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Hexachlorobenzene distribution in tissues of swine
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
51,
1-7 (1979)
Hexachlorobenzene Distribution in Tissues of Swine LARRY G. HANSEN, JOSEPH SIMON, STEVEN B. DORN, AND RICHARD
H. TESKE’
Division of Pharmacology and Toxicology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801, and Division of Veterinary Medical Research, Food and Drug Administration, Beltsville, Maryland 20705 Received September
18, 1978; accepted July 9, 1979
Hexachlorobenzene Distribution in Tissues of Swine. HANSEN, L. G., SIMON, J., DORN, B., AND TESKE, R. H. (1979). Tox~col. Appl. Pharmacol. 51, 1-7. Purified hexachlorobenzene (HCB) was administered to third-litter sows at dietary concentrations of 0, 1, or 20 ppm throughout gestation and nursing. Swine receiving 1 ppm were not adversely affected and residue concentrations in tissues other than fat and bone marrow remained at or below the dietary concentration. Residues of HCB in the dissectable fat of these pigs accumuiated to concentrations five- to seven-fold higher than the dietary concentration. Piglets accumulated fat residues, through both placental transfer and nursing, that were higher than those of the sows. A similar proportional accumulation of HCB in fat occurred in sows receiving 20 ppm in the diet. Other toxic signs included neutrophiha, gastric irritation, fatty replacement of Brunner’s gland, and pancreatic periductal fibrosis. Hepatotoxicity was not apparent, perhaps because of age-related changes in both control and treatment groups. S.
Hexachlorobenzene (HCB) contamination results from both agricultural use and industrial discharge, and residues are found in humans, animals, and animal feeds (Booth and McDowell, 1975; Burns and Miller, 1975; Yang et al., 1976). Toxic manifestations include hepatomegaly, porphyria, immunosuppression, and interference with hematopoiesis and central nervous system function (Schmid, 1960; Booth and McDowell, 1975; Gralla ethl., 1977; Hansen et al., 1977; Loose et al., 1977). The particular toxicosis that occurs is highly dependent on species, sex, dose, and time of exposure. The present study is undertaken to determine the effects of HCB on swine and their offspring and to define further the accumulation and distribution of HCB residues in food-producing animals.
METHODS Fifteen crossbred (Hampshire/Yorkshire) sow? of proven performance (third litter) were weighed and randomly assigned to individual pens (1.8 x 2.1 m) which were grouped according to diet treatment. They were fed standard 16% protein ration for a ‘J-day acclimation period. To prepare the experimental diets, purified HCB” was added to the standard ration by way of a concentrated premix to attam levels of 1 and 20 ppm (Hansen er al., 1977). Controls were continued on the uncontaminated ration. Analyses of four batches of feed from each dietary level -by electron capture gasliquid chromatography showed that the HCB content was ~0.01, 1.36+(SD) 0.19, and 21.50+1.12ppm for the control, “1-ppm” and “20-ppm” diets, respectively. The sows received 2-2.5 kg of feed twice daily 2 Animal Science Department, University of Illinois at Urbana. 3 Hexachlorobenzene, 95 o/opure, obtained from the Aldrich Chemical Co., Milwaukee, Wis., was washed with warm ethanol, and recrystallized three times from benzene. The resulting product was greater than 99.5 % pure (gas-liquid chromatography).
’ Division of Veterinary Medical Research, Food and Drug Administration, Beltsville, Md. 20705. 1
All
0041-008x/79/13ooo1-07$02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
2
HANSEN
and fresh water was provided ud libit~nr by an automatic watering system. After the sows had been given the treated feed for I week, they were checked daily for signs of estrus and hand-mated to one of two purebred Berkshire boars.” Three to five days before farrowing, they were weighed and the pens were equipped with farrowing crates, straw, and heat lamps. Small amounts of alfalfa hay were offered at this time for bulk and there were no problems with postpartum constipation. All farrowings were supervised and assistance was rendered when necessary. The pigs were individually identified at birth, and records made of birth order. A standard protocol of uterine infusion with tetracycline hydrochloride, needle tooth clipping, iron dextran injection, and castration was followed. Pigs were weaned at 6 weeks of age, at which time the sows were weighed and killed. At 7 weeks of age (7 days after weaning), the five pigs born first from each litter were also killed. Heparinized blood samples were collected from ear veins in sows and by vena cava puncture in pigs. The animals were killed by electrical stun and exsanguination and were examined for gross pathological changes. Major organs were removed, weighed, and subdivided for residue analysis and microscopic examination. Hematological and histological data were collected by standard procedures. For HCB analysis, 2.00-g tissue samples were transferred to 50-ml test tubes with 15 ml of glass-distilled hexane. Samples were homogenized with a Polytron-20T ultrasonic grinder. The shaft of the grinder was rinsed thoroughly with hexane, which was added to the sample tube. The sample and solvent were then added simultaneously with 40 ml of sand : sodium sulfate (1 : 1) to a chromatography column already containing 20 ml of the sand : sodium sulfate. The column was eluted with three 50-ml portions of hexane, which were first used to rinse the sample tube, and the eluate was collected in a 300-ml round-bottom flask. The eluate was concentrated to 5 ml by rotary evaporation and transferred to a chromatography column containing IO g of 5 % deactivated alumina covered by 4 g of anhydrous sodium sulfate. The flask was rinsed two times with 5-ml portions of hexane and the column was eluted with the rinses and an additional 20 ml of hexane. The eluate was collected in a KudernaDanish evaporator, and the volume was then adjusted for electron capture gas-liquid chromatography as described previously (Hansen et al., 1977).
RESULTS Becauseof differences in the estrous cycles, the duration of treatment before farrowing ranged from 141 to 170 days for sows
ET AL.
receiving 1 ppm HCB and from 157 to I90 days for sows receiving 20 ppm; controls farrowed from 140 to 325 days after the beginning of the experiment. Individual treatment periods and reproductive performance are presented in Table 1. One sow from the I-ppm group did not conceive. HCB had, in general, no adverse effects on the sows. Weight gains were greatest in the high-dose group, which nursed fewer pigs (Table 2). Erythrocyte counts were paraliel in all three groups before and after weaning. The mean corpuscular volume declined in all groups; values were 61-64 pm3 before breeding and 53-55 pm3 at the time of weaning. Although total leukocytes increased, lymphocytes declined by 17, 18, and 26% in the control, low-dose, and high-dose groups, respectively. Neutrophilia was most pronounced in the high-dose group. Relative organ weights were similar in all groups; however, the brains, which should have been the most constant, tended to be relatively smaller in the treated groups, probably because these sows were not as lean as the controls (Table 2). Liver-to-brain ratios were 17.8, 19.3, and 21.3 in the control, low-dose, and high-dose groups, respectively, indicating a slight hepatic enlargement that may have been masked by the heavier body weight (fat) of the treated sows. Significant gross pathologic findings were limited to the stomach and consisted of a catarrhal exudation to mild ulceration in four sows of the low-dose group and in two of five sows in the high-dose group. Signs of gastric irritation were absent in controls. Microscopically, a catarrhal gastritis was evident in two sows each from the control and low-dose groups and in three sows from the high-dose group. Mild gastric ulceration was apparent only in sections from two sows of the low-dose group. Splenic white pulp was mildly decreasedin three sowsgiven the low doseand in one given the high dose. Some degree of arteriolar thickening and fatty replacement of the myocardium was present in all groups; the
HEXACHLOROBENZENE
RESIDUES
TABLE
3
IN SWINE
1
REPRODUCTIVEPERFORMANCEOF Sows RECEIVINGDIETARY HEXACHLOROBENZENETHROUGH BREEDING, GESTATION, AND NURSING Farrowing Days treated”
Live born
Control 5 4 1 3 2
140 144 157 226 325
7 16 7 10 9
I ppm HCB IO 9 7 6 8’
141 149 167 167 170
20 ppm HCB 11 13 12 15 14
157 160 161 180 190
Sow No.
Not full term
Stillborn
0 0 0 0 -
0 2 0 0 -
Mummified
1 0 I 0 -
6 3 12 7 10
Mortality (Week)
Pig production”
1 2
No.
0
0
I I 1 I
2 0 I 0
7 12 6 8 8
0 1 4 4 --
0 0 0 0
12 9 8 7
9.5 + 2.4 8.9kO.9 7.0 f 2.3 10.9+ 3.4 -
0 0 5 2 2
0 0 0 0
6 3 7 5
1
1
13.5+ 1.8 15.5 +4.6 12.2+0.8 13.85 1.7 7.9k1.3
0 Prior to farrowing. b Number weaned at 6 weeks, weight taken at 7 weeks. Values are means + SD. e Sow did not conceive. TABLE
2
WEIGHT CHANGE IN Sows RECEIVING DIETARY HEXACHLOROBENZENE Dietary HCB (ppm) P
lb
20”
Initial weight (kg) (at weaning of previous litter)
141.3k6.4
140.5+21.0
139.8+ 12.0
Weaning weight (kg) (at weaning of current litter)
167.2k7.2
167.0+ 13.3
178.6+ 11.7
20.1* 13.0
28.1 f7.7
Weight gain (%)
18.6+ 10.2
0 Values are given as means f SD for five sows. b Values are given as means It SD for four sows.
Weight (kg) 14.3k2.0 9.8+ 1.5 11.7+
1.0
9.8 5 3.5 11.2+ 1.6
4
HANSEN
sarcolemma nuclei were very prominent in two sows given the high dose. Renal interstitial fibroplasia and fatty replacement of Brunner’s gland in the duodenum were seen in two sows of the high-dose group and in the sow of the low-dose group that had the highest kidney HCB residue. Pancreatic periductal fibrosis was present in two sows of the high-dose group. Ovaries generally contained some cystic follicles, but the sow of the low-dose group that did not conceive was the only one with extensive large cystic follicles. Other microscopic abnormalities that were equally distributed among all sows were mild to moderate degenerative changes, bile duct hyperplasia, septum and capsular thickening, and lymphocyte infiltration in the liver; mild to moderate glomerular disease, tubular degenerative changes, and arteriolar thickening in the kidneys; fatty replacement of parenchyma and some fatty necrosis in the pancreas ; and some hyperplasia and occasional extensive eosinophilic infiltration in the lymph nodes. Other tissues examined but having no consistent notable lesions included lung, large intestine, adrenal glands, and thyroid gland. Five control piglets and five piglets from the high-dose group were examined for microscopic lesions at 7 weeks; there were no differences between the groups in the mild changes observed. There were no significant differences in erythrocyte or leukocyte populations between the three groups, but there was a slight tendency for piglets from the treated groups to have fewer and larger erythrocytes; the mean corpuscular volumes (MCV) were 42.2, 50.4, and 52.3 pm3 for the control, low-dose, and high-dose groups, respectively. As noted previously, this trend was absent in the sows. Residues. HCB accumulated to the highest concentrations in fat and bone marrow (primarily in fat) of the sows (Table 3). The residues were more evenly distributed between dissectable fat and other tissues in sows of the low-dose group. Sows of the high-dose group,
ET AL. TABLE
3
HEXACHLOROBENZENE RESIDUES IN TISSUES OF Sows RECEIVING DIETARY HEXACHLOROBENZENE THROUGH GESTATION AND NURSING (183-232 DAYS) Dietary Tissue Mesenteric
fat
marrow
Adrenal
gland
(ppm)
I’
Oh
Backfatd Bone
HCB
0.02+0.01
LO&
0.02jzO.01 -
6.7k1.9
20 1.4
78.6k58.7 89.4k35.6
6.8k1.2
71.6k31.7
2.1*
Brain
-
1.1 kO.7
2.6-10.6
Muscle’
-
0.7kO.5
2.3 &- 0.7
0.6+0.8
2.5+ 1.7kO.5
Liver Kidney
-
0.2fO.l
Ovary
-
1.3+
Placenta
-
0.2kO.2
1.2
7.5k5.8
1.1
1.3 + 0.9
1.1
0.4kO.2
L?Values (ppm fresh weight) are given as means + SD for five sows. h Because of high background and low dilution factor, initial results from control sows were unreliable. The contamination problem was solved before analysis of samples from HCB-treated sows. Analyses of mesenteric fat and backfat from control sows were repeated after correction of the problem. c Includes the sow that did not conceive. d Means of bilateral samples from three sows in each group. ’ Adductor group in ham.
TABLE
4
MEAN HEXACHLOROBENZENE RESIDUES IN FAT (MESENTERIC FAT, BACK FAT, AND BONE MARROW) OF SOWS RECEIVING DIETARY HEXACHLOROBENZENE Dietary Days treated
I
183-191
6.1 + 1.6b
199-209
6.1 + I .3”
2 12-232
6.4 4 2.8”
HCB
(ppm) 20 52.9 + 14.9’ 123.2k23.3’
a Duration of treatment days was dependent on variation in breeding dates. b Mean+SD of six samples (three samples from each of two sows). c Mean + SD of nine samples (three samples from each of three sows). d Mean + SD of three samples from one sow.
HEXACHLOROBENZENE
in which fat concentrations did not reach a peak until later (Table 4), accumulated a greater proportion of the HCB in fat relative to other tissues (Table 3). Even with shorter HCB exposure time and a 7-day elimination period, piglets accumulated higher concentrations of HCB in their fat that did the sows (Table 5). Fat samples were difficult to obtain from the piglets and a considerable amount of connective tissue was frequently included; those samples which contained less than 20 % hexane-extractable fat were not included in the means. The remaining samples (50) from the piglets TABLE
RESIDUES IN SWINE
5
contained (mean 2 SD) 54 + 13 y0 fat, whereas the mesenteric fat samples from the sows contained 80 -t 18 ‘A fat (one sample from a sow contained only 32% fat and therefore a second subsample was analyzed). Three stillborn pigs from sows of the low-dose group had a mean ( t SD) HCB concentration of 9.9 + 7.1 ppm in fat; three stillborns from sows of the high-dose group had only 8.7 + 3.3 ppm in their fat; and two piglets of the high-dose group (from the same sow) that died at 3 days of age had accumulated 35.4 + 20.4 ppm in their fat.
5
HEXACHLOROBENZENE RESIDUES IN FAT AND BRAIN IN FIGS LITERS OF Sows FED DIETARY HEXACHLOROBENZENE’
Fat Litter No.
No. of Pigs
Control 1 3 4 5
1 ppm HCB 6 7 9 10 Average
20 ppm HCB Ii 12 13 14 15 Average
5 4 3 2 2 16
Brain No. of
HCB @pm)
Pigs
HCB (pm)
5
-
5 5 I5
0.08 f 0.02 0.10+0.01 0.09 f. 0.01
7.31+ 1.00 9.53 + I .47 8.33 + 2.22 7.10+_ 1.25 8.23 f I .66
5 4 5 5 19
0.39 + 0.04 0.39kO.14 0.30&-0.08 0.29?0.10 0.34f0.10
92+52 112*20 96+31 172+ 10 99+44 108 + 42
5 3 3 5 5 21
2.19kO.46 3.1OkO.98 3.56 f 2.20 5.57* 1.20 1.23 + 0.30 3.09+ 1.89
0.43 + 0.33 0.34kO.06 0.55 kO.65 0.14+0.05 0.37kO.38
Average
FROM
-
a Differences in the number of pigs represented vs the number killed were generally due to insufficiency of samples (i.e., when processed for analysis, some fat samples were found to be primarily connective tissue).
b
HANSEN
DISCUSSION Hansen et al. (1977) observed a tendency toward reduced weight gain in feeder pigs receiving up to 100 ppm dietary HCB when compared with controls; this was not seen in sows of the present study. There was a mild indication of liver enlargement among the sows, but it was not as pronounced as might be expected from the feeder pig study (Hansen et al., 1977). The 31 (10ppm HCB) and 61 “/, (100 ppm HCB) declines in lymphocytes observed in the feeder pig study were also greater than the 26% decline observed with the sows that received 20 ppm HCB in the present study. There was a tendency toward neutrophilia in sows of the high-dose group; this effect has also been observed in beagle dogs, but at higher HCB doses (Gralla et a/., 1977).
The propensity toward gastric irritation and ulceration has been previously observed in swine (Hansen ef a/., 1976) and nonhuman primates (Allen, 1975) given PCB-contaminated diets. The lack of precise correlation between gross and microscopic observations with respect to gastric lesions was due to the fact that the lesion was not always present when tissues were trimmed for embedding. The significance of other lesions is uncertain because of low experimental numbers and low incidence; however, the involvement of Brunner’s gland may be significant because of the double exposure of the duodenum through dietary source and biliary excretion. Pancreatic periductal fibrosis may indicate an HCB-megiated effect because of the high accumulation in the pancreas (Hansen er a/., 1977).
The sows receiving 1 ppm HCB accumulated comparable residues in fat, adrenal, and kidney but somewhat higher residues in liver, brain, muscle, and ovary during the 183 to 232 days of feeding than did feeder pigs receiving the same dietary concentration for only 91 days (Hansen et al., 1977). In addition, the sows given 20 ppm dietary HCB did not reach the anticipated (based on
ET AL.
results in feeder pigs) 100-140 ppm equilibrium residue in fat until after 200 days of exposure. This longer time was probably due to a combination of factors: (1) the sows had a greater proportion of body fat available for dispersal and dilution of the HCB; (2) the older animals had a larger proportion of fatty replacement in the tissues, permitting them to sequester more HCB; and (3) farrowing and nursing provided added excretory routes. The piglets tended to accumulate higher HCB residues in fat and brain than did the sows. Some placental transfer had occurred, as shown by the presence of residues in the stillborn pigs, but the pigs in the low-dose group were able to limit accumulation of HCB in fat to less than 10 ppm, which was the approximate residue they were born with. Although analysis of fat from ‘the 3-day-old piglets of the high-dose group indicated the accumulation of HCB from milk, residue concentrations in pigs from the low-dose group were apparently reduced by the interaction between intake, elimination, and dilution by growth (Hansen and Welborn, 1977). The eventual higher residues observed in pigs of the high-dose group could have been exaggerated somewhat by their access to the sow’s feed, even though control feed was provided for the piglets. Swine receiving feed containing 1 ppm HCB throughout gestation and nursing (up to 212 days) were not adversely affected; at the same time residue concentrations in most tissues generally remained at or below the dietary concentration, although HCB accumulated in the fat at five to seven times the dietary concentration. This dietary concentration, equivalent to about 0.025 mg/kg/ day, at which toxicity was not observed, is still well above the upper human limit of 0.6 @g/kg/day suggested by FAO/WHO (1974). However, the accumulation of HCB in fat above dietary concentrations cautions against exposure of food-producing animals to higher levels. At a dietary concentration of 20 ppm HCB (ca. 0.5 mg/kg/day), fat residues accu-
HEXACHLOROBENZENE
mulated to values considered to be hazardous. The effects were very similar to those observed when 20 ppm of Aroclor 1242 was fed under the same conditions (Hansen et al., 1973, but HCB was accumulated to concentrations that were several fold higher than those of PCB. Kuiper-Goodman et al. (1977) have found that 0.5 mg/kg/day for 15 weeks is a “no-effect level” for HCB in rats and a similar dose level was not toxic to dogs (Gralla et a/., 1977), but swine appear to be somewhat more susceptible, even with their large fat reserve for storage. The fat-stored HCB, however, may have been responsible for some of the toxic signs if it was mobilized during the stress of farrowing. ACKNOWLEDGMENTS Supported Bureau of 223-74-7084. from Sandra L. Shipley, Wilson.
by Food and Drug Administration, Veterinary Medicine, Contract FDA The authors are grateful for assistance M. Sundlof, Ronald J. Gardner, David Dennis W. Wilson, and William C.
REFERENCES ALLEN, J. R. (1975). Response of the nonhuman primate to polychlorinated biphenyl exposure. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 34, 1675-1679. BOOTH, N. H., AND MCDOWELL, J. R. (1975). Toxicity of hexachlorobenzene and associated residues in edible animal tissues. .I. Amer. Vet. Med. Ass. 166,591~595.
BURNS,J. E., AND MILLER, F. M. (1975). Hexachlorobenzene contamination: Its effect in a Louisiana population. Arch. Environ. Health 30, 4448.
7
RESIDUES IN SWINE
FAO/WHO (1974). 1973 Evaluations of some pesticide residues in food. FAO/AGP/1973/M/9/1, WHO Pesticide Residue Series 3, pp. 291-305. Food and Agricultural Organization/World Health Organization, Rome, Italy. GRALLA, E. J., FLEISCHMAN, R. W., LUTHRA, Y. K., HAGOPIAN, M., BAKER, J. R., ESBER, H., AND MARCUS, W. (1977). Toxic effects of hexachlorobenzene after daily administration to beagle dogs for one year. Toxicol. Appl. Pharmacol. 40,227-239. HANSEN, BEVILL,
L. G.,
HANSEN,
L.
BYERLY,
C. S., METCALF,
R. L.,
AND
R. F. (1975). Effect of a polychlorinated biphenyl mixture on swine reproduction and tissue residues. Amer. J. Vet. Res. 36, 23-26. G.,
WIEKHORST,
W.
B.,
AND
SIMON,
J.
(1976). Effects of dietary Aroclor 1242 on channel catfish (Ictn/urus punctatus) and the selective accumulation of PCB components. J. Fish. Res. Board Can. 33, 1343-1352. HANSEN, L. G., AND WELBORN, M. E. (1977). Distribution, dilution and elimination of polychlorinated biphenyl analogs in growing swine. J. Pharm. Sci. 66,497-501. HANSEN, L. G., WILSON, D. W., BYERLY, C. S., SUNDLOF, S. F., AND DORN, S. B. (1977). Effects and residues of dietary hexachlorobenzene in growing swine. J. Toxicol. Environ. Health 2, 557-567. KUIPER-GOODMAN, KORSRUD, G.
T., GRANT,
D.
L.,
MOODIE,
C. A.,
O., AND MUNRO, I. C. (1977). Subacute toxicity of hexachlorobenzene in the rat. Toxicol.
Appl.
Pharmacol.
40, 529-549.
LOOSE, L. D., PITTMAN, K. SILKWORTH, J. B. (1977).
A., BENITZ, K. F., AND Polychlorinated biphenyl induced humoral immuno-
and hexachlorobenzene suppression. J. Reticuloendothel. Sot. 22, 253-271. SCHMID, R. (1960). Cutaneous porphyria in Turkey. IV. Engl. YANG, R. GOLBERG,
J. Med.
263, 397-398.
S. H.,
MUELLER, W. AND COULSTON,
L.,
F., GRACE, H. K., F. (1976). Hexain laboratory monkey
chlorobenzene contamination chow. J. Agr. Food Chem. 24, 563-565.
AND
APPLIED
PHARMACOLOGY
51,
1-7 (1979)
Hexachlorobenzene Distribution in Tissues of Swine LARRY G. HANSEN, JOSEPH SIMON, STEVEN B. DORN, AND RICHARD
H. TESKE’
Division of Pharmacology and Toxicology, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801, and Division of Veterinary Medical Research, Food and Drug Administration, Beltsville, Maryland 20705 Received September
18, 1978; accepted July 9, 1979
Hexachlorobenzene Distribution in Tissues of Swine. HANSEN, L. G., SIMON, J., DORN, B., AND TESKE, R. H. (1979). Tox~col. Appl. Pharmacol. 51, 1-7. Purified hexachlorobenzene (HCB) was administered to third-litter sows at dietary concentrations of 0, 1, or 20 ppm throughout gestation and nursing. Swine receiving 1 ppm were not adversely affected and residue concentrations in tissues other than fat and bone marrow remained at or below the dietary concentration. Residues of HCB in the dissectable fat of these pigs accumuiated to concentrations five- to seven-fold higher than the dietary concentration. Piglets accumulated fat residues, through both placental transfer and nursing, that were higher than those of the sows. A similar proportional accumulation of HCB in fat occurred in sows receiving 20 ppm in the diet. Other toxic signs included neutrophiha, gastric irritation, fatty replacement of Brunner’s gland, and pancreatic periductal fibrosis. Hepatotoxicity was not apparent, perhaps because of age-related changes in both control and treatment groups. S.
Hexachlorobenzene (HCB) contamination results from both agricultural use and industrial discharge, and residues are found in humans, animals, and animal feeds (Booth and McDowell, 1975; Burns and Miller, 1975; Yang et al., 1976). Toxic manifestations include hepatomegaly, porphyria, immunosuppression, and interference with hematopoiesis and central nervous system function (Schmid, 1960; Booth and McDowell, 1975; Gralla ethl., 1977; Hansen et al., 1977; Loose et al., 1977). The particular toxicosis that occurs is highly dependent on species, sex, dose, and time of exposure. The present study is undertaken to determine the effects of HCB on swine and their offspring and to define further the accumulation and distribution of HCB residues in food-producing animals.
METHODS Fifteen crossbred (Hampshire/Yorkshire) sow? of proven performance (third litter) were weighed and randomly assigned to individual pens (1.8 x 2.1 m) which were grouped according to diet treatment. They were fed standard 16% protein ration for a ‘J-day acclimation period. To prepare the experimental diets, purified HCB” was added to the standard ration by way of a concentrated premix to attam levels of 1 and 20 ppm (Hansen er al., 1977). Controls were continued on the uncontaminated ration. Analyses of four batches of feed from each dietary level -by electron capture gasliquid chromatography showed that the HCB content was ~0.01, 1.36+(SD) 0.19, and 21.50+1.12ppm for the control, “1-ppm” and “20-ppm” diets, respectively. The sows received 2-2.5 kg of feed twice daily 2 Animal Science Department, University of Illinois at Urbana. 3 Hexachlorobenzene, 95 o/opure, obtained from the Aldrich Chemical Co., Milwaukee, Wis., was washed with warm ethanol, and recrystallized three times from benzene. The resulting product was greater than 99.5 % pure (gas-liquid chromatography).
’ Division of Veterinary Medical Research, Food and Drug Administration, Beltsville, Md. 20705. 1
All
0041-008x/79/13ooo1-07$02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
2
HANSEN
and fresh water was provided ud libit~nr by an automatic watering system. After the sows had been given the treated feed for I week, they were checked daily for signs of estrus and hand-mated to one of two purebred Berkshire boars.” Three to five days before farrowing, they were weighed and the pens were equipped with farrowing crates, straw, and heat lamps. Small amounts of alfalfa hay were offered at this time for bulk and there were no problems with postpartum constipation. All farrowings were supervised and assistance was rendered when necessary. The pigs were individually identified at birth, and records made of birth order. A standard protocol of uterine infusion with tetracycline hydrochloride, needle tooth clipping, iron dextran injection, and castration was followed. Pigs were weaned at 6 weeks of age, at which time the sows were weighed and killed. At 7 weeks of age (7 days after weaning), the five pigs born first from each litter were also killed. Heparinized blood samples were collected from ear veins in sows and by vena cava puncture in pigs. The animals were killed by electrical stun and exsanguination and were examined for gross pathological changes. Major organs were removed, weighed, and subdivided for residue analysis and microscopic examination. Hematological and histological data were collected by standard procedures. For HCB analysis, 2.00-g tissue samples were transferred to 50-ml test tubes with 15 ml of glass-distilled hexane. Samples were homogenized with a Polytron-20T ultrasonic grinder. The shaft of the grinder was rinsed thoroughly with hexane, which was added to the sample tube. The sample and solvent were then added simultaneously with 40 ml of sand : sodium sulfate (1 : 1) to a chromatography column already containing 20 ml of the sand : sodium sulfate. The column was eluted with three 50-ml portions of hexane, which were first used to rinse the sample tube, and the eluate was collected in a 300-ml round-bottom flask. The eluate was concentrated to 5 ml by rotary evaporation and transferred to a chromatography column containing IO g of 5 % deactivated alumina covered by 4 g of anhydrous sodium sulfate. The flask was rinsed two times with 5-ml portions of hexane and the column was eluted with the rinses and an additional 20 ml of hexane. The eluate was collected in a KudernaDanish evaporator, and the volume was then adjusted for electron capture gas-liquid chromatography as described previously (Hansen et al., 1977).
RESULTS Becauseof differences in the estrous cycles, the duration of treatment before farrowing ranged from 141 to 170 days for sows
ET AL.
receiving 1 ppm HCB and from 157 to I90 days for sows receiving 20 ppm; controls farrowed from 140 to 325 days after the beginning of the experiment. Individual treatment periods and reproductive performance are presented in Table 1. One sow from the I-ppm group did not conceive. HCB had, in general, no adverse effects on the sows. Weight gains were greatest in the high-dose group, which nursed fewer pigs (Table 2). Erythrocyte counts were paraliel in all three groups before and after weaning. The mean corpuscular volume declined in all groups; values were 61-64 pm3 before breeding and 53-55 pm3 at the time of weaning. Although total leukocytes increased, lymphocytes declined by 17, 18, and 26% in the control, low-dose, and high-dose groups, respectively. Neutrophilia was most pronounced in the high-dose group. Relative organ weights were similar in all groups; however, the brains, which should have been the most constant, tended to be relatively smaller in the treated groups, probably because these sows were not as lean as the controls (Table 2). Liver-to-brain ratios were 17.8, 19.3, and 21.3 in the control, low-dose, and high-dose groups, respectively, indicating a slight hepatic enlargement that may have been masked by the heavier body weight (fat) of the treated sows. Significant gross pathologic findings were limited to the stomach and consisted of a catarrhal exudation to mild ulceration in four sows of the low-dose group and in two of five sows in the high-dose group. Signs of gastric irritation were absent in controls. Microscopically, a catarrhal gastritis was evident in two sows each from the control and low-dose groups and in three sows from the high-dose group. Mild gastric ulceration was apparent only in sections from two sows of the low-dose group. Splenic white pulp was mildly decreasedin three sowsgiven the low doseand in one given the high dose. Some degree of arteriolar thickening and fatty replacement of the myocardium was present in all groups; the
HEXACHLOROBENZENE
RESIDUES
TABLE
3
IN SWINE
1
REPRODUCTIVEPERFORMANCEOF Sows RECEIVINGDIETARY HEXACHLOROBENZENETHROUGH BREEDING, GESTATION, AND NURSING Farrowing Days treated”
Live born
Control 5 4 1 3 2
140 144 157 226 325
7 16 7 10 9
I ppm HCB IO 9 7 6 8’
141 149 167 167 170
20 ppm HCB 11 13 12 15 14
157 160 161 180 190
Sow No.
Not full term
Stillborn
0 0 0 0 -
0 2 0 0 -
Mummified
1 0 I 0 -
6 3 12 7 10
Mortality (Week)
Pig production”
1 2
No.
0
0
I I 1 I
2 0 I 0
7 12 6 8 8
0 1 4 4 --
0 0 0 0
12 9 8 7
9.5 + 2.4 8.9kO.9 7.0 f 2.3 10.9+ 3.4 -
0 0 5 2 2
0 0 0 0
6 3 7 5
1
1
13.5+ 1.8 15.5 +4.6 12.2+0.8 13.85 1.7 7.9k1.3
0 Prior to farrowing. b Number weaned at 6 weeks, weight taken at 7 weeks. Values are means + SD. e Sow did not conceive. TABLE
2
WEIGHT CHANGE IN Sows RECEIVING DIETARY HEXACHLOROBENZENE Dietary HCB (ppm) P
lb
20”
Initial weight (kg) (at weaning of previous litter)
141.3k6.4
140.5+21.0
139.8+ 12.0
Weaning weight (kg) (at weaning of current litter)
167.2k7.2
167.0+ 13.3
178.6+ 11.7
20.1* 13.0
28.1 f7.7
Weight gain (%)
18.6+ 10.2
0 Values are given as means f SD for five sows. b Values are given as means It SD for four sows.
Weight (kg) 14.3k2.0 9.8+ 1.5 11.7+
1.0
9.8 5 3.5 11.2+ 1.6
4
HANSEN
sarcolemma nuclei were very prominent in two sows given the high dose. Renal interstitial fibroplasia and fatty replacement of Brunner’s gland in the duodenum were seen in two sows of the high-dose group and in the sow of the low-dose group that had the highest kidney HCB residue. Pancreatic periductal fibrosis was present in two sows of the high-dose group. Ovaries generally contained some cystic follicles, but the sow of the low-dose group that did not conceive was the only one with extensive large cystic follicles. Other microscopic abnormalities that were equally distributed among all sows were mild to moderate degenerative changes, bile duct hyperplasia, septum and capsular thickening, and lymphocyte infiltration in the liver; mild to moderate glomerular disease, tubular degenerative changes, and arteriolar thickening in the kidneys; fatty replacement of parenchyma and some fatty necrosis in the pancreas ; and some hyperplasia and occasional extensive eosinophilic infiltration in the lymph nodes. Other tissues examined but having no consistent notable lesions included lung, large intestine, adrenal glands, and thyroid gland. Five control piglets and five piglets from the high-dose group were examined for microscopic lesions at 7 weeks; there were no differences between the groups in the mild changes observed. There were no significant differences in erythrocyte or leukocyte populations between the three groups, but there was a slight tendency for piglets from the treated groups to have fewer and larger erythrocytes; the mean corpuscular volumes (MCV) were 42.2, 50.4, and 52.3 pm3 for the control, low-dose, and high-dose groups, respectively. As noted previously, this trend was absent in the sows. Residues. HCB accumulated to the highest concentrations in fat and bone marrow (primarily in fat) of the sows (Table 3). The residues were more evenly distributed between dissectable fat and other tissues in sows of the low-dose group. Sows of the high-dose group,
ET AL. TABLE
3
HEXACHLOROBENZENE RESIDUES IN TISSUES OF Sows RECEIVING DIETARY HEXACHLOROBENZENE THROUGH GESTATION AND NURSING (183-232 DAYS) Dietary Tissue Mesenteric
fat
marrow
Adrenal
gland
(ppm)
I’
Oh
Backfatd Bone
HCB
0.02+0.01
LO&
0.02jzO.01 -
6.7k1.9
20 1.4
78.6k58.7 89.4k35.6
6.8k1.2
71.6k31.7
2.1*
Brain
-
1.1 kO.7
2.6-10.6
Muscle’
-
0.7kO.5
2.3 &- 0.7
0.6+0.8
2.5+ 1.7kO.5
Liver Kidney
-
0.2fO.l
Ovary
-
1.3+
Placenta
-
0.2kO.2
1.2
7.5k5.8
1.1
1.3 + 0.9
1.1
0.4kO.2
L?Values (ppm fresh weight) are given as means + SD for five sows. h Because of high background and low dilution factor, initial results from control sows were unreliable. The contamination problem was solved before analysis of samples from HCB-treated sows. Analyses of mesenteric fat and backfat from control sows were repeated after correction of the problem. c Includes the sow that did not conceive. d Means of bilateral samples from three sows in each group. ’ Adductor group in ham.
TABLE
4
MEAN HEXACHLOROBENZENE RESIDUES IN FAT (MESENTERIC FAT, BACK FAT, AND BONE MARROW) OF SOWS RECEIVING DIETARY HEXACHLOROBENZENE Dietary Days treated
I
183-191
6.1 + 1.6b
199-209
6.1 + I .3”
2 12-232
6.4 4 2.8”
HCB
(ppm) 20 52.9 + 14.9’ 123.2k23.3’
a Duration of treatment days was dependent on variation in breeding dates. b Mean+SD of six samples (three samples from each of two sows). c Mean + SD of nine samples (three samples from each of three sows). d Mean + SD of three samples from one sow.
HEXACHLOROBENZENE
in which fat concentrations did not reach a peak until later (Table 4), accumulated a greater proportion of the HCB in fat relative to other tissues (Table 3). Even with shorter HCB exposure time and a 7-day elimination period, piglets accumulated higher concentrations of HCB in their fat that did the sows (Table 5). Fat samples were difficult to obtain from the piglets and a considerable amount of connective tissue was frequently included; those samples which contained less than 20 % hexane-extractable fat were not included in the means. The remaining samples (50) from the piglets TABLE
RESIDUES IN SWINE
5
contained (mean 2 SD) 54 + 13 y0 fat, whereas the mesenteric fat samples from the sows contained 80 -t 18 ‘A fat (one sample from a sow contained only 32% fat and therefore a second subsample was analyzed). Three stillborn pigs from sows of the low-dose group had a mean ( t SD) HCB concentration of 9.9 + 7.1 ppm in fat; three stillborns from sows of the high-dose group had only 8.7 + 3.3 ppm in their fat; and two piglets of the high-dose group (from the same sow) that died at 3 days of age had accumulated 35.4 + 20.4 ppm in their fat.
5
HEXACHLOROBENZENE RESIDUES IN FAT AND BRAIN IN FIGS LITERS OF Sows FED DIETARY HEXACHLOROBENZENE’
Fat Litter No.
No. of Pigs
Control 1 3 4 5
1 ppm HCB 6 7 9 10 Average
20 ppm HCB Ii 12 13 14 15 Average
5 4 3 2 2 16
Brain No. of
HCB @pm)
Pigs
HCB (pm)
5
-
5 5 I5
0.08 f 0.02 0.10+0.01 0.09 f. 0.01
7.31+ 1.00 9.53 + I .47 8.33 + 2.22 7.10+_ 1.25 8.23 f I .66
5 4 5 5 19
0.39 + 0.04 0.39kO.14 0.30&-0.08 0.29?0.10 0.34f0.10
92+52 112*20 96+31 172+ 10 99+44 108 + 42
5 3 3 5 5 21
2.19kO.46 3.1OkO.98 3.56 f 2.20 5.57* 1.20 1.23 + 0.30 3.09+ 1.89
0.43 + 0.33 0.34kO.06 0.55 kO.65 0.14+0.05 0.37kO.38
Average
FROM
-
a Differences in the number of pigs represented vs the number killed were generally due to insufficiency of samples (i.e., when processed for analysis, some fat samples were found to be primarily connective tissue).
b
HANSEN
DISCUSSION Hansen et al. (1977) observed a tendency toward reduced weight gain in feeder pigs receiving up to 100 ppm dietary HCB when compared with controls; this was not seen in sows of the present study. There was a mild indication of liver enlargement among the sows, but it was not as pronounced as might be expected from the feeder pig study (Hansen et al., 1977). The 31 (10ppm HCB) and 61 “/, (100 ppm HCB) declines in lymphocytes observed in the feeder pig study were also greater than the 26% decline observed with the sows that received 20 ppm HCB in the present study. There was a tendency toward neutrophilia in sows of the high-dose group; this effect has also been observed in beagle dogs, but at higher HCB doses (Gralla et a/., 1977).
The propensity toward gastric irritation and ulceration has been previously observed in swine (Hansen ef a/., 1976) and nonhuman primates (Allen, 1975) given PCB-contaminated diets. The lack of precise correlation between gross and microscopic observations with respect to gastric lesions was due to the fact that the lesion was not always present when tissues were trimmed for embedding. The significance of other lesions is uncertain because of low experimental numbers and low incidence; however, the involvement of Brunner’s gland may be significant because of the double exposure of the duodenum through dietary source and biliary excretion. Pancreatic periductal fibrosis may indicate an HCB-megiated effect because of the high accumulation in the pancreas (Hansen er a/., 1977).
The sows receiving 1 ppm HCB accumulated comparable residues in fat, adrenal, and kidney but somewhat higher residues in liver, brain, muscle, and ovary during the 183 to 232 days of feeding than did feeder pigs receiving the same dietary concentration for only 91 days (Hansen et al., 1977). In addition, the sows given 20 ppm dietary HCB did not reach the anticipated (based on
ET AL.
results in feeder pigs) 100-140 ppm equilibrium residue in fat until after 200 days of exposure. This longer time was probably due to a combination of factors: (1) the sows had a greater proportion of body fat available for dispersal and dilution of the HCB; (2) the older animals had a larger proportion of fatty replacement in the tissues, permitting them to sequester more HCB; and (3) farrowing and nursing provided added excretory routes. The piglets tended to accumulate higher HCB residues in fat and brain than did the sows. Some placental transfer had occurred, as shown by the presence of residues in the stillborn pigs, but the pigs in the low-dose group were able to limit accumulation of HCB in fat to less than 10 ppm, which was the approximate residue they were born with. Although analysis of fat from ‘the 3-day-old piglets of the high-dose group indicated the accumulation of HCB from milk, residue concentrations in pigs from the low-dose group were apparently reduced by the interaction between intake, elimination, and dilution by growth (Hansen and Welborn, 1977). The eventual higher residues observed in pigs of the high-dose group could have been exaggerated somewhat by their access to the sow’s feed, even though control feed was provided for the piglets. Swine receiving feed containing 1 ppm HCB throughout gestation and nursing (up to 212 days) were not adversely affected; at the same time residue concentrations in most tissues generally remained at or below the dietary concentration, although HCB accumulated in the fat at five to seven times the dietary concentration. This dietary concentration, equivalent to about 0.025 mg/kg/ day, at which toxicity was not observed, is still well above the upper human limit of 0.6 @g/kg/day suggested by FAO/WHO (1974). However, the accumulation of HCB in fat above dietary concentrations cautions against exposure of food-producing animals to higher levels. At a dietary concentration of 20 ppm HCB (ca. 0.5 mg/kg/day), fat residues accu-
HEXACHLOROBENZENE
mulated to values considered to be hazardous. The effects were very similar to those observed when 20 ppm of Aroclor 1242 was fed under the same conditions (Hansen et al., 1973, but HCB was accumulated to concentrations that were several fold higher than those of PCB. Kuiper-Goodman et al. (1977) have found that 0.5 mg/kg/day for 15 weeks is a “no-effect level” for HCB in rats and a similar dose level was not toxic to dogs (Gralla et a/., 1977), but swine appear to be somewhat more susceptible, even with their large fat reserve for storage. The fat-stored HCB, however, may have been responsible for some of the toxic signs if it was mobilized during the stress of farrowing. ACKNOWLEDGMENTS Supported Bureau of 223-74-7084. from Sandra L. Shipley, Wilson.
by Food and Drug Administration, Veterinary Medicine, Contract FDA The authors are grateful for assistance M. Sundlof, Ronald J. Gardner, David Dennis W. Wilson, and William C.
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BURNS,J. E., AND MILLER, F. M. (1975). Hexachlorobenzene contamination: Its effect in a Louisiana population. Arch. Environ. Health 30, 4448.
7
RESIDUES IN SWINE
FAO/WHO (1974). 1973 Evaluations of some pesticide residues in food. FAO/AGP/1973/M/9/1, WHO Pesticide Residue Series 3, pp. 291-305. Food and Agricultural Organization/World Health Organization, Rome, Italy. GRALLA, E. J., FLEISCHMAN, R. W., LUTHRA, Y. K., HAGOPIAN, M., BAKER, J. R., ESBER, H., AND MARCUS, W. (1977). Toxic effects of hexachlorobenzene after daily administration to beagle dogs for one year. Toxicol. Appl. Pharmacol. 40,227-239. HANSEN, BEVILL,
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R. F. (1975). Effect of a polychlorinated biphenyl mixture on swine reproduction and tissue residues. Amer. J. Vet. Res. 36, 23-26. G.,
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SIMON,
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(1976). Effects of dietary Aroclor 1242 on channel catfish (Ictn/urus punctatus) and the selective accumulation of PCB components. J. Fish. Res. Board Can. 33, 1343-1352. HANSEN, L. G., AND WELBORN, M. E. (1977). Distribution, dilution and elimination of polychlorinated biphenyl analogs in growing swine. J. Pharm. Sci. 66,497-501. HANSEN, L. G., WILSON, D. W., BYERLY, C. S., SUNDLOF, S. F., AND DORN, S. B. (1977). Effects and residues of dietary hexachlorobenzene in growing swine. J. Toxicol. Environ. Health 2, 557-567. KUIPER-GOODMAN, KORSRUD, G.
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LOOSE, L. D., PITTMAN, K. SILKWORTH, J. B. (1977).
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and hexachlorobenzene suppression. J. Reticuloendothel. Sot. 22, 253-271. SCHMID, R. (1960). Cutaneous porphyria in Turkey. IV. Engl. YANG, R. GOLBERG,
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S. H.,
MUELLER, W. AND COULSTON,
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chlorobenzene contamination chow. J. Agr. Food Chem. 24, 563-565.