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Neurotoxic behavioral effects of Lake Ontario salmon diets in rats
Neurotoxicologyand Teratology, Vol. 12, pp. 139-143. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0892-0362/90 $3.00 + .00
Neurotoxic Behavioral Effects of Lake Ontario Salmon Diets in Rats D A V I D R. H E R T Z L E R
Department o f Psychology, S U N Y at Oswego, Oswego, N Y 13126 R e c e i v e d 19 February 1988
HERTZLER, D. R. Neurotoxic behavioral effects of Lake Ontario salmon diets in rats. NEUROTOXICOL TERATOL 12(2) 139-143, 1990.--Six experiments were conducted to examine possible neurotoxic effects of the exposure to contaminants in Lake Ontario salmon administered through the diets of rats. Rats were fed different concentrations of fish (8%, 15% or 30%) in one of three diet conditions: Lake Ontario salmon, Pacific Ocean salmon, or laboratory rat chow only. Following 20 days on the diets, rats were tested for five minutes per day in a modified open field for one or three days. Lake Ontario salmon diets consistently produced significantly lower activity, rearing, and nosepoke behaviors in comparison with ocean salmon or rat chow diet conditions. A dose-response effect for concentration of lake salmon was obtained, and the attenuation effect occurred in males, females, adult or young animals, and postweaning females, with fish sampled over a five-year period. While only two of several potential contaminants were tested, both fish and brain analyses of rnirex and PCBs relate to the behavioral effects. Lake Ontario salmon
Mirex
PCBs
Locomotor activity
A potential source of toxic contamination of human food resources involves fish which have been obtained from polluted oceans, lakes and rivers. Such polluted waterways are a national problem, varying only in identification and quantification of the toxic chemicals involved. A graphic example of the severity of this problem exists in the Great Lakes where, despite their size and depth, high concentrations of several contaminants have been reported. The contaminants of primary concern may vary (e.g., PCB's in Lake Michigan, mirex in Lake Ontario), however, the issue is a universal one: that of demonstrating the nature and degree of toxicity and dangers of human consumption of contaminated lake fish. As a specific example, analyses by the New York State Department of Environmental Conservation have repeatedly indicated that salmonid fish from Lake Ontario routinely contain high levels of organochiorides such as mirex, photomirex, elevated levels of PCB's, and low levels of dieldrin, hexachlorobenzene and DDT (15). In addition, dioxin has been identified in fish, turtles and gulls in Lake Ontario (19). The organochloride contaminants are of special concern due to their toxicity, stability, and longevity. They are nonvolatile, hydrophobic, and lipophilic, and are concentrated in suspended lake sediments, where they enter the food chain (13). Despite state advisories against human consumption, sports fishing continues to be developed through the annual stocking of 5.2 million salmonids annually. In addition to year round lake fishing, spring and fall spawning runs of large salmon up the tributary rivers and streams produces a fishing frenzy, as large numbers of adult fish (10 to 35 Ib) are caught to be eaten. Through the consumption of fish, humans are being exposed to concentrations of a variety of contaminants which may produce synergistic toxicity. Previous research and state advisories have utilized dose-response studies involving chronic or acute doses of single contaminants. Toxic, carcinogenic, and reproductive effects of several individual pollutants have been well documented (1, 2, 24, 25).
Open field
Behavior
Rat
Since the central nervous system may be more sensitive to such toxic effects, the initial threat to humans may involve more subtle CNS effects of chronic low-level exposure to several interactive contaminants which bioaccumulate over time. Controlled laboratory studies of single contaminants have reported traces of organochloride pesticides in brain tissue, as well as CNS behavioral effects for PCB's (1, 3, 4, 20, 22) and mirex (11, 14, 21, 24). Possible synergistic CNS effects of multiple toxic substances in the contaminated fish have not been investigated. Synergistic effects, however, have been reported for other physiological functions by several investigators. Chu et al. (6) reported that hepatic enzyme induction identified in rats fed Lake Ontario salmon was due to several contaminants, because the level of PCB's in the fish diet was far below PCB levels necessary for similar PCB dose effects. Villeneuve et al. (23) reported that hepatic changes in rats fed Lake Ontario salmon were similar to effects of mirex, PCB's and DDT observed by others. They argue that the best method of evaluation of potential risk is to test for the combination of chemicals that occur naturally and have been environmentally altered (p. 651). Hornshaw, Auerlich and Johnson (9) support this approach in their report that PCB's derived from animals and, therefore, altered by exposure to the environment have more toxic effects than technical grade PCB mixtures. Support for possible synergistic effects has been reported by Martin (13), who demonstrated that feeding microtine rodents diets of Lake Ontario salmonids caused a greater decline in reproductive ability than did diets which had mirex or PCB's in the same concentration as the fish diet. Since Chu et al. (6) report the existence of more than 2,800 chemicals in the Great Lakes region, the specific contaminants and their interactive nature will be difficult to establish. The critical question, which is the focus of this paper, is whether consumption of environmentally contaminated Lake Ontario salmon produces behavioral differences that can serve as indices of neurotoxicity. 139
1~
HERTZLER
TABLE 1 CONDITIONS
Experiment 1 2 3 4 5 6
UTILIZED IN EXPERIMENTS
100 1~
Age
Fish Conditions*
n/Group
Sex
53-56 53-56 53-56 107-111 42~-4 80-85
C, O 30%, L 30% C, O 30%, L 15%, L 30% C, O 08%, L 08% O 30%, L 30% O 30%, L 30% O 30%, L 30%
7 7 7 8 8 4
M F F M M Ft
*C = chow, O = ocean salmon except 1 (tuna), L = Lake Ontario salmon. tMaternal rats 2 days postweaning.
The present experiments were designed to investigate whether rats that consumed diets containing different concentrations of Lake Ontario salmon would show behavioral indices of CNS toxicity. Several studies are reported in which dose-response effects are demonstrated. Treatments also included sex, age, and different manipulations. The highest diet fish condition (30%) was chosen to determine whether behavioral effects would be evident at diet levels below which toxic effects to the reproductive system have been previously reported (13).
80
A
= -- Chow C~'-"O Ocean H Lake
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60
e-
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~_ 20
I-0
0
I
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1
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DAYS FIG. 1. Mean three-day activity scores for rats fed 30% lake fish, 30% ocean fish or rat chow diets (Experiment 1).
Laboratory Rat Chow. The mixture was then combined with water to make a wet mash.
METHOD
Procedures Subjects and Diets The subjects were Holtzman strain rats bred in the Oswego colony. The colony has controlled temperature, humidity and a 12-hour daily dark-light cycle. Following weaning, litters were reduced to eight animals, which then were maintained in group cages until the study began. The studies utilized animals of different sex and age as illustrated in Table 1. Diets Subjects in each study were fed a wet ground rat chow mash in which the appropriate amount of Lake Ontario salmon or control salmon was thoroughly mixed. Fresh daily portions large enough to provide food at all times were provided in petri dishes. Approximately 75 g (dry weight) of the ground chow/fish mixture was mixed with 75 cc of water (more water was sometimes added to adjust for the liquid content of fish). Uneaten food was discarded and the dishes were cleaned each day. The three feeding conditions included two fish conditions, Lake Ontario salmon (LF) and ocean salmon (OF), and ground laboratory chow (C). All Lake Ontario fish were obtained in fall spawning runs in the Salmon River. Three fish for Experiments 1, 2, 3 were from the Spring Brook Weir (1982), three fish for Experiments 4 and 5 (1984) and for Experiment 6 (1986) were from the Altmar Hatchery located 19 kilometers upriver from the lake. Control ocean salmon, which was used in all studies except Experiment 1 (tuna control), was obtained from local markets packaged for human consumption. Aurlich and Ringer (2) report that ocean salmon contain negligible levels of PCB's, mirex, and other hydrocarbon pesticides, which our analyses confh'm. The .salmon was filleted according to the guidelines of the NY Department of Environmental Conservation and was ground, homogenized with a Hobart Grinder, and was frozen in packages sized for daily use. In Experiment 6 the skin was removed from the fillets. When needed, the fish was defrosted and mixed in appropriate proportions by weight with dry Purina
At the beginning of each study, the rats were weighed and placed individually in double-sized cages, with water available ad lib. For the next 20 days, they were given the appropriate diet condition. On Day 21 they were weighed, recorded for blind testing, and returned to their normal diet of unground Purina Laboratory Rat chow. Following the appropriate behavioral testing, animals in Experiments 2, 5, and 6 were sacrificed and their brains removed for analyses. Open field behavioral testing was accomplished in a modified open field (10). The black box measured 68 x 68 cm with 56 cm high walls. Silver grid lines designated 16 identical squares. A 3 cm hole was centered in each square. A white subfloor was located 4 cm below the floor. A behavioral trial was initiated by transporting the subject individually in a carrying cage to the dimly lit experimental room where it was placed in the center of the open field. The frequency of four behaviors was then counted during each minute of a five-minute trial session. The behaviors included activity (crossing of a grid line by the right forepaw), rearing (raising both forepaws off the floor), nosepoke (putting nose below floor surface in a hole) and grooming [paw, tail, or body licking, face rubbing, scratching, or body vibration (8)]. After each session the rat was returned to its home cage and the floor was cleaned with a dilute detergent solution. Animals in Experiments 1, 2, and 3 were tested for three consecutive days, while the remaining studies involved one day of testing. All studies except for Experiment 1 used blind control in behavioral testing. RESULTS
The rats willingly consumed the various diets and no significant differences in weight gains between diet groups were obtained in any of the studies. In Experiment 1, 3 x 3 analyses of variance were used to evaluate the effects of diets and days of testing for each of the four behaviors. Mean values, illustrated in Fig. 1, demonstrate a
NEUROTOXIC EFFECTS OF LAKE ONTARIO FISH
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BEHAVIORS ACTIVITY
FIG. 2. Mean activity scores for rats fed 30%, 15%, or 8% lake fish, ocean fish or rat chow diets (Experiments 2 and 3). reduced activity level for the LF group across days. The ANOVA for activity indicates significant effects for diets, F(2,18) = 4.52, p < 0 . 0 5 , across days, F(2,36) = 44.84, p < 0 . 0 5 , as well as significant interaction, F(4,36)= 2.99, p<0.05. A test for simple main effects (24) for Day 1 showed significant activity differences between LF and C, F(1,18)= 11.16, p<0.05, and LF vs. OF, F(1,18) = 5.21, p<0.05, however, no differences between OF and C. Differences were not significant for Days 2 and 3. Figure 1 illustrates that this is not unexpected, since the two control conditions show a marked reduction over Days 2 and 3, which is typical of normal rats, while the LF condition is depressed over all days. Experiment 2, which utilized ocean salmon as the control diet and double blind procedures, shows evidence of the reduced performance produced by the 30% LF diet condition. Matched 15% diet conditions were also included. In the 30% conditions, 3 x 4 A N O V A ' s were significant for days and diets, for activity, F(2,48)=29.72, p < 0 . 0 5 , F(3,24)=3.16, p<0.05, and nosepokes, F(2,48)= 16.41, p < 0 . 0 5 , F(3,24)= 4.20, p < 0 . 0 5 , F(3,24)=4.20, p<0.05. Individual comparisons showed no significant differences between OF and C for any comparison. The 30% LF group was significantly reduced on Day 1 compared with both OF and C groups on activity, t(12) = 10.05, 6.21, ps<0.05, rearing, t(12) = 3.58, 2.12, ps<0.05, and with OF only on nosepokes, t(12) = 3.02, p<0.05. Performance of the 30% LF group continued to be lower on Day 2, however, only the nosepoke condition was significantly different from the OF group, t(12) = 2.43, p<0.05. Figure 2 shows the mean activity scores for the three test days. Figure 3 illustrates the mean values for the nosepoke, rearing and grooming behaviors on Day 1. Figure 3 demonstrates that the group means for the LF 15% diet condition were lower than either the OF or C groups in all Day 1 conditions, however, only in activity did it approach significance against the OF group, t(l 2) = 2.10, p = 0.05. Two days after the final test session, animals were evaluated in a blind procedure by an independent observer for motor coordination. The test utilized a 2 cm × 1.5 m board elevated 30 cm above a table. The rat was placed on the center of the balance beam, and its motor coordination and balancing activity was observed for one min as it moved from to one end or the other.
FIG. 3. Day one mean frequency scores for three behaviors for rats fed either 30% or 15% lake fish, ocean fish or rat chow diets (Experiment 2). Each rat was rated either normal, slightly impaired, or greatly impaired. No animal was judged to be greatly impaired. Observations of the individual animals indicated variability between animals, however, this was distributed over all groups. Thus, motor coordination impairment was not responsible for the LF diet groups reduced activity or behavior effects. The rats in the LF 15% condition showed greater variability in their Day 1 performance, with two of the rats having lower activity scores than any other animal in any condition. Since this suggested the possibility of a dose threshold, Experiment 3 introduced matched 8% fish diets. The results of this study indicated no significant difference between the three diet conditions for any of the behavioral measures or days. The mean activity scores for these groups are shown in Fig. 4. In Experiments 4 and 5 other manipulations were accomplished. Fish collected in October 1984 were used, with one day of open field testing accomplished prior to the use of subjects in other research measures. Older (108 days) male rats were tested in Experiment 4 after exposure to the 30% diet conditions. A significant reduction was again obtained for the LF against the OF condition for activity, t(14) = 5.20, p<0.01. Experiment 5 utilized 42-44-day-old males which were exposed to manipulations designed to evaluate the resilience of the LF depression effect. Following the standard 20-day feeding paradigm, these animals were placed on a 80% food deprivation feeding schedule, and were handled and weighed daily for 7 days. The Day 8 open field test again showed a reduced activity level which approached significance, t(14)=1.83, p = 0 . 0 8 . A difference in the pattern of responses (per min) over the 5-min test was obtained in which the OF group showed a high min 1 rate which habituated over 5 min, while the LF group produced lower scores which did not habituate. The average activity for Experiments 4 and 5 is included in Fig. 4, which illustrates composite Day 1 activity means for all reported studies. Experiment 6 data were obtained from 8 female rats, from a teratological study, which were tested two days after the weaning of their litters. Two groups had consumed either 30% LF or 30% OF for at least 21 days during pregnancy and 15 days following birth of their pups. Thus, a longer diet exposure and postweaning behavior were variables of interest. They were tested on Day 23
142
HERTZLER
140
TABLE 2 ANALYSES OF FISH AND POOLED WHOLE BRAIN SAMPLES
120
Mirex Experiment 4
PCB 'S Experiment 2
Diet Analyses (ppb dry wt.)
(ppm dry wt.)
Experiment 2 100
>I'-
80
I-0
60
6 5
4
Ocean fish Lake fish
0.0 374.8720.8
2 10
I
CHOW
I
OCEAN
I
LAKE 8
I
|
LAKE LAKE 15 30
CONDITIONS FIG. 4. Composite Day one mean activity scores for all diet conditions for all experiments.
postpartum prior to being sacrificed for brain analyses. Fish were obtained in October 1986. Again, the LF diet group showed a decrease in behavioral measures, with significant differences in activity, t(6)= 4.10, p<0.01. The mean activity scores are shown in Fig. 4.
Diet and Brain Analysis The analyses reported in this paper were done by the Toxicology Bioassay Laboratory of the State University Research Center at Oswego. The laboratory was involved in a large scale research program involving analyses of mirex and PCB levels in Lake Ontario fish for a variety of projects. The standardized laboratory procedures were utilized for the analyses in these studies to evaluate levels of PCB's and mirex in the fish and brain samples. The PCB analyses utilized a modified method for quantitative PCB electron capture gas chromatography reported by Webb and McCall (27). Mirex was analyzed using methods from the EPA guidelines [Watts (26)]. Samples of the ground, lake and ocean salmon and pooled brain samples were analyzed. The rats were deeply anesthetized with ether and decapitated. The whole brains were extracted, pooled by diet condition and homogenized. Results of the analyses for Experiments 2 and 4 are shown in Table 2. Both the samples of Lake Ontario salmon and the brains of the rats in the Lake Ontario diet conditions have higher levels of PCBs and mirex than controls. DISCUSSION
These experiments provide convincing evidence that the consumption of Lake Ontario salmonids produces a significantly altered pattern of behavior in laboratory rats. When placed in a novel situation, such as the modified open field, male and female rats, of various ages, in various states including nonhandled,
502.05
10.33 21.4092.70
Pooled Whole Brain Analyses (all ppb dry wt.)
1
20
1.81
Chow Ocean Lake (15%) Lake (30%)
0.0 0.0 30.8 22.3
0.0 1.16 -23.46
220.3 150.6 251.4 389.6
handled, and food deprived conditions, show a consistent behavioral deficit when compared to rats fed comparable ocean salmon or control diets. This behavioral deficit was obtained in a doseresponse pattern with diets (30% and 15%, but not 8%) which are lower than those previously reported to produce toxic reproductive effects. The effect was consistently obtained for salmon obtained in 1981-82, 1984, and 1986. It was also replicated with maternal rats directly after weaning. Since motor performance deficits were not observed on the balance beam, these results support the hypothesis that the consumption of Lake Ontario salmon does produce a changed behavioral profile in rats, which may be due to CNS neurotoxic effects. In order for a CNS effect to occur, the toxic substance must cross the blood-brain barrier, and be identified in brain tissue. The analyses of rat brains in these studies indicate that elevated levels of both mirex and PCB's did occur. Mirex and PCB's were chosen as test contaminants because they represent a group of organochloride contaminants that are found in high concentrations in Lake Ontario. It is not to be concluded that either or both of these contaminants are solely responsible for the behavioral deficits. If the problem was that straightforward, the classical method of matched laboratory doses of the two contaminants could be employed. Martin (13) has already demonstrated that this approach does not apply in reproductive deficiencies caused by Lake Ontario salmon diets. A recent analysis of Lake Ontario salmon (conducted by NYS DEC, 1988) being used in our current studies reported detectable levels for 13 of 14 tested contaminants, while ocean salmon showed trace amounts for only 3. Again, this may not include all contaminants responsible for the behavioral effects. These studies consistently demonstrate a reduction in the various behavioral open field measures. Previous studies have reported both increased activity (16, 18, 20, 21) and decreased activity (15) by animals receiving specific organochloride treatments. A similar reduction to novel stimulation was reported for mice exposed to mirex and kepone by Reiter and Kidd (18). In the current studies the effect persisted across conditions, such as handling, deprivation, or maternal postweaning testing, even though these conditions did change the baseline activity levels. It can also be noted in Fig. 2 that the 30% lake fish diet group shows the greatest reduction on Day 1 when the rats were placed in the open field for their first exposure. The control groups then habituated over Days 2 and 3, while the experimental group produced low activity levels on Days 1 and 2, with a marked increase on Day 3. This pattern of responding, along with the
NEUROTOXIC EFFECTS OF LAKE ONTARIO FISH
143
persistent reduction of activity in open field tests, serves as preliminary evidence for a proposed explanation of the neurotoxic effects. It is hypothesized that the persistent reduction of activity in the open field tests may be due to a change in the manner in which these animals respond emotionally to aversive situations. An increased reactivity to aversive or novel (which may be aversive) stimulation would thus produce reduced behavior in the novel open field test situation. This explanation has been supported by evidence from four experiments reported by Daly, Hertzler and Sargent (7), which were designed to evaluate the influence of Lake Ontario salmon diets on rats' reaction to aversive events. The results from each of these studies indicate that ingestion of Lake Ontario salmon increases the reactivity of rats to aversive events. It is concluded, therefore, that the novel exposure to the open field was more aversive to the lake fish diet groups, and thus produced the lower activity scores. It is unlikely that humans would consume fish in their diets for
20 consecutive days, however, the procedure was used to accelerate the possible long-term bioaccumulation of the contaminants which can occur over an extended period of exposure. In humans the consumption of contaminated fish is probably only one contributing source of such pollutants. Due to their extreme stability, once absorbed, these contaminants accumulate and biomagnify over long periods of time. These studies provide additional evidence that support the thesis that repeated consumption of fish from contaminated waters such as Lake Ontario poses a potential health risk for humans. ACKNOWLEDGEMENTS The fish and brain analyses were conducted by the Toxicology Bioassay Laboratory of the State University Research Center at Oswego. I would like to thank Stephanie Popowich, Hope Joyce and Patricia Kelleher for their assistance in data collection, and Helen Daly for her suggestions during the research project and manuscript preparation.
REFERENCES 1. Allen, J. R.; Carstens, L. A.; Barsotti, D. A. Residual effects of short-term low-level exposure of nonhuman primates to polychlorihated biphenyls. Toxicol. Appl. Pharmacol. 30:440--451; 1974. 2. Auerlich, R. D.; Ringer, R. K. Current status of PCB toxicity to mink and effect on reproduction. Arch. Environ. Contam. Toxicol. 6: 279-292; 1977. 3. Bowman, R. E.; Heironimus, M. P. Hypoactivity in adolescent monkeys perinatally exposed to PCBs and hyperactive as juveniles. Neurobehav. Toxicol. Teratol. 3:15-18; 1981. 4. Bowman, R. E.; Heironimus, M. P.; Allen, J. R. Correlation of PCB body burden with behavioral toxicology in monkeys. Pharmacol. Biochem. Behav. 9:49-56; 1978. 5. Chou, S. M.; Miike, T.; Payne, W. M.; Davis, G. J. Neuropathology of 'spinning' syndrome induced by prenatal intoxication with a PCB in mice. Ann. NY Acad. Sci. 320:373-395; 1979. 6. Chu, I.; Villeneuve, D. C.; Valli, V. E.; Ritter, L.; Norstrom, R. J.; Ryan, J. J.; Becking, G. C. Toxicological response and its reversibility in rats fed Lake Ontario or Pacific coho salmon for 13 weeks. J. Environ. Sci. Health [B] 19:713-732; 1984. 7. Daly, H. B.; Hertzler, D. R.; Sargent, D. Ingestion of environmentally contaminated Lake Ontario salmon by laboratory rats increases avoidance of unpredictable aversive nom'eward and mild electric shock. Behav. Neurosci. 103:1356-1365; 1989. 8. Gispen, W. H.; Weigant, V. M.; Greven, H. M.; deWeid, D. The induction of excessive grooming in the rat by intraventricular applications of peptides derived from ACTH. Life Sci. 17:645-652; 1975. 9. Homshaw, T. C.; Aulerich, R. J.; Johnson, H. E. Feeding Great Lakes fish to mink: Effects on mink and accumulation and elimination of PCBs by mink. J. Toxicol. Environ. Health 11:933-946; 1983. 10. Isaacson, R. L.; Yongue, B.; McClearn, D. Dopamine agonists: their effect on locomotion and exploration. Behav. Biol. 23:163-179; 1978. 11. Ivie, G. W.; Gibson, J. R.; Bryant, H. E.; Begin, J. J.; Barnett, J. R.; Dorough, H. W. Accumulation, distribution and excretion of Mirex14C in animals exposed for long periods to the insecticides in the diet. J. Agric. Food Chem. 22(4):646--653; 1974. 12. Jacobson, J.; Jacobson, S.; Fein, G.; Schwartz, P.; Dowler, J. Prenatal exposure to an environmental toxin: a test of the multiple effects model. Dev. Psychol. 20(4):523-532; 1984.
13. Martin, K. Reproductive and tissue response in prairie voles fed mirex and lake ontario salmon. NYSEA Grant (NOAA) Report; 1982. 14. Mehendale, H. M.; Fishbein, L.; Fields, M.; Matthews, H. B. Fate of Mirex-14C in rats and plants. Bull. Environ. Contam. Toxicol. 8:200-207; 1972. 15. NYSDEC. Toxic substances in fish and wildlife; 1984. 16. Peeler, D. F. Open field activity as a function of preweaning generational exposure to mirex. Miss. Acad. Sci. J. 21:58-63; 1976. 17. Reiter, L. Behavioral toxicology: effects of early postnatal exposure to neurotoxins on development of locomotor activity in the rat. J. Occup. Med. 19:201-204; 1977. 18. Reiter, L.; Kidd, K. Behavioral effects of sub-acute exposure to Kepone or Mirex on the weanling rat. Toxicol. Appl. Pharmacol. 45:357; 1978. 19. Stone, W. B. Levels ofPCBs, DDE and Mirex in waterfowl collected in New York State, 1978-80. Arch. Environ. Contam. Toxicol. 13:372-382; 1984. 20. Storm, J. E.; Hart, J. L.; Smith, R. F. Behavior of mice after pre- and postnatal exposure to Arochlor 1254. Neurobehav. Toxicol. Teratol. 3:5-9; 1981. 21. Thorne, B. M.; Rallor, E.; Wallace, T. Mirex and behavior in the long evans rat. Bull. Environ. Contam. Toxicol. 19:351-359; 1978. 22. Tilson, M. A.; Davis, G. J.; McLachland, J. A.; Lucier, G. W. The effects of polychlorinated biphenyls given prenatally on the neurobehavioral development of mice. Environ. Res. 18:466--474; 1979. 23. Villeneuve, D. C.; Valle, V.; Norstrom, R.; Freeman, H.; Sanglang, G. B.; Ritter, L.; Becking, G. Toxicological response of rats fed Lake Ontario or Pacific Coho salmon for 28 days. J. Environ. Health [B] 16:649-689; 1981. 24. Warren, R. J.; Kirkpatrick, R. L.; Young, R. W. Barbituate-induced sleeping times, liver weights, and reproduction of cottontail rabbits after Mirex ingestion. Bull. Environ. Contam. Toxicol. 19:223-227; 1978. 25. Waters, E. M.; Huff, J. E.; Gerstner, H. B. Mirex: an overview. Environ. Res. 14:212-222; 1977. 26. Watts, R., ed. Analyses of pesticide residues in human and environmental samples. EPA Manual; June 1980. 27. Webb, R. G.; McCall, A. C. Quantitative PCBs standards for electron capture gas chromatography. J. Chromatogr. Sci. 11:366--373; 1973.
0892-0362/90 $3.00 + .00
Neurotoxic Behavioral Effects of Lake Ontario Salmon Diets in Rats D A V I D R. H E R T Z L E R
Department o f Psychology, S U N Y at Oswego, Oswego, N Y 13126 R e c e i v e d 19 February 1988
HERTZLER, D. R. Neurotoxic behavioral effects of Lake Ontario salmon diets in rats. NEUROTOXICOL TERATOL 12(2) 139-143, 1990.--Six experiments were conducted to examine possible neurotoxic effects of the exposure to contaminants in Lake Ontario salmon administered through the diets of rats. Rats were fed different concentrations of fish (8%, 15% or 30%) in one of three diet conditions: Lake Ontario salmon, Pacific Ocean salmon, or laboratory rat chow only. Following 20 days on the diets, rats were tested for five minutes per day in a modified open field for one or three days. Lake Ontario salmon diets consistently produced significantly lower activity, rearing, and nosepoke behaviors in comparison with ocean salmon or rat chow diet conditions. A dose-response effect for concentration of lake salmon was obtained, and the attenuation effect occurred in males, females, adult or young animals, and postweaning females, with fish sampled over a five-year period. While only two of several potential contaminants were tested, both fish and brain analyses of rnirex and PCBs relate to the behavioral effects. Lake Ontario salmon
Mirex
PCBs
Locomotor activity
A potential source of toxic contamination of human food resources involves fish which have been obtained from polluted oceans, lakes and rivers. Such polluted waterways are a national problem, varying only in identification and quantification of the toxic chemicals involved. A graphic example of the severity of this problem exists in the Great Lakes where, despite their size and depth, high concentrations of several contaminants have been reported. The contaminants of primary concern may vary (e.g., PCB's in Lake Michigan, mirex in Lake Ontario), however, the issue is a universal one: that of demonstrating the nature and degree of toxicity and dangers of human consumption of contaminated lake fish. As a specific example, analyses by the New York State Department of Environmental Conservation have repeatedly indicated that salmonid fish from Lake Ontario routinely contain high levels of organochiorides such as mirex, photomirex, elevated levels of PCB's, and low levels of dieldrin, hexachlorobenzene and DDT (15). In addition, dioxin has been identified in fish, turtles and gulls in Lake Ontario (19). The organochloride contaminants are of special concern due to their toxicity, stability, and longevity. They are nonvolatile, hydrophobic, and lipophilic, and are concentrated in suspended lake sediments, where they enter the food chain (13). Despite state advisories against human consumption, sports fishing continues to be developed through the annual stocking of 5.2 million salmonids annually. In addition to year round lake fishing, spring and fall spawning runs of large salmon up the tributary rivers and streams produces a fishing frenzy, as large numbers of adult fish (10 to 35 Ib) are caught to be eaten. Through the consumption of fish, humans are being exposed to concentrations of a variety of contaminants which may produce synergistic toxicity. Previous research and state advisories have utilized dose-response studies involving chronic or acute doses of single contaminants. Toxic, carcinogenic, and reproductive effects of several individual pollutants have been well documented (1, 2, 24, 25).
Open field
Behavior
Rat
Since the central nervous system may be more sensitive to such toxic effects, the initial threat to humans may involve more subtle CNS effects of chronic low-level exposure to several interactive contaminants which bioaccumulate over time. Controlled laboratory studies of single contaminants have reported traces of organochloride pesticides in brain tissue, as well as CNS behavioral effects for PCB's (1, 3, 4, 20, 22) and mirex (11, 14, 21, 24). Possible synergistic CNS effects of multiple toxic substances in the contaminated fish have not been investigated. Synergistic effects, however, have been reported for other physiological functions by several investigators. Chu et al. (6) reported that hepatic enzyme induction identified in rats fed Lake Ontario salmon was due to several contaminants, because the level of PCB's in the fish diet was far below PCB levels necessary for similar PCB dose effects. Villeneuve et al. (23) reported that hepatic changes in rats fed Lake Ontario salmon were similar to effects of mirex, PCB's and DDT observed by others. They argue that the best method of evaluation of potential risk is to test for the combination of chemicals that occur naturally and have been environmentally altered (p. 651). Hornshaw, Auerlich and Johnson (9) support this approach in their report that PCB's derived from animals and, therefore, altered by exposure to the environment have more toxic effects than technical grade PCB mixtures. Support for possible synergistic effects has been reported by Martin (13), who demonstrated that feeding microtine rodents diets of Lake Ontario salmonids caused a greater decline in reproductive ability than did diets which had mirex or PCB's in the same concentration as the fish diet. Since Chu et al. (6) report the existence of more than 2,800 chemicals in the Great Lakes region, the specific contaminants and their interactive nature will be difficult to establish. The critical question, which is the focus of this paper, is whether consumption of environmentally contaminated Lake Ontario salmon produces behavioral differences that can serve as indices of neurotoxicity. 139
1~
HERTZLER
TABLE 1 CONDITIONS
Experiment 1 2 3 4 5 6
UTILIZED IN EXPERIMENTS
100 1~
Age
Fish Conditions*
n/Group
Sex
53-56 53-56 53-56 107-111 42~-4 80-85
C, O 30%, L 30% C, O 30%, L 15%, L 30% C, O 08%, L 08% O 30%, L 30% O 30%, L 30% O 30%, L 30%
7 7 7 8 8 4
M F F M M Ft
*C = chow, O = ocean salmon except 1 (tuna), L = Lake Ontario salmon. tMaternal rats 2 days postweaning.
The present experiments were designed to investigate whether rats that consumed diets containing different concentrations of Lake Ontario salmon would show behavioral indices of CNS toxicity. Several studies are reported in which dose-response effects are demonstrated. Treatments also included sex, age, and different manipulations. The highest diet fish condition (30%) was chosen to determine whether behavioral effects would be evident at diet levels below which toxic effects to the reproductive system have been previously reported (13).
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Laboratory Rat Chow. The mixture was then combined with water to make a wet mash.
METHOD
Procedures Subjects and Diets The subjects were Holtzman strain rats bred in the Oswego colony. The colony has controlled temperature, humidity and a 12-hour daily dark-light cycle. Following weaning, litters were reduced to eight animals, which then were maintained in group cages until the study began. The studies utilized animals of different sex and age as illustrated in Table 1. Diets Subjects in each study were fed a wet ground rat chow mash in which the appropriate amount of Lake Ontario salmon or control salmon was thoroughly mixed. Fresh daily portions large enough to provide food at all times were provided in petri dishes. Approximately 75 g (dry weight) of the ground chow/fish mixture was mixed with 75 cc of water (more water was sometimes added to adjust for the liquid content of fish). Uneaten food was discarded and the dishes were cleaned each day. The three feeding conditions included two fish conditions, Lake Ontario salmon (LF) and ocean salmon (OF), and ground laboratory chow (C). All Lake Ontario fish were obtained in fall spawning runs in the Salmon River. Three fish for Experiments 1, 2, 3 were from the Spring Brook Weir (1982), three fish for Experiments 4 and 5 (1984) and for Experiment 6 (1986) were from the Altmar Hatchery located 19 kilometers upriver from the lake. Control ocean salmon, which was used in all studies except Experiment 1 (tuna control), was obtained from local markets packaged for human consumption. Aurlich and Ringer (2) report that ocean salmon contain negligible levels of PCB's, mirex, and other hydrocarbon pesticides, which our analyses confh'm. The .salmon was filleted according to the guidelines of the NY Department of Environmental Conservation and was ground, homogenized with a Hobart Grinder, and was frozen in packages sized for daily use. In Experiment 6 the skin was removed from the fillets. When needed, the fish was defrosted and mixed in appropriate proportions by weight with dry Purina
At the beginning of each study, the rats were weighed and placed individually in double-sized cages, with water available ad lib. For the next 20 days, they were given the appropriate diet condition. On Day 21 they were weighed, recorded for blind testing, and returned to their normal diet of unground Purina Laboratory Rat chow. Following the appropriate behavioral testing, animals in Experiments 2, 5, and 6 were sacrificed and their brains removed for analyses. Open field behavioral testing was accomplished in a modified open field (10). The black box measured 68 x 68 cm with 56 cm high walls. Silver grid lines designated 16 identical squares. A 3 cm hole was centered in each square. A white subfloor was located 4 cm below the floor. A behavioral trial was initiated by transporting the subject individually in a carrying cage to the dimly lit experimental room where it was placed in the center of the open field. The frequency of four behaviors was then counted during each minute of a five-minute trial session. The behaviors included activity (crossing of a grid line by the right forepaw), rearing (raising both forepaws off the floor), nosepoke (putting nose below floor surface in a hole) and grooming [paw, tail, or body licking, face rubbing, scratching, or body vibration (8)]. After each session the rat was returned to its home cage and the floor was cleaned with a dilute detergent solution. Animals in Experiments 1, 2, and 3 were tested for three consecutive days, while the remaining studies involved one day of testing. All studies except for Experiment 1 used blind control in behavioral testing. RESULTS
The rats willingly consumed the various diets and no significant differences in weight gains between diet groups were obtained in any of the studies. In Experiment 1, 3 x 3 analyses of variance were used to evaluate the effects of diets and days of testing for each of the four behaviors. Mean values, illustrated in Fig. 1, demonstrate a
NEUROTOXIC EFFECTS OF LAKE ONTARIO FISH
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FIG. 2. Mean activity scores for rats fed 30%, 15%, or 8% lake fish, ocean fish or rat chow diets (Experiments 2 and 3). reduced activity level for the LF group across days. The ANOVA for activity indicates significant effects for diets, F(2,18) = 4.52, p < 0 . 0 5 , across days, F(2,36) = 44.84, p < 0 . 0 5 , as well as significant interaction, F(4,36)= 2.99, p<0.05. A test for simple main effects (24) for Day 1 showed significant activity differences between LF and C, F(1,18)= 11.16, p<0.05, and LF vs. OF, F(1,18) = 5.21, p<0.05, however, no differences between OF and C. Differences were not significant for Days 2 and 3. Figure 1 illustrates that this is not unexpected, since the two control conditions show a marked reduction over Days 2 and 3, which is typical of normal rats, while the LF condition is depressed over all days. Experiment 2, which utilized ocean salmon as the control diet and double blind procedures, shows evidence of the reduced performance produced by the 30% LF diet condition. Matched 15% diet conditions were also included. In the 30% conditions, 3 x 4 A N O V A ' s were significant for days and diets, for activity, F(2,48)=29.72, p < 0 . 0 5 , F(3,24)=3.16, p<0.05, and nosepokes, F(2,48)= 16.41, p < 0 . 0 5 , F(3,24)= 4.20, p < 0 . 0 5 , F(3,24)=4.20, p<0.05. Individual comparisons showed no significant differences between OF and C for any comparison. The 30% LF group was significantly reduced on Day 1 compared with both OF and C groups on activity, t(12) = 10.05, 6.21, ps<0.05, rearing, t(12) = 3.58, 2.12, ps<0.05, and with OF only on nosepokes, t(12) = 3.02, p<0.05. Performance of the 30% LF group continued to be lower on Day 2, however, only the nosepoke condition was significantly different from the OF group, t(12) = 2.43, p<0.05. Figure 2 shows the mean activity scores for the three test days. Figure 3 illustrates the mean values for the nosepoke, rearing and grooming behaviors on Day 1. Figure 3 demonstrates that the group means for the LF 15% diet condition were lower than either the OF or C groups in all Day 1 conditions, however, only in activity did it approach significance against the OF group, t(l 2) = 2.10, p = 0.05. Two days after the final test session, animals were evaluated in a blind procedure by an independent observer for motor coordination. The test utilized a 2 cm × 1.5 m board elevated 30 cm above a table. The rat was placed on the center of the balance beam, and its motor coordination and balancing activity was observed for one min as it moved from to one end or the other.
FIG. 3. Day one mean frequency scores for three behaviors for rats fed either 30% or 15% lake fish, ocean fish or rat chow diets (Experiment 2). Each rat was rated either normal, slightly impaired, or greatly impaired. No animal was judged to be greatly impaired. Observations of the individual animals indicated variability between animals, however, this was distributed over all groups. Thus, motor coordination impairment was not responsible for the LF diet groups reduced activity or behavior effects. The rats in the LF 15% condition showed greater variability in their Day 1 performance, with two of the rats having lower activity scores than any other animal in any condition. Since this suggested the possibility of a dose threshold, Experiment 3 introduced matched 8% fish diets. The results of this study indicated no significant difference between the three diet conditions for any of the behavioral measures or days. The mean activity scores for these groups are shown in Fig. 4. In Experiments 4 and 5 other manipulations were accomplished. Fish collected in October 1984 were used, with one day of open field testing accomplished prior to the use of subjects in other research measures. Older (108 days) male rats were tested in Experiment 4 after exposure to the 30% diet conditions. A significant reduction was again obtained for the LF against the OF condition for activity, t(14) = 5.20, p<0.01. Experiment 5 utilized 42-44-day-old males which were exposed to manipulations designed to evaluate the resilience of the LF depression effect. Following the standard 20-day feeding paradigm, these animals were placed on a 80% food deprivation feeding schedule, and were handled and weighed daily for 7 days. The Day 8 open field test again showed a reduced activity level which approached significance, t(14)=1.83, p = 0 . 0 8 . A difference in the pattern of responses (per min) over the 5-min test was obtained in which the OF group showed a high min 1 rate which habituated over 5 min, while the LF group produced lower scores which did not habituate. The average activity for Experiments 4 and 5 is included in Fig. 4, which illustrates composite Day 1 activity means for all reported studies. Experiment 6 data were obtained from 8 female rats, from a teratological study, which were tested two days after the weaning of their litters. Two groups had consumed either 30% LF or 30% OF for at least 21 days during pregnancy and 15 days following birth of their pups. Thus, a longer diet exposure and postweaning behavior were variables of interest. They were tested on Day 23
142
HERTZLER
140
TABLE 2 ANALYSES OF FISH AND POOLED WHOLE BRAIN SAMPLES
120
Mirex Experiment 4
PCB 'S Experiment 2
Diet Analyses (ppb dry wt.)
(ppm dry wt.)
Experiment 2 100
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2 10
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LAKE LAKE 15 30
CONDITIONS FIG. 4. Composite Day one mean activity scores for all diet conditions for all experiments.
postpartum prior to being sacrificed for brain analyses. Fish were obtained in October 1986. Again, the LF diet group showed a decrease in behavioral measures, with significant differences in activity, t(6)= 4.10, p<0.01. The mean activity scores are shown in Fig. 4.
Diet and Brain Analysis The analyses reported in this paper were done by the Toxicology Bioassay Laboratory of the State University Research Center at Oswego. The laboratory was involved in a large scale research program involving analyses of mirex and PCB levels in Lake Ontario fish for a variety of projects. The standardized laboratory procedures were utilized for the analyses in these studies to evaluate levels of PCB's and mirex in the fish and brain samples. The PCB analyses utilized a modified method for quantitative PCB electron capture gas chromatography reported by Webb and McCall (27). Mirex was analyzed using methods from the EPA guidelines [Watts (26)]. Samples of the ground, lake and ocean salmon and pooled brain samples were analyzed. The rats were deeply anesthetized with ether and decapitated. The whole brains were extracted, pooled by diet condition and homogenized. Results of the analyses for Experiments 2 and 4 are shown in Table 2. Both the samples of Lake Ontario salmon and the brains of the rats in the Lake Ontario diet conditions have higher levels of PCBs and mirex than controls. DISCUSSION
These experiments provide convincing evidence that the consumption of Lake Ontario salmonids produces a significantly altered pattern of behavior in laboratory rats. When placed in a novel situation, such as the modified open field, male and female rats, of various ages, in various states including nonhandled,
502.05
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Pooled Whole Brain Analyses (all ppb dry wt.)
1
20
1.81
Chow Ocean Lake (15%) Lake (30%)
0.0 0.0 30.8 22.3
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handled, and food deprived conditions, show a consistent behavioral deficit when compared to rats fed comparable ocean salmon or control diets. This behavioral deficit was obtained in a doseresponse pattern with diets (30% and 15%, but not 8%) which are lower than those previously reported to produce toxic reproductive effects. The effect was consistently obtained for salmon obtained in 1981-82, 1984, and 1986. It was also replicated with maternal rats directly after weaning. Since motor performance deficits were not observed on the balance beam, these results support the hypothesis that the consumption of Lake Ontario salmon does produce a changed behavioral profile in rats, which may be due to CNS neurotoxic effects. In order for a CNS effect to occur, the toxic substance must cross the blood-brain barrier, and be identified in brain tissue. The analyses of rat brains in these studies indicate that elevated levels of both mirex and PCB's did occur. Mirex and PCB's were chosen as test contaminants because they represent a group of organochloride contaminants that are found in high concentrations in Lake Ontario. It is not to be concluded that either or both of these contaminants are solely responsible for the behavioral deficits. If the problem was that straightforward, the classical method of matched laboratory doses of the two contaminants could be employed. Martin (13) has already demonstrated that this approach does not apply in reproductive deficiencies caused by Lake Ontario salmon diets. A recent analysis of Lake Ontario salmon (conducted by NYS DEC, 1988) being used in our current studies reported detectable levels for 13 of 14 tested contaminants, while ocean salmon showed trace amounts for only 3. Again, this may not include all contaminants responsible for the behavioral effects. These studies consistently demonstrate a reduction in the various behavioral open field measures. Previous studies have reported both increased activity (16, 18, 20, 21) and decreased activity (15) by animals receiving specific organochloride treatments. A similar reduction to novel stimulation was reported for mice exposed to mirex and kepone by Reiter and Kidd (18). In the current studies the effect persisted across conditions, such as handling, deprivation, or maternal postweaning testing, even though these conditions did change the baseline activity levels. It can also be noted in Fig. 2 that the 30% lake fish diet group shows the greatest reduction on Day 1 when the rats were placed in the open field for their first exposure. The control groups then habituated over Days 2 and 3, while the experimental group produced low activity levels on Days 1 and 2, with a marked increase on Day 3. This pattern of responding, along with the
NEUROTOXIC EFFECTS OF LAKE ONTARIO FISH
143
persistent reduction of activity in open field tests, serves as preliminary evidence for a proposed explanation of the neurotoxic effects. It is hypothesized that the persistent reduction of activity in the open field tests may be due to a change in the manner in which these animals respond emotionally to aversive situations. An increased reactivity to aversive or novel (which may be aversive) stimulation would thus produce reduced behavior in the novel open field test situation. This explanation has been supported by evidence from four experiments reported by Daly, Hertzler and Sargent (7), which were designed to evaluate the influence of Lake Ontario salmon diets on rats' reaction to aversive events. The results from each of these studies indicate that ingestion of Lake Ontario salmon increases the reactivity of rats to aversive events. It is concluded, therefore, that the novel exposure to the open field was more aversive to the lake fish diet groups, and thus produced the lower activity scores. It is unlikely that humans would consume fish in their diets for
20 consecutive days, however, the procedure was used to accelerate the possible long-term bioaccumulation of the contaminants which can occur over an extended period of exposure. In humans the consumption of contaminated fish is probably only one contributing source of such pollutants. Due to their extreme stability, once absorbed, these contaminants accumulate and biomagnify over long periods of time. These studies provide additional evidence that support the thesis that repeated consumption of fish from contaminated waters such as Lake Ontario poses a potential health risk for humans. ACKNOWLEDGEMENTS The fish and brain analyses were conducted by the Toxicology Bioassay Laboratory of the State University Research Center at Oswego. I would like to thank Stephanie Popowich, Hope Joyce and Patricia Kelleher for their assistance in data collection, and Helen Daly for her suggestions during the research project and manuscript preparation.
REFERENCES 1. Allen, J. R.; Carstens, L. A.; Barsotti, D. A. Residual effects of short-term low-level exposure of nonhuman primates to polychlorihated biphenyls. Toxicol. Appl. Pharmacol. 30:440--451; 1974. 2. Auerlich, R. D.; Ringer, R. K. Current status of PCB toxicity to mink and effect on reproduction. Arch. Environ. Contam. Toxicol. 6: 279-292; 1977. 3. Bowman, R. E.; Heironimus, M. P. Hypoactivity in adolescent monkeys perinatally exposed to PCBs and hyperactive as juveniles. Neurobehav. Toxicol. Teratol. 3:15-18; 1981. 4. Bowman, R. E.; Heironimus, M. P.; Allen, J. R. Correlation of PCB body burden with behavioral toxicology in monkeys. Pharmacol. Biochem. Behav. 9:49-56; 1978. 5. Chou, S. M.; Miike, T.; Payne, W. M.; Davis, G. J. Neuropathology of 'spinning' syndrome induced by prenatal intoxication with a PCB in mice. Ann. NY Acad. Sci. 320:373-395; 1979. 6. Chu, I.; Villeneuve, D. C.; Valli, V. E.; Ritter, L.; Norstrom, R. J.; Ryan, J. J.; Becking, G. C. Toxicological response and its reversibility in rats fed Lake Ontario or Pacific coho salmon for 13 weeks. J. Environ. Sci. Health [B] 19:713-732; 1984. 7. Daly, H. B.; Hertzler, D. R.; Sargent, D. Ingestion of environmentally contaminated Lake Ontario salmon by laboratory rats increases avoidance of unpredictable aversive nom'eward and mild electric shock. Behav. Neurosci. 103:1356-1365; 1989. 8. Gispen, W. H.; Weigant, V. M.; Greven, H. M.; deWeid, D. The induction of excessive grooming in the rat by intraventricular applications of peptides derived from ACTH. Life Sci. 17:645-652; 1975. 9. Homshaw, T. C.; Aulerich, R. J.; Johnson, H. E. Feeding Great Lakes fish to mink: Effects on mink and accumulation and elimination of PCBs by mink. J. Toxicol. Environ. Health 11:933-946; 1983. 10. Isaacson, R. L.; Yongue, B.; McClearn, D. Dopamine agonists: their effect on locomotion and exploration. Behav. Biol. 23:163-179; 1978. 11. Ivie, G. W.; Gibson, J. R.; Bryant, H. E.; Begin, J. J.; Barnett, J. R.; Dorough, H. W. Accumulation, distribution and excretion of Mirex14C in animals exposed for long periods to the insecticides in the diet. J. Agric. Food Chem. 22(4):646--653; 1974. 12. Jacobson, J.; Jacobson, S.; Fein, G.; Schwartz, P.; Dowler, J. Prenatal exposure to an environmental toxin: a test of the multiple effects model. Dev. Psychol. 20(4):523-532; 1984.
13. Martin, K. Reproductive and tissue response in prairie voles fed mirex and lake ontario salmon. NYSEA Grant (NOAA) Report; 1982. 14. Mehendale, H. M.; Fishbein, L.; Fields, M.; Matthews, H. B. Fate of Mirex-14C in rats and plants. Bull. Environ. Contam. Toxicol. 8:200-207; 1972. 15. NYSDEC. Toxic substances in fish and wildlife; 1984. 16. Peeler, D. F. Open field activity as a function of preweaning generational exposure to mirex. Miss. Acad. Sci. J. 21:58-63; 1976. 17. Reiter, L. Behavioral toxicology: effects of early postnatal exposure to neurotoxins on development of locomotor activity in the rat. J. Occup. Med. 19:201-204; 1977. 18. Reiter, L.; Kidd, K. Behavioral effects of sub-acute exposure to Kepone or Mirex on the weanling rat. Toxicol. Appl. Pharmacol. 45:357; 1978. 19. Stone, W. B. Levels ofPCBs, DDE and Mirex in waterfowl collected in New York State, 1978-80. Arch. Environ. Contam. Toxicol. 13:372-382; 1984. 20. Storm, J. E.; Hart, J. L.; Smith, R. F. Behavior of mice after pre- and postnatal exposure to Arochlor 1254. Neurobehav. Toxicol. Teratol. 3:5-9; 1981. 21. Thorne, B. M.; Rallor, E.; Wallace, T. Mirex and behavior in the long evans rat. Bull. Environ. Contam. Toxicol. 19:351-359; 1978. 22. Tilson, M. A.; Davis, G. J.; McLachland, J. A.; Lucier, G. W. The effects of polychlorinated biphenyls given prenatally on the neurobehavioral development of mice. Environ. Res. 18:466--474; 1979. 23. Villeneuve, D. C.; Valle, V.; Norstrom, R.; Freeman, H.; Sanglang, G. B.; Ritter, L.; Becking, G. Toxicological response of rats fed Lake Ontario or Pacific Coho salmon for 28 days. J. Environ. Health [B] 16:649-689; 1981. 24. Warren, R. J.; Kirkpatrick, R. L.; Young, R. W. Barbituate-induced sleeping times, liver weights, and reproduction of cottontail rabbits after Mirex ingestion. Bull. Environ. Contam. Toxicol. 19:223-227; 1978. 25. Waters, E. M.; Huff, J. E.; Gerstner, H. B. Mirex: an overview. Environ. Res. 14:212-222; 1977. 26. Watts, R., ed. Analyses of pesticide residues in human and environmental samples. EPA Manual; June 1980. 27. Webb, R. G.; McCall, A. C. Quantitative PCBs standards for electron capture gas chromatography. J. Chromatogr. Sci. 11:366--373; 1973.