Journal of Toxicology and Environmental Health, Part A, 67:845–850, 2004
Copyright© Taylor & Francis Inc.
ISSN: 1528–7394 print / 1087–2620 online
DOI: 10.1080/15287390490441265
RAPID COMMUNICATION: BACKGROUND
CONCENTRATIONS OF DIOXINS, FURANS, AND PCBs
IN SPRAGUE-DAWLEY RATS AND JUVENILE SWINE
Michael V. Ruby,1 Stan W. Casteel,2 Timothy J. Evans,2
Kurt A. Fehling,3 Dennis J. Paustenbach,3 Robert A. Budinsky,4
John P. Giesy,5 Lesa L. Aylward,6 Bryce D. Landenberger4
1
Exponent, Inc., Boulder, Colorado, USA
College of Veterinary Medicine, University of Missouri-Columbia,
Columbia, Missouri, USA
3
ChemRisk, Inc., San Francisco, California, USA
4
Dow Chemical Company, Midland, Michigan, USA
5
Department of Zoology, Michigan State University, East Lansing,
Michigan, USA
6
Exponent, Inc., Alexandria, Virginia, USA
2
In preparation for a study of the relative oral bioavailability of polychlorinated dibenzo-p-dioxins
and dibenzofurans (PCDD/Fs) in soils (typically containing less than 1 ppb 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD] toxic equivalents [TEQ]), the background concentrations of PCDD/
Fs and selected polychlorinated biphenyls (PCBs) were measured in liver and adipose tissue
from female Sprague-Dawley rats and juvenile swine after 30 d of ingesting laboratory chow.
The measured concentrations of TCDD and other PCDD/Fs in rat livers were severalfold less
than previously reported in the literature for control (unexposed) laboratory rodents. The concentrations of PCDD/Fs and selected PCBs in livers of swine were three- to fourfold lower than
those reported for rats. The lower concentrations found in this study compared to previous
findings may be due to inadvertent laboratory contamination in previous studies or to declining
levels of PCDD/Fs in laboratory feed, which parallel the declines in emissions, general environmental levels, and human food and tissue levels of PCDD/Fs.
Soils in the Midland, MI, area were reported to contain levels of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), including 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), above typical background levels in soils
but typically less than 1 ppb TCDD toxic equivalents (TEQ) (Dow Chemical
Company, 2000). A study of the relative oral bioavailability of PCDD/Fs from
these soils compared to administration of PCDD/F in the diet is planned. However, because of the relatively low PCDD/F levels in the soils and the difficulties of studying tissue burdens at these levels, such an investigation will be
We appreciate the assistance of Dr. Hon-Wing Leung and Terri Horvath. Funding was provided by the
Dow Chemical Company.
Address correspondence to Lesa L. Aylward, Exponent, Inc., 1800 Diagonal Road, Suite 355, Alexandria,
VA 22314, USA. E-mail: laylward@exponent.com
845
846
M. V. RUBY ET AL.
feasible only if tissue concentrations resulting from absorbed doses from soil are
detectable above background tissue concentrations of these compounds.
Studies of standard laboratory rat feed and tissues of “unexposed” laboratory rats have demonstrated certain PCDD/Fs to be present (Vanden Heuvel et al.,
1994; Walker et al., 1999; Schrenk et al., 1994). Vanden Heuvel et al. (1994)
found average liver concentrations of TCDD in female Sprague-Dawley rats of
approximately 6 pg TCDD/g lipid; total PCDD/F TEQ was 21 and 78 pg TEQ/g
lipid in 60- and 200-d-old rats, respectively (detected compounds only). These
average concentrations correspond to approximately 0.2 pg TCDD/g of liver
tissue and up to 2.9 pg TEQ/g liver tissue (assuming liver lipid content of 3.67%
for a generic rat; Nelson et al., 1986). Other investigators reported greater
concentrations in control rats. Walker et al. (1999; samples taken from female
Sprague-Dawley rats during a 1993 experiment) and Schrenk et al. (1994;
female Wistar rats) found mean concentrations of 20 and 110 pg TCDD/g liver
(wet weight), respectively, in control rats.
Background tissue concentrations of this magnitude could present a serious
challenge for conducting a dioxin bioavailability study with Midland-area soils,
because anticipated absorbed doses from these soils would be low compared
to these background tissue concentrations. The objective of this study was to
establish the concentrations of PCDD/Fs and coplanar polychlorinated biphenyls (PCBs) in feed and tissue samples from “control” Sprague-Dawley rats and
juvenile swine under the laboratory conditions to be used in the oral bioavailability study of Midland-area soils.
METHODS
All in vivo work was conducted at the College of Veterinary Medicine,
University of Missouri–Columbia (authors SWC and TJE). Ten female SpragueDawley rats (4 m of age) were obtained from Harlan (Indianapolis, Indiana)
and placed in individual cages. The rats were maintained on Purina laboratory
rodent diet 5001 and deionized water ad libitum for 30 d, after which they
were euthanized by CO2 inhalation in a sealed cage. Body weights were
recorded weekly. Immediately after being euthanized, livers were removed
and as much fatty tissue as possible was collected from each rat (primarily from
the abdominal cavity). Liver and adipose tissue samples from pairs of rats were
combined (to provide sufficient sample mass for analysis), resulting in five
composite samples each of liver and adipose tissue.
Five intact male juvenile swine (Sus scrofa) at 6 wk of age were obtained
from Chinn Farms, a commercial swine operation in Clarence, MO. The swine
were held in quarantine for 7 d while gradually being acclimatized to the study
diet (Ziegler Bros., Inc., Gardners, PA), a specially formulated swine diet previously determined to contain low dioxin/furan concentrations. Feeding
occurred twice daily in equal portions (total daily feed equivalent to 4% of
body weight, adjusted every 3 d to account for increased body weights), and
deionized water was provided ad libitum. At the end of 30 d, the swine were
BACKGROUND DIOXINS IN LAB ANIMAL TISSUES AND FEED
847
electrically stunned and then electrocuted according to methods approved by
the American Veterinary Medical Association (AVMA). Immediately after the
swine were euthanized, the entire liver was removed and weighed, and
approximately 75 g of subcutaneous fat was collected from the abdominal wall
of each animal.
Tissue samples were placed immediately into Ziploc bags, frozen (−80° C),
and shipped to Alta Analytical Laboratory, Inc. (Alta), in Eldorado Hills, CA, for
homogenization and analysis. Samples (50 g) of rodent and swine diets were
shipped to Alta for analysis of PCDDs/Fs and selected PCBs. At Alta, the liver
and fat samples were extracted in methylene chloride/hexane and analyzed
for lipid content (U.S. EPA method 1613), and PCDD/F and PCB concentrations were determined by high-resolution gas chromatography/mass spectrometry (HR-GC/MS) by U.S. EPA methods 1613 and 1668, respectively.
RESULTS
Rat weights averaged 250 g at study initiation and 274 g at termination. Rat
liver weights ranged from 7.5 to 10.6 g (average of 9.5 g). Swine weights averaged 15.8 kg at study initiation, and 36.6 kg at termination, a gain of 132%
over the 30-d maintenance on the Ziegler Bros. swine diet (typical for juvenile
swine at 6 wk of age). Swine liver weights ranged from 789 to 1097 g (average
953 g).
Concentrations of PCDD/F and PCB in rat liver and adipose tissues and
swine adipose tissues are presented (Table 1). Only OCDD (2/5 samples, trace
levels) and PCB-106/118 (5/5 samples, mean concentration of 2.6 pg/g) were
detected in swine liver (data not shown). Few PCDD/F or PCB congeners were
detected in any tissues, and the calculated mean TEQ concentrations for the
tissue samples were influenced by the detection limits. The low concentrations
of PCDD/Fs and PCBs observed in the tissue samples are consistent with the
minimal concentrations observed in feed (Table 1).
DISCUSSION
Background concentrations of PCDD/Fs observed in tissues of both rats
and swine were consistently low and support the feasibility of using these two
animal models to assess the relative oral bioavailability of PCDD/Fs from
Midland-area soils. The concentrations of TCDD in rat livers (<0.059 pg/g)
were considerably less than the range of 0.2–110 pg/g TCDD previously
reported in the literature for control (unexposed) rats (Vanden Heuvel et al.,
1994; Walker et al., 1999; Schrenk et al., 1994). Similarly, detected concentrations of PCDD/Fs in swine adipose tissue (mean 0.142 pg/g TEQ; 0.31 pg/g
lipid) were approximately four-fold lower than values reported in the fat of U.S.
pork animals in 1997 (overall mean 1.3 pg TEQ/g lipid; Lorber et al., 1997).
The discrepancy between the concentrations of PCDD/F in control rat
livers in this study compared to previous studies (Vanden Heuvel et al., 1994;
848
PCDD/Fs TEQb
(pg/g wet weight)
PCDD/Fs (pg/g
wet weight)
2,3,7,8-TCDD
1,2,3,7,8-PeCDD
1,2,3,4,7,8-HxCDD
1,2,3,6,7,8-HxCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,
8-HpCDD
OCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
1,2,3,4,7,8-HxCDF
1,2,3,6,7,8-HxCDF
2,3,4,6,7,8-HxCDF
1,2,3,7,8,9-HxCDF
1,2,3,4,6,7,
8-HpCDF
1,2,3,4,7,8,
9-HpCDF
OCDF
Analyte
0.278 U
0.455
—
0.0807 U
0.0404 U
1.84
26.4
0.0493 U
0.0970 U
1.28 A
0.592 A
0.550 A
0.108 U
0.123 U
1.52 A
0.0594 U
0.480 A
0.332 A
1.35 A
0.491 A
7.30
Minimum
29.3
0.626
0.0960
1.43
0.715
0.582
0.282
0.0615 U
1.56
0.0297 U
0.521
0.375
1.54
0.551
7.71
Mean
—
0.970 A
0.0807 U
33.7
0.807 A
0.286 A
1.52 A
0.780 A
0.656 A
0.484 A
0.123 U
1.63 A
0.0594 U
0.614 A
0.433 A
1.66 A
0.625 A
8.10
Maximum
Rat, Liver
—
2
0
5
4
1
5
5
5
3
0
5
0
5
5
5
5
5
Number
of
detects
(n = 5)a
0.364
0.139 U
0.0404 U
4.33
0.687
0.0485 U
0.0454 U
0.0467 U
0.0540 U
0.0540 U
0.0615 U
0.0387 U
0.0297 U
0.175
0.0510 U
0.192
0.0665 U
1.18
Mean
—
0.278 U
0.0807 U
3.60 A
0.0493 U
0.0970 U
0.0907 U
0.0933 U
0.108 U
0.108 U
0.123 U
0.0774 U
0.0594 U
0.0879 U
0.102 U
0.130 U
0.133 U
0.0811 U
Minimum
Rat, Adipose
—
0.278 U
0.0807 U
5.12 A
1.03 A
0.0970 U
0.0907 U
0.0933 U
0.108 U
0.108 U
0.123 U
0.0774 U
0.0594 U
0.697 A
0.102 U
0.702 A
0.133 U
1.91 A
Maximum
—
0
0
5
4
0
0
0
0
0
0
0
0
1
0
1
0
4
Number
of
detects
(n = 5)a
0.142
0.478
0.0404 U
1.23
0.0247 U
0.0485 U
0.0454 U
0.0467 U
0.0540 U
0.0540 U
0.0615 U
0.0387 U
0.0297 U
0.0440 U
0.0510 U
0.0650 U
0.0665 U
0.0406 U
Mean
—
0.278 U
0.0807 U
0.607 A
0.0493 U
0.0970 U
0.0907 U
0.0933 U
0.108 U
0.108 U
0.123 U
0.0774 U
0.0594 U
0.0879 U
0.102 U
0.130 U
0.133 U
0.0811 U
Minimum
—
1.45 A
0.0807 U
2.10 A
0.0493 U
0.0970 U
0.0907 U
0.0933 U
0.108 U
0.108 U
0.123 U
0.0774 U
0.0594 U
0.0879 U
0.102 U
0.130 U
0.133 U
0.0811 U
Maximum
Swine, Adipose
TABLE 1. Concentrations of PCDD/Fs and Selected PCBs in Rat and Swine Tissues and Samples of Rat and Swine Feed
—
2
0
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Number
of
detects
(n = 5)
0.150b
0.278 U
0.0807 U
8.53 A
0.0493 U
0.0970 U
0.0907 U
0.0933 U
0.108 U
0.108 U
0.123 U
0.0774 U
0.0594 U
0.0879 U
0.102 U
0.130 U
0.133 U
0.717 A
Purina
rodent diet
0.531b
0.403 A,B
0.336 U
0.709 U
0.139 U
0.326 U
0.336 U
0.336 U
0.395 U
0.287 U
0.380 U
0.217 U
0.234 U
0.374 U
0.202 U
0.615 U
0.253 U
0.268 U
Zeigler
Bros.
swine
diet
849
2.32
Total TEQb
(pg/g wet weight)
—
—
1.09 U
1.36 U
16.6
0.574 U
39.3
0.509 U
1.01 U
0.902 U
0.886 U
0.951 U
1.66 U
15.5
48.7
0.848 U
Minimum
—
—
1.09 U
1.36 U
20.1
0.574 U
49.2
0.509 U
8.31
6.94
0.886 U
0.951 U
1.66 U
18.6
55.7
0.848 U
Maximum
—
—
0
0
5
0
5
0
3
4
0
0
0
5
5
0
Number
of
detects
(n = 5)a
0.818
0.454
0.545 U
0.68 U
665
33.7
1,700
28.6
0.505 U
214
56.7
97.4
0.830 U
511
1,434
26.0
Mean
—
—
1.09 U
1.36 U
580
29.2
1,540
24.0
1.01 U
196
52.8
92.6
1.66 U
472
1,320
24.4
Minimum
Rat, Adipose
—
—
1.09 U
1.36 U
804
40.2
2,030
32.3
1.01 U
242
63.4
112
1.66 U
570
1,580
30.0
Maximum
—
—
0
0
5
5
5
5
0
5
5
5
0
5
5
5
Number
of
detects
(n = 5)a
0.218
0.0752
1.67
0.680 U
26.9
0.812
75.1
0.255 U
0.505 U
9.31
1.54
2.41
0.83 U
14.8
43.4
0.424 U
Mean
—
—
1.09 U
1.36 U
16.6
0.574 U
50.1
0.509 U
1.01 U
6.78
0.886 U
0.951 U
1.66 U
10.8
31.2
0.848 U
Minimum
—
—
6.17
1.36 U
55.1
2.91
142
0.509 U
1.01 U
17.5
4.37
5.29
1.66 U
25.8
77.9
0.848 U
Maximum
Swine, Adipose
—
—
1
0
5
1
5
0
0
5
2
4
0
5
5
0
Number
of
detects
(n = 5)
0.225b
0.0751b
1.09 U
1.36 U
29.9
0.574 U
78.4
0.509 U
1.01 U
9.75
0.886 U
0.951 U
1.66 U
26.8
81.6
0.848 U
Purina
rodent diet
0.594b
0.0633 Ub
1.24 U
1.21 U
1.60 U
1.47 U
2.10 U
1.66 U
1.15 U
1.60 U
1.20 U
0.899 U
0.848 U
1.47 U
4.99 U
1.38 U
Zeigler
Bros.
swine
diet
Note. —, Not applicable; A, the amount detected is below the lower calibration limit of the instrument; TEQ, toxicity equivalence concentration; U, undetected (value represents method detection limit); means
calculated assuming non-detects equal to ½ detection limit, and means are considered undetected if all concentrations averaged were undetected; B, detected in method blank.
a
Each analysis was conducted on composited tissue from two animals.
b
TEQ estimated with non-detects at one-half of method detection limit using World Health Organization 1998 TEF values.
0.480
0.545 U
0.680 U
18.3
0.287 U
45.1
0.255 U
4.62
5.22
0.443 U
0.476 U
0.830 U
16.8
52.1
0.424 U
PCBs
(pg/g wet weight)
PCB-77
PCB-81
PCB-105
PCB-114
PCB-106/118
PCB-123
PCB-126
PCB-156
PCB-157
PCB-167
PCB-169
PCB-170
PCB-180
PCB-189
PCBs TEQb
(pg/g wet weight)
Mean
Analyte
Rat, Liver
TABLE 1. Concentrations of PCDD/Fs and Selected PCBs in Rat and Swine Tissues and Samples of Rat and Swine Feed (Continued)
850
M. V. RUBY ET AL.
Walker et al., 1999; Schrenk et al., 1994) could be due to one or a combination of several factors. Previous reports of greater concentrations in control
animals could have been the result of inadvertent laboratory contamination.
However, it is more likely that the substantial general decline in emissions and
environmental levels of PCDD/Fs (Hays & Aylward, 2003) resulted in a decline
in levels of PCDD/Fs in laboratory rat feed and therefore in control rat tissues
(paralleling the observed approximately 10-fold declines in human food and
tissue levels since the 1970s; Hays & Aylward, 2003; Lorber, 2002). A comparison of the levels of detected, quantitated PCDD/Fs in rat feed (Agway,
NIH31), as reported by Vanden Heuvel et al. (1994) (HpCDD, OCDD, and
OCDF), with those found in the current study shows approximately 10-fold
lower levels in feed in the current study.
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