80.1 ppm polychlorinated biphenyl content.
L'alimentation de quatre groupes de huit truites arc-en-ciel (Salmo gairdneri) avec des rations contenant respectivement 0, 1, 10 et 100 ppm de diphenyle -1 polychlorure, Aroclor 1254, au cours d'une periode allant jusqu'a 330 jours, n'affecta pas le taux de croissance de ces poissons. On decela des changements pathologiques dans les reins de 13 truites experimentales. Ces changements consistaient en des foyers de nephrose avec formation de cylindres cellulaires ou granuleux. Le plus grand nombre de cas de nephrose survint chez les truites dont la ration contenait 10 ppm d'Aroclor. L'augmentation du nombre d'hepatocytes par unite d'aire s'avera proportionnelle a la teneur de la ration en Aroclor. On nota une diminution de la quantite de pulpe blanche splenique chez 23 des 27 truites dent la ration contenait 10 et 100 ppm d'Aroclor. La quantite de pulpe blanche splenique et le nombre de leucocytes s'avererent aussi moins eleves chez les truites qui presentaient des lesions de nephrose. La moyenne des residus tissulaires des truites dont la ration contenait 1, 10 et 100 ppm d'Aroclor s'eleva respectivement 'a 1.4, 2.3 et 80.1 ppm de cette substance.
*Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. From a thesis submitted by the senior author to the Graduate Faculty, University of Guelph, in partial fulfilment of the requirements for the degree of Master
INTRODUCTION
of Science.
Supported in part by the Ontario Department of Health Grant #PR257 and by funds from the Ontario Ministry of Agriculture and Food. Present address of senior author: I.D.R.C., Apartado Aereo, 53106 Bogota, D.E. Columbia. Address reprint requests to Dr. Joan Budd.
Submitted July 29, 1974.
208
Polychlorinated biphenyls (PCBs) have been identified as widespread contaminants in aquatic and terrestrial ecosystems (16, 23, 27) and high levels of PCBs have been found in fish from European (16, 18) and Ncrth American waters (26, 31, 32, 35). In the aquatic environment these compounds enter the biota by way of the water and food (10). Invertebrates associated
Can. J. comp. Med.
with contaminated sediments contain high levels of PCBs (21). Biological magnification can occur as these contaminants move up the food chain (11, 28, 36). Acute toxicity studies in several species of fish have established lethal levels for some of the more common chlorobiphenyls (31) but sublethal exposure to these compounds over a long period of time may present the greatest hazard. Fish and fish products are utilised as a source of protein by man and his domestic animals, thus man becomes the end point on the food chain. Pathological effects of PCBs on fish have received little attention. Degeneration of the liver and lowered reproduction in salmon (Salmo salar) were noted by Jensen et al (17). Retarded growth and stimulation of thyroid activity (12, 30) were seen in lake trout (Salvelinus namaycush) fed PCBs and it was suggested that this might affect viability and reproductive success. This is a report of the effects in fish of oral exposure to sublethal levels of a polychlorinated biphenyl for up to 330 days. Rainbow trout (Salmo gairdneri) were selected for study because of their increasing use as a food fish. Aroclor 1254 was used because it appears to be one of the common contaminants in tissues of aquatic species (10, 32).
MATERIALS AND METHODS Four groups, each of eight fish, were placed in 15 gallon capacity glass tanks. A charcoal filtered water supply provided a flow of 12 changes every 24 hours and artificial light was provided for 12 hours daily. The environment was monitored and controlled to maintain close to optimal conditions for maintenance of fish. A replicate experiment was set up 60 days after the first. The basic ration was composed of two parts raw frozen pig liver and one part dry commercial trout food'. Prior to use an aliquot from each batch of food was analysed and found to be free of detectable levels of PCBs. A stock solution of the PCB was prepared by the addition of 10 g of commercial Aroclor 1254' to 90 g of olive oil. This
PCB stock solution was further diluted with olive oil (w/w) and added to the basic ration to give concentrations of 100 parts per million (ppm), 10 ppm and 1 ppm. The control ration contained olive oil but no PCB. To each 500 g of mixed food 15.5 g of gelatin in 100 ml hot water was added. Rations were stored in air tight aluminum containers at -25°C until required. Fish were fed once daily on a calculated intake of 4% of body weight initially but later were fed according to demand. Each group of fish was weighed as a unit at monthly intervals. Fork lengths were measured at the time of killing. Satisfactory rate of gain in the experimental groups was judged by comparison with the control groups. Food intake per day expressed as a percentage of body weight was plotted against the average weight of each group. Haematological examinations were performed every two weeks on one fish randomly selected from each test group and on all fish at the time of killing. Individual fish were anaesthetised with methane tricaine sulfonate3 and 0.2 ml of blood was withdrawn from the dorsal aorta into a heparinised syringe. Total erythrocyte and leukocyte counts were made using the dilution method of Yokoyama (34) and an improved Neubauer haemocytometer. Two fish from each group were killed in carbon dioxide saturated water at 21 day intervals from day 229 to day 330. The following tissues were examined for histological lesions: gill, spleen, gonad, liver, heart, pancreas, head and midkidney and brain. Tissues for routine examination were placed immediately in Susa fixative and after 24 hours were transferred to 65% alcohol. All sections were stained with haematoxylin and eosin (H & E). Liver specimens to be examined for fat were fixed in 10% neutral buffered formalin and frozen sections were stained with oil red 0. The liver glycogen content was assessed using fixation by the Lisson Vokear technique and staining with periodic acid Schiff (PAS) reaction. The brains were fixed in formalin as above or formalin/ammonium bromide and frozen sections were stained by Penfield's, Ortega's and Ramon y Cajal's
2Aroclor 1254 (a chlorinated biphenyl containing 54% chlorine) was generously supplied by Monsanto Canada Ltd., Toronto, Ontario.
'Martin Feed Mills, Elmira, Ontario.
Vol. 39
April, 1975
3MS222, Sandoz (Canada) Ltd., Dorval, Quebec.
209
methods for cell identification (6). A stage micrometer and a 0.8 x 0.2 mm field was used to document the number of hepatocytes per unit area. PCBs were extracted from homogenized whole fish by the method of Grant et al (13) and analysed by the method of Saschenbrecker and Ecobichon (29) using a Micro-Tek model MT-2204 gas chromatograph equipped with a 63Ni high temperature electron capture detector. The quantitation and separation of PCBs was obtained by the method of Armour and Burke (3). Total fat content of three specimens from each group was extracted by a 24 hr ether extraction process in a soxhlet apparatus followed by a gravimetric method using Bloor's reagent (2).
the 10 ppm group and two from the 100 ppm group). Most of the fish affected were killed after day 258 (Table I). There was no nephrosis in kidneys from 13 control fish. The earliest recognized change was an increase in the size of epithelial lining cells of the tubule and vacuolation of the nucleus in some cells. The cytoplasm of affected cells frequently contained vacuoles some of which had eosinophilic proteinaceous inclusions. In what appeared to be a later stage there was a loss of cytoplasmic volume and a flattening of the cells. Pycnotic nuclei were present in many of the flattened cells. In affected tubules many of the epithelial cells were detached from the basement membrane and were visible in the lumen. Adjacent cells seemed to have covered the exposed but intact basement membrane so RESU LTS that severely affected tubules appeared as structures filled with proteinaceAt the termination of both trials in the ousdilated by a single layer of low casts and experiment there were 13 fish in each of epithelial cellslined 1). (Fig. the control and 1 ppm groups and 14 fish to identify It was not always in each of the IO ppm and 100 ppm groups. the exact location of thepossible in lesion the renal the exThe fish died during the course of a which The distal segments tubule. periment from causes unrelated to the diet. larger lumen lined by low columnar have epitheTen fish died during the course of the exseemed to be mainly involved. Howweight throughout the entire experimental lium in ever the more extensive lesions other segperiod and there were no significant dif- ments of the tubules were affected also. ferences among the growth rates of control The severity of the nephrosis varied but and principal groups. Occasional behavioureven in the most severely affected fish some al changes were noted in the fish fed the of the kidney tubules were unaffected. 10 ppm and 100 ppm PCB diets. Sporadic Data obtained from the 13 fish which of two duration days periods of one to lesions were examined for had nephrotic increased aggression and hyperexcitability from normal values. The deviations other were evident in these groups. There was an were all within the normal counts red cell increase in the degree of pigmentation in the the mean values obto close being range fish on all rations containing PCB after 50 days. Initially the entire body was darker but later the pigmentation became concentrated in the fins and caudal peduncle. Mean blood cell counts of principals did not differ from control fish. Renal tubular degeneration (nephrosis) and changes in the liver and spleen were found in some fish fed PCBs compared with tissues from fish on the control ration. All other tissues appeared to be normal. KIDNEY
Renal lesions were present in 13 fish (four from the 1 ppm group, seven from 4Tracor Inc., Augustin, Texas, U.S.A.
210
Fig. 1. Renal tubule with granular cast (C). Flattened epithelial cells (arrow) line the affected tubule. Normal haematopoictic tissue (H) and tubules (N) on the periphery. X545.
Can. J. comp. Med.
TABLE I. Number of Cases of Nephrosis Related to Time and the Level of PCB in the Diet of Rainbow Trout
Days on Experiment 229 ...................................... 243 250 ....................................
258 ................................... 264 ................................... 285 ................... 307 ................................... 330 .................................. Total number of affected fish per group
...
0 ppm 0/2&
0/2 0/2 0/1 0/3 0/2 0/1 0/13
Level of PCB in the Ration 1 ppm 100 ppm 10 ppm b 1/2 0/2 _ 0/2
0/2 0/2 1/2 2/4 0/1 1/1 4/14
0/2 0/2 0/2 1/4 0/1 0/1 2/14
1/4 2/3 3/4 1/1 7/14
aNumber of fish affected/total number of fish bNo fish were killed
TABLE II. A Comparison of White Blood Cell (WBC) Counts at Time of Killing From Rainbow Trout With and Without Nephrosis
Mean WBC of all (56) Fish................. Standard Error............................ Mean WBC of 13 Fish with Nephrosis....... Standard Error............................
tained. The total white cell counts at the time of killing were below the mean values obtained from fish on the same treatment which had been bled previously (Table II). However those differences were not statistically significant at the 5% level. LIVER There was considerable variation evident in the degree of vacuolation and the apparent density of the cytoplasm of hepatocytes. In some sections from both controls and principals most cells appeared vacuolated, in others the cytoplasma was dense in appearance, occasionally more basophilic and there was an increase in the number of cells per unit area. No other changes were noted in the livers. Hepatocytes were counted in ten randomly selected fields from 4 micron sections of livers from each group. A significant increase in the number of cells per unit area was related to the higher levels of PCB in the diet (Table III). There was no association between the hepatocyte cell count and the prevalence of nephrosis. Mean cell density values were 36.91 for controls and 47.40, 51.00 and 54.50 for the 1 ppm, 10 ppm and 100 ppm PCB fed fish respectively.
Vol. 39-April, 1975
Level of PCB in the Ration l ppm 0 ppm 10 ppm 34.3 33.5 34.3 -+d 5.6 :1 6.5 0 20.5 23.8 6.5 + 8.7
100 ppm 42.4
9.1 27.3 + 9.8 +
In the PAS stained sections of liver from fish in the control groups the hepatocytes contained abundant glycogen-positive material. In the livers from the groups on 1 ppm and 10 ppm PCB containing rations there were variable quantities of glycogen. This appeared to correlate with the degree of vacuolation present in H&E stained sections. The hepatocytes with dense cytoplasm had only small amounts of glycogen near the sinusoidal margins of some cells and around the blood vessels. These liver sections had a high hepatocyte cell count per unit area (Table III). Fat, identified with the oil red 0 stain, was present only in variable but relatively small quantity in the livers of 53 fish examined. A comparison of the liver weight to body weight ratio was made from the data collected at the time of killing. The liver weight was expressed as a percentage of the total body weight and a mean value was obtained for each PCB fed group killed between days 229 and 330. The mean percent liver weight to body weight for fish on the control ration was 1.27. The means were 1.24, 1.44 and 1.53% respectively for the 1, 10 and 100 ppm PCB fed fish. These differences were not significant at the P > 0.5
level.
211
TABLE III. Hepatocyte Cell Counts per 0.8 mm x 0.2 mm Unit Area of Rainbow Trout Livers (Sections 41 thick) 0 ppm
Number of Livers .......................... Mean Cell Count ........... Standard Error ............................ .
11 36.91 i. . 1.84
Level of PCB in the Ration 10 ppm 100 ppm 1 ppm 10 10 10 54.501 51.001 47.40a i 1.29 i 4.02 4 3.50
'Significant at P > 0.05
SPLEEN
Changes in the spleen included variation in degree of pigmentation, the number of corpuscles and the amount of white pulp (lymphoid elements) present. To evaluate these observations spleen sections were examined without an identification i.e. "blind evaluation". As a base for comparison the white pulp was considered "normal" when it was present in amounts approximately equal to that of the red pulp and "reduced" when the white pulp was less than the red. The amount of pigment and number of corpuscles visible were classified into three overlapping categories, abundant, moderate or sparse. These criteria were used to compare the effects of the various levels of PCB in the diet. Spleens from fish on the higher levels of PCB in the diet had less pigment, less corpuscles per unit area and reduced amounts of white pulp compared with spleens from control fish (Table IV). Of the 13 fish with nephrosis 11 had low total white cell counts and reduced splenic corpuscles and white pulp. In fish which had low white cell counts but no nephrosis there was no association with
splenic change. Whole fish were analysed for total PCB concentration. The PCB concentrations found in the tissues of the fish on both the 1 ppm and 10 ppm PCB containing diets were much lower (mean values 1.3 and 2.3 ppm respectively) than those from the fish fed at the 100 ppm level of PCB (mean value 81.1 ppm) (Table V). Analysis of whole blood for PCB content indicated a similar trend with mean values from 0.7, 0.5 to 3.7 ppm respectively (Table V). There was no observable difference in the PCB concentrations found in fish killed on day 229 compared with those killed on day 330. The analysis of total fat content showed
212
no significant variation between the groups. The mean for all groups was 57.8% with a range from 39.8 to 65.9% wet weight of whole fish.
DISCUSSION Renal physiological processes in freshwater fishes differ from that of mammals in that only about 5% of the total glomerular filtrate is reabsorbed (15). Renal tubules of healthy fish are almost impervious to fluid in both the proximal and distal regions. One of the early signs of the PCB lesion in the kidney was a swelling, apparently due to fluid intake, of the epithelial lining cells of the affected tubules. One might speculate that the PCB or its break down products in the glomerular filtrate exert a deleterious effect on cell membranes resulting in increased permeability and thus allowing fluid to enter the cells. At the cellular level PCBs are reported to reduce enzyme activity (7, 9, 18, 22, 33) especially that concerned with energy production. The cells of the proximal renal tubule are rich in mitochondria as would be expected from the amount of energy required for the positive absorption of materials from the glomerular filtrate against the osmotic gradient. Diminution of the activity of these mitochondria would lessen their effectiveness, adversely affecting the permeability of the cell membrane. Damage to the upper nephron allowing absorption of fluid would result in a concentration of PCB in the filtrate. This may explain why the distal segment of the tubules appeared to be more severely affected than the proximal ones. The concentrations of PCBs in tissue from the fish on the 1 ppm PCB containing ration were low in relation to the blood PCB levels and considering the duration
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TABLE IV. Relationship Between Splenic Change and PCB Intake Levels in Rainbow Trout Levels of PCB in Ration O ppm 1 ppm 10 ppm 13 13 14
Number of Fish per Group .................. Pigment: Abundant............................. Moderate .............................
lOppm 13
Sparse ................................
7 3 3
4 6 3
1 3 10
2 4 7
Corpuscles: Abundant............................. Moderate ............................. Sparse ............................... Reduced White Pulp .......................
8 3 2 5
6 7 0 8
1 4 9 12
2 10 1 11
TABLE V. PCB Concentrations in Whole Fish and Whole Blood of Rainbow Trout
Level of PCB in Ration 1 ppm 10 ppm
O ppm
Number of Whole
Fish Sampled....
Mean PCB Value...
Range .............
Number of Blood Samples. . . Mean PCB Value ..
Range .............
3 N.D.N.D. 2
N.D. (0.0 - 0.0)
4 1.3 (1.1
-
1.4)
4 2.3 (1.9
-
3
4
0.7 (0.24 - 0.89)
0.5 (0.28
3.1)
-
0.77)
100 ppm
4 81.1
(70.1
-
112.0)
3 3.7
(2.37 - 5.27)
&N.D. = Nondetectable
of feeding (Table V). The concentrations of PCBs in whole fish on the 10 ppm intake were less than double those of fish on the 1 ppm PCB diet but the concentrations in the fish on the 100 ppm PCB diet were
much higher (Table V). Detoxifying enzymes are present at a low rate of efficiency in fish (4, 5) and it would seem that these rainbow trout were able to detoxify or eliminate the PCB to a degree and that it was the process of elimination which resulted in a high prevalence of nephrosis but at 100 ppm the system was overloaded and a greater proportion of the PCB was absorbed to be deposited in the tissues. Rehfeld et al (25) found that dosage was critical in the amount of PCB (Aroclor 1248) retained in the tissues of chicks. The ability of the surviving nephrons in mammalian kidneys to compensate in the event of loss of function of many of their number has been well documented. It seems probable that the kidneys of fishes may be able to do the same as they function in-
Vol. 39 -April, 1975
termittently in the normal control of urine production (15). Extensive damage would have to occur therefore before the fish would suffer from physiological incapacity. The significance of the kidney lesions alone in the health of these fish was probably limited since all had some unaffected tubules. The increased numbers of cells per unit area seen in the livers of fish on the 100 ppm ration with a concomitant depletion of glycogen may indicate that some physiological processes were becoming diminished. Other workers have demonstrated that faecal excretion of PCBs is minimal in mammals and birds which suggests that the biliary system is not a factor in its elimination (2, 8, 24). In this investigation the chromatographic peaks obtained from the food samples containing Aroclor 1254 were almost identical with those obtained from the whole fish tissue residues which suggests that little degradation of the compound occurred in the body. 213
The changes in the spleen, more marked Platonow was invaluable. Mrs. K. Hodson's with the higher PCB intakes and commonly technical assistance is gratefully acknowlpresent in those fish with nephrosis, as well edged. as the lowered white cell counts in these cases, suggest another potential threat to the health of the fish. It appeared that REFERENCES many of the affected spleens might have 1. ALBRO, P. W. and L. FISHBEIN. Intestinal abreduced ability to respond to antigenic sorption of polychlorinated biphenyls in rats. Bull. stimulation and that this might be an imenvir. Contam. Toxic. 8: 26-31. 1972. portant factor in considering the effect of 2. ANNINO, J. S. Clinical Chemistry. p. 270. Boston: Brown & Co. 1964. PCBs. Further investigations are required 3. Little, ARMOUR, J. A. and J. A. BURKE. Method for to evaluate the role of PCBs in conjunction separating polychlorinated biphenyls from DDT and its analogs. J. Ass. off Analyt. Chem. 53: 761-768. with physiological stresses and infectious 1970. agents before the importance of PCBs in 4. BUHLER, D. Hepatic drug metabolism in fishes. Fedn. Proc. 25: 343. 1966. the environment can be fully understood. P. J., D. V. PARKE and R. T. WILThe relatively low tissue residues from 5. CREAVEN, LIAMS. A fluorometric study of the hydroxylation of biphenyl in vitro by liver preparations of various the fish fed the diets containing 1 ppm or Biochem. J. 96: 879-885. 1965. 10 ppm PCB suggest that the trout were 6. species. CULLING, C. F. A. Handbook of Histopathological capable of eliminating the PCB at those Techniques. London: Butterworths. 1963. CUTKOMP, L. K., H. H. YAP, E. V. VEA and levels in the diet even when it was adminis- 7. R. B. KOCH. Inhibition of oligomycin sensitive (mitotered over a long period (Table V). Mayer chondrial) Mg2 ATPase by DDT and selected analogues in fish and insect tissues. Life Sci. 10: 1201. ('20) has stated that rainbow trout re1971. quire an oral dose of PCB in excess of 8. DAHLGREN, R. B., Y. A. GREICHUS and R. L. LINDER. Storage and excretion of polychlorinated 300 mg per kg of body weight for five days biphenyls in the pheasant. J. Wildl. Mgmt. 35: 823that states he 828. However, 1971. lethal. before it is D., L. K. CUTKOMP, H. H. YAP and deaths occur after exposure to low levels 9. DESAIAH, R. B. KOCH. Inhibition of oligomycin-sensitive and of PCBs in the water. The rainbow trout insensitive magnesium adenosine triphosphatase activity in fish by polychlorinated biphenyls. Biochem. in this study survived an oral intake of Pharmac. 21: 857-866. 1972. 4 PCB at the rates of 0.04 mg, 0.4 mg and 10. DUKE, T. W., J. I. LOWE and A. J. WILSON, JR. A polychlorinated biphenyl (Aroclor 1254) in the mg per kg body weight for 330 days. water, sediment and biota of Escambia Bay, Florida. Bull. envir. Contam. Toxic. 5: 171-180. 1970. The importance of fish as a link in the E. H., L. F. STICKEL, L. J. BLUS, transfer of PCBs from an aqueous to a ter- 11. DUSTMAN, W. L. REICHEL and S. N. WEIMEYER. The occurrestrial environment was stressed by Hamrence and significance of polychlorinated biphenyls in the environment. Trans. N. Am. Wildl. and Nat. mond (14). The tissue residues in fish on Resources Conf. 36: 118-131. 1971. the two lower levels of PCB in this study 12. GRANT, B. and D. SWEDBERG. Physiological effects of PCBs on fish. In Progress in Sport Fishery indicate that, although the danger of Research. p. 36. Washington, D.C.: United States Dept. of the Interior. 1970. magnification exists, rainbow trout feedGRANT, D. L., W. E. J. PHILLIPS and B. C. ing on the biota with a concentration of 13. VILLENEUVE. Metabolism of a PCB (Arochlor not more than 10 ppm of Aroclor 1254 1254) mixture in the rat. Bull. envir. Contam. Toxic. 6: 102-112. 1971. would be unlikely to contain concentrations 14. HAMMOND, A. Research topics, chemical pollution: of the PCB acutely toxic to man or other polychlorinated biphenyls. Science 175: 155-156. 1972. HICKMAN, C. P. and B. F. TRUMP. The kidney. In predators but these trout may be unable 15. Fish Physiology, Vol. I. W. S. Hoar and D. J. Ranto withstand the rigours of a natural envidall, Eds. New York and London; Academic Press. 1969. ronment.
ACKNOWLEDGMENTS The authors wish to express appreciation to the faculty and staff of the Departments of Pathology and Biomedical Science
(Toxicology), Ontario Veterinary College and to the personnel of the Veterinary Services Branch, (Poultry Pathology), Ontario Ministry of Agriculture and Food for their support and assistance. Throughout the course of this study the advice of Dr. N.
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29. SASCHENBRECKER, P. W. and D. J. ECOBICHON. Extraction and gas chromatographic analysis of chlorinated insecticides from animal tissues. Agric. Food Chem. 15: 168-170. 1967. 30. SCHOETTGER, R. A. In Progress in Sport Fishery Research. Resource Publication 121. p. 2. Washington, D.C.: Bureau of Sport Fisheries and Wildlife. 1973. 31. STALLING, D. L. and F. L. MAYER, JR. Toxicities of POBs to fish and environmental residues. Environmental Health Perspectives. Expl. Issue 1: 159-164. 1972. 32. VEITH, G. D. and G. F. LEE. Chlorobiphenyls PCBs) in fish from the Milwaukee region. Proc. 14th Conf. Great Lakes Res. Internat. Ass. Great Lakes Res. pp. 157-169. 1971. 33. YAP, H. H., D. DESAIAH, L. K. CUTKOMP and R. B. KOCH. Sensitivity of fish ATPases to polychlorinated byphenyls. Nature, Lond. 233: 61-62. 1971. 34. YOKOYAMA, H. 0. Studies on the origin, development and seasonal variations in the blood cells of the perch (P. flavescens). Bull. Jap. Soc. Sc. Fish. 34: 1066. 1960. 35. ZITKO, V. PCB's and organochlorine pesticides in some freshwater and marine fishes. Bull. envir. Contam. Toxic. 6; 464-471. 1971. 36. ZITKO, V. and P. M. CHOI. PCB and pp-DDE in eggs of cormorants, gulls and ducks from the Bay of Fundy, Canada. Bull. envir. Contam. Toxic. 7: 63-67. 1972.
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