BIOCHEMICAL, HISTOLOGICAL AND REPRODUCTIVE EFFECTS IN MINK (MUSTELA
VISON) EXPOSED TO POLYCHLORINATED DIBENZOFURANS (PCDFs) AND 2,3,7,8
TETRACHLORODIBENZO-P-DIOXIN (TCDD)
By
Jeremy Noel Moore

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Animal Science – Master of Science
2013

ABSTRACT
BIOCHEMICAL, HISTOLOGICAL AND REPRODUCTIVE EFFECTS IN MINK (MUSTELA
VISON) EXPOSED TO POLYCHLORINATED DIBENZOFURANS (PCDFs) AND 2,3,7,8
TETRACHLORODIBENZO-P-DIOXIN (TCDD)
By
Jeremy Noel Moore
In the Tittabawassee River basin, the major proportion of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD)-like exposure to mammals is from 2,3,7,8-tetrachlorodibenzofuran (TCDF) and
2,3,4,7,8-pentachlorodibenzofuran (PeCDF). Mink tissues collected from the Tittabawassee
River had concentrations of TCDF and PeCDF that exceeded toxicity reference values (TRV),
suggesting the potential for adverse effects. However, field evaluation of mink residing in the
area indicated that the population was healthy. Two mink feeding studies were conducted to
investigate the toxic potencies of TCDF and PeCDF in attempt to explain the unexpected lack of
effects in the field. The first study was a toxicokinetic study that indicated hepatic cytochrome
P450 activity can be used as an index of exposure to TCDF and PeCDF. The second study
assessed the effects of feeding TCDD, TCDF or PeCDF at doses expected to cause adverse
effects on reproduction and offspring viability and growth. The lack of significant effects on
reproduction and offspring viability was unexpected based on TRVs established from other
mammalian studies. Results suggest that the World Health Organization (WHO) Toxic
Equivalency Factor (TEF) for TCDF requires further evaluation, and in the case of mink, the
TEF for PCB 126 is underestimated or should be standardized outside the TCDD-centric TEF
approach.

DEDICATION

In Memory of
Greg Markham
&
Robert “Bio Bob” Collins
&
Marie B. Moore

iii

ACKNOWLEDGMENTS

Dr. Steven Bursian
Dr. Scott Fitzgerald
Dr. John Newsted
Dr. Matthew Zwiernik
Dr. John P. Giesy
Ms. Jane Link
Mr. Angelo Napolitano
Mr. Patrick Bradley
Dr. Marcus Hecker
Dr. Kerrie Beckett
Dr. Ben Yamini
Dr. Karl Strause
Mr. Andrew Christofferson
Mr. Michael Kramer
Ms. Nozomi Ikeda
Ms. Jessica Hudon
Dr. Xaowei Zhang
Mr. Eric Higley
Ms. Melissa Shotwell
Dr. Denise Kay
Mr. David Hamman

iv

Ms. Molly Wiemersma
Mr. Joost Van Dam
Mr. Joel Griffith
Ms. Brittany Denison
Ms. Stephanie Plautz
Ms. Karla Masterhazy
Ms. Megan Barker
Mr. Dustin Tazelaar
Mr. Michael Nadeau
Mr. Jeff Greenlee
Mr. Andrew Cohen-Barnhouse
Mr. Anthony Satkowiak
Dr. Robert Budinsky
Ms. Lesa Aylward
Dr. Nora Bello
Mr. Cyrus Park
Dr. Kelly Wessell
Mr. Mike Fales
Dr. Sigrid Dixon

Mrs. Sara Lima-Sousa Moore

v

TABLE OF CONTENTS

LIST OF TABLES…………………………………………………………………………….....vi
LIST OF FIGURES………………………………………………………...…………………...viii
ABBREVIATIONS………………………………………………………………………………ix
CHAPTER 1
INTRODUCTION
TCDD and TCDD-like compounds……………………………………………..1
TCDD, PCDF and PCB configuration and mechanism of action……….…....2
PCDD, PCDF and PCB classification……………………………………….….4
Model for examining toxic effects…….……………………..………………….5
The Tittabawassee River and a sentinel species and laboratory model….......6
CHAPTER 2
HEPATIC P450 ENZYME ACTIVITY, TISSUE MORPHOLOGY AND HISTOLOGY OF
MINK (MUSTELA VISON) EXPOSED TO POLYCHLORINATED DIBENZOFURANS
(PCDFS)
ABSTRACT……………………………………………………………………..10
INTRODUCTION…………………………………………………………….....11
MATERIALS AND METHODS………………………………………………..13
Mink husbandry, exposure and necropsy…………………………….13
Chemicals and reagents………………………………………………..16
EROD and MROD Quantification……………………………..……..16
Quantification of PCDD, PCDF and TEQ……………………………17
Data analysis……………………………………………………….…...18
RESULTS……………………………………………………………………….18
PCDF concentrations in liver………………………………………….18
Gross morphology and histology………………………………………19
EROD and MROD activities…………………………………………...22
DISCUSSION……………………………………………………………………32
PCDF concentrations in liver………………………………………….32
Histology………………………………………………………………...33
Enzyme induction……………………………………………………....35
ACKNOWLEDGEMENTS……………………………………………………..36

vi

CHAPTER 3
EFFECTS OF DIETARY EXPOSURE OF MINK (MUSTELA VISON) TO 2,3,7,8 –
TETRACHLORODIBENZO-P-DIOXIN (TCDD), 2,3,4,7,8PENTACHLORODIBENZOFURAN (PECDF) AND 2,3,7,8TETRACHLORODIBENZOFURAN (TCDF) ON REPRODUCTON AND OFFSPRING
VIABILITY AND GROWTH

ABSTRACT…………………………………………………………..................38
INTRODUCTION……………………………………………………………….39
MATERIALS AND METHODS………………………………………………...41
Chemical and reagents…………………………………………………41
Dietary treatments……………………………………………………...42
Animals………………………………………………………………….46
Housing……………………………………………………………..…...46
Exposure period………………………………………………………...46
Chemical analysis……………………………………………………....48
Histological analysis…………………………………………………....48
Statistical analysis……………………………………………………...49
RESULTS…………………..……………………………………………….......49
Reproductive performance and offspring viabiltity…………………49
Body mass……………………………………………………………....53
Organ mass……………………………………………………………..56
Pathology………………………………………………………………..61
Hepatic and adipose TCDD/PeCDF/TCDF concentrations………….63
DISCUSSION…………………………………………………………………....68
Reproductive performance and offspring viabiltity………………….68
Body mass…………………………………………………………….....72
Organ mass……………………………………………………………...73
Pathology………………………………………………………………..75
Hepatic and adipose TCDD/PeCDF/TCDF concentrations……….....75
Conclusions……………………………………………………………...76
ACKNOWLEDGEMENT……………………………………………………….77
APPENDIX……………………………………….……………………………...78
SUPPLEMENTAL DATA……………………………………………....79

CHAPTER 4
CONCLUSIONS AND RECOMMENDATIONS FOLLOWING TWO LABORATORY
FEEDING STUDIES EVALUATING THE EFFECTS OF TCDD AND TCDD-LIKE
COMPOUNDS ON MINK (MUSTELA VISON)
CONCLUSIONS………………………………………………………………115
vii

RECOMMENDATIONS……………………………………………………...120
Evaluate TCDD-like compound interactions and the relative
toxicity of TCDD-like compounds in mink………..…………......…120

REFERENCES……………………………………………………………………..………...122

viii

LIST OF TABLES

Table 2.1. Daily dose and concentrations of 2,3,7,8-tetrachlorodibenzofuran (TCDF) and/or
2,3,4,7,8-pentachlorofdibenzofuran (PeCDF) in the liver of mink (Mustela vison)
………………………………………………………………………………………………...14
Table 2.2. Incidence of gross and histological effects in female mink exposed to either TCDF,
PeCDF singly or as a mixture through the diet for up to 180
…………………………………………………………………………………………………20
Table 3.1. Composition and nutrient analysis of basal experimental diets (as fed basis)
………………………………………………………………………………………………..43
Table 3.2. Dietary concentrations and corresponding doses of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8tetrachlorodibenzofuran
(TCDF)………………………………………………………………………………………45
Table 3.3. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,34,7,8pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) on reproduction
and kit growth and survivability through six weeks of
age……………………………………………………………………………………………51
Table 3.4. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) on adult female
pre-whelping mass (g) and juvenile male and female mass (g) from 14 to 27 weeks of
age……………………………………………………………………………………………54
Table 3.5. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzo
(PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) on juvenile male and female relative
organ mass (% of body mass) at 27 weeks of
age…………………………………………………………………………………………....58
Table 3.6. Effects of dietary 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) on organ
histology of juvenile
mink………………………………………………………………………………………….62
Table 3.7. Hepatic and adipise concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
2,3,4,7,8-pentaclorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) and
bioaccumulation factors in adult female mink and their juvenile
offspring……………………………………………………………………………………..64

ix

Table S1. Effects of TCDD, PeCDF, and TCDF on adult female mink absolute organ mass (g)
…………………………………………………………………………………………..........79
Table S2. Effects of TCDD, TCDF, and PeCDF on adult female mink relative organ mass (% of
body
mass)………………………………………………………………………………………….83
Table S3. Effects of TCDD, PeCDF, and TCDF on female mink kit absolute organ mass (g) at
six weeks
ofage…………………………………………………………………………….....................87
Table S4. Effects of TCDD, PeCDF, and TCDF on female mink kit relative organ mass (% of
body mass) at six weeks of
age……………………………………………………………..………………………….….89
Table S5. Effects of TCDD, PeCDF, and TCDF on male mink kit absolute organ mass (g) at
six weeks of
age……………………………………………………………………………........................93
Table S6. Effects of TCDD, PeCDF, and TCDF on male mink kit relative organ mass (% of
body mass) at six weeks of
age…………………………………………………………………………………………....95
Table S7. Effects of TCDD, PeCDF, and TCDF on juvenile female mink absolute organ mass
(g) at 27 weeks of
age……………………………………………………………………………………………99
Table S8. Effects of TCDD, PeCDF, and TCDF on juvenile female mink relative organ mass
(% of body mass) at 27 weeks of age……………………………………………………….103
Table S9. Effects of TCDD, TCDF, and PeCDF on juvenile male mink absolute organ mass
(g) at 27 weeks of age…………………………………..………………………………..…107
Table S10. Effects of TCDD, PeCDF, and TCDF on juvenile male mink relative organ mass
(% of body mass) at 27 weeks of age…………………………………………………...….111

x

LIST OF FIGURES

Figure 1.1. Configuration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)……......................2
Figure 1.2a. Configuration of 2,3,4,7,8-pentachlorodibenzofuran (PeCDF)……….…………3
Figure 1.2b. Configuration of 2,3,7,8-tetrachlorodibenzofuran (TCDF)…………….………..3
Figure 1.3. Configuration of 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126)….………………....3
Figure 1.4. The TCDD Toxic Equivalents (TEQ) equation ………………….……………….5
Figure 2.1. Single cyst consisting of squamous epithelial cells …………………...…………21
Figure 2.2. TCDF exposure and EROD and MROD activity in mink ………..…..………… 23
Figure 2.3. TCDF liver concentration and EROD and MROD activity in mink (Moore et al.,
2009)…………………………………………………………………………… ….................25
Figure 2.4. PeCDF exposure and EROD and MROD activity in mink …………...…………27
Figure 2.5. PeCDF liver concentrations and EROD and MROD activity in mink (Moore et al.,
2009)……………………………………………………………………………….………….29
Figure 2.6. TCDF and PeCDF exposure and EROD and MROD activity in mink …………31

xi

ABBREVIATIONS

AhR

Aryl hydrocarbon receptor

ATL

Aquatic Toxicology Laboratory

EROD

Ethoxyresorufin-O-deethylase

EFF

Experimental Fur Farm

CYP1A1

Cytochrome P450, family 1, member A1

CYP1A2

Cytochrome P450, family 1, member A2

LOAEL

Lowest observed adverse effect level

MROD

Methoxy resorufin-o-deethylase

MSU

Michigan State University

NOAEL

No observed adverse effect level

PeCDF

2,3,4,7,8-Polychlorinated dibenzofuran

PCBs

Polychlorinated biphenyls

PCDD

Polychlorinated dibenzo-p-dioxin

PCDF

Polychlorinated dibenzofuran

PCB 126

3,3’,4,4’,5-Pentachlorobiphenyl

PAHs

Polycyclic aromatic hydrocarbons

SE

Standard error

TCDD

2,3,7,8-Tetrachlorodibenzo-p-dioxin

TCDF

2,3,7,8-Tetrachlorodibenzofuran

TEF

Toxic equivalency factor

TEQ

TCDD Toxic equivalents

TR

Tittabawasssee River

TRV

Toxicity reference value
xii

CHAPTER 1
INTRODUCTION

TCDD and TCDD-like compounds
Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans
(PCDFs) are primarily byproducts of commercial processes, but they are also naturally
occurring compounds (Safe, 1990). Polychlorinated biphenyls (PCBs) are man-made
structurally related compounds that along with PCDDs and PCDFs are ubiquitous, persistent,
and toxic (Safe, 1998, Van den Berg et al., 1994). 2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD) is the most studied, and considered the most potent, of these structurally related
compounds (Van den Berg et al., 1998, 2006). TCDD-like compounds are chemicals with
structures and mechanism of action similar to TCDD. There are 17 TCDD-like PCDDs and
PCDFs, and 12 TCDD-like PCB congeners. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and
TCDD-like compounds are located throughout the food chain (Giesy and Kannan, 1998).
The persistence of these compounds is due to their lipophilic nature, which allows them to
bioaccumulate (Safe, 1990) in tissues of fish, wildlife and humans. Effects of TCDD and
TCDD-like chemicals in living organisms include enzyme induction, developmental
deformities, reproductive failure, liver damage, wasting syndrome, and death (Giesy et al.,
1994, Blankenship and Giesy, 2002). Further study of these compounds is warranted due to
their ubiquitous presence, persistence and toxicity so that humans may avoid, minimize
and/or manage the impacts that these compounds pose to all living organisms.

1

TCDD, PCDF and PCB configuration and mechanism of action
2,3,7,8-Tetrachlorodibenzo-p-dioxin and TCDD-like compounds are classified as
polycyclic aromatic hydrocarbons (PAHs). These compounds are distinguished by two sixcarbon ring structures connected by one or two “bridge bonds”. TCDD has two “bridge
bonds” each containing a single oxygen atom (Figure 1.1). TCDF and PeCDF have one
“bridge bond” containing a single oxygen atom and another “bridge bond” linking two
carbons from each ring (Figure 1.2a and 1.2b). The two rings comprising the PCBs are
directly linked by a single carbon-to-carbon bond between the two rings (Figure 1.3). The
various PCDD, PCDF and PCB congeners are further distinguished by the location and
number of chlorine atoms on the carbon atoms of the rings.
There are eight positions on the carbon ring skeletons of PCDDs and PCDFs to which
chlorine atoms may bind and there are ten potential binding sites on PCBs. Compounds that
differ from one another only by number and/or location of chlorine atoms are called
congeners. There are 209 possible PCB congeners, 135 PCDF congeners, and 75 PCDD
congeners (Erickson, 1997). The chemical properties and toxic potencies of individual
congeners are dependent on the number and positions of chlorine atoms on the two rings.

Figure 1.1. Configuration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

2

Figure 1.2a. Configuration of 2,3,4,7,8-pentachlorodibenzofuran (PeCDF)

Figure 1.2b. Configuration of 2,3,7,8-tetrachlorodibenzofuran (TCDF)

Figure 1.3. Configuration of 3,3’,4,4’,5-pentachlorobiphenyl (PCB 126)

3

Due to their co-planar structure, these TCDD-like compounds are known to induce a
common suite of effects through a shared mechanism of action. This mechanism is mediated
by binding of each planar ligand to a specific high-affinity cytosolic protein, known as the
aryl hydrocarbon receptor (AhR) (Zwiernik et al., 2012). Once bound, the ligand and
receptor complex translocate into the nucleus, and activates transcription of several genes
including cytochrome P450 1A1 (CYP1A1) (Denison and Nagy, 2003).

PCDD, PCDF and PCB classification
The coplanar structure and lipophilic nature of PCDDs, PCDFs and PCBs place these
contaminants in a class of compounds that are environmentally persistent and toxic to living
organisms. The World Health Organization (WHO) developed a standardized approach
known as the Toxic Equivalence (TEQ) method (Van den Berg et al., 2006) to quantify risk
of harm to living organisms when these compounds are present. Since TCDD is believed to
be the most toxic of these compounds, the toxicity of specific PCDD, PCDF and PCB
congeners are evaluated relative to TCDD. Each congener is assigned a Toxic Equivalency
Factor (TEF) value. 2,3,7,8-Tetrachlorodibenzo-p-dioxin is assigned a TEF value of 1.0,
while TCDF is assigned a TEF value of 0.1, indicating that this furan is considered to be 10%
as potent as TCDD. 2,3,4,7,8-Pentachlorodibenzofuran is assigned a TEF value of 0.3, thus
it is thought to be 30% as potent as TCDD. 3,3’,4,4’,5-Pentachlorobiphenyl (PCB 126) is
assigned a TEF value of 0.1, implying it is 10% as potent as TCDD. The TCDD-like activity
contributed by each congener in a mixture expressed as TEQ is determined by multiplying
the concentration of the congener by its TEF value. The total TEQ present in a mixture is the
sum of the products of each congener’s specific concentration and its specific TEF value

4

(Figure 1.4). For example, if a sample of liver contains concentrations of 10 ng TCDF/kg
and 10 ng PeCDF/kg, then the TEQ contributed by TCDF and PeCDF are 1 ng (10 ng x 0.1)
TEQ/kg and 3 ng (10 ng x 0.3) TEQ/kg, respectively, or a total TEQ sum of 4 ng/kg (1 ng
TEQ/kg + 3 ng TEQ/kg).

TEQ = ∑i →n [(Congeneri × TEFi ) + .......( Congenern × TEFn )]
Figure 1.4. The TCDD Toxic Equivalents (TEQ) equation (Zwiernik et al., 2008)

Model for examining toxic effects
Because humans and other vertebrate species that share the same environment often
have similar responses to toxic substances, mink (Mustela vison) may be used as surrogates
to monitor environmental contaminant exposure and effects (Zwiernik et al., 2011). Basu et
al. (2007) defines mink as a sentinel species because they meet certain criteria, these include:
a (1) widespread distribution, (2) high trophic status, the (3) ability to accumulate
contaminants, may be (4) maintained and studied in captivity, (5) captured in sufficient
numbers, reside within (6) restricted home ranges, have a (7) well-known biology, and are
(8) sensitive to contaminants. Laboratory and field studies of mink exposed to TCDD and/or
TCDD-like compounds has shown adverse effects based on examination of morphological,
histological, biochemical and reproductive characteristics. Mink are a model mammal to
evaluate the risk of harm caused by TCDD and TCDD-like compounds because: (1) they are
among the most sensitive species to PCBs (Aulerich and Ringer, 1977, Beckett et al., 2008)
and related PCDDs (Hochstein et al., 1988, 1998); (2) their nutritional requirements are well
5

documented (National Research Council, 1982); a (3) stock of known genetic origin is
readily available; (4) all stages of their life cycle can be successfully perpetuated in the
laboratory; and (5) mink have a large biological and toxicological response data base (Shump
et al., 1976, Scientifur, 1987, 1992; Sundqvist, 1989; Aulerich et al., 1999).

The Tittabawassee River and a sentinel species and laboratory model
The Tittabawassee River (TR) is the largest tributary of the Saginaw River/Bay
watershed, Michigan, USA. The city of Midland is a major industrial and population center
on the TR, where significant concentrations of polychlorinated dibenzofurans (PCDFs) and
polychlorinated dibenzo-p-dioxins (PCDDs) have been found in sediments and floodplain
soils (Hilsherova et al., 2003). The Dow Chemical Company (Dow) is a corporation
headquartered in Midland since 1867 with the history of producing vast amounts of
chemicals that have been exported throughout the world. Products of Dow have included
agricultural chemicals, caustic soda, elemental chlorine, and bleach, as well as Agent Orange
produced during the Vietnam War. The improper disposal of graphite anodes used in the
chloralkali process has led to environmental contamination by 2,3,7,8-tetrachloridbenzo-pdioxin (TCDD) and other TCDD-like compounds, including PCDFs.
Zwiernik et al. (2008a) reported concentrations of PCDDs and PCDFs in tissues of
mammals collected in the TR (Michigan, USA) basin in 2003, 2004, and 2005. The average
TEQ-adjusted TCDD-like concentrations in the livers from 22 wild mink, harvested
downstream of Midland, Michigan averaged 400 ng TEQ/kg, of which 290 ng TEQ/kg was
contributed by PCDFs and 21 ng TEQ/kg was contributed by PCDDs. The upstream control
mink had average liver concentrations of 20 ng TEQ/kg that were more evenly distributed

6

among the PCDDs, PCDFs, and TCDD-like PCBs (Zwiernik et al., 2008a). Upstream
median dietary exposure was 0.68 ng TEQ/kg and median dietary exposure in a downstream
study area was 31 ng TEQ/kg (Zwiernik et al., 2008a). As one of the most highly exposed
and most sensitive species based on the toxicological potency of these furan mixtures,
dietary- and tissue-based exposure data suggested that mink residing in the TR basin, should
be experiencing adverse effects. However, no pathology was reported for any of the 22 wild
mink collected from within the study area and population measures including abundance and
demographics, indicated that mink populations were stable and at, or close to, carrying
capacity for the TR. Mink did not exhibit any adverse effects despite exposure to TEQ
concentrations that exceeded dietary and hepatic concentration toxicity reference values
(TRVs). In light of this disparity, it was concluded that additional information on the
potency of the environmentally relevant toxic mixture of compounds found in the TR soils,
sediments, and wildlife were needed. While it may be feasible to trap mink in order to
evaluate morphological, histological and population characteristics, trapping live mink, and
then studying their reproduction and the viability of their offspring is not. To provide risk
managers with the best possible information pertaining to the potency of the site-specific
contaminant mixture, two controlled feeding studies were conducted in which ranch mink
were exposed to relevant PCDD and PCDF congeners at concentrations bracketing those
observed in the field to determine dose-and-time dependent effects and to examine whether
these congeners effect reproduction and offspring viability and growth.
Two mink feeding studies were conducted at the Michigan State University
Experimental Fur Farm (EFF) to elucidate the toxic potencies of the two most prevalent
TCDD-like PCDF compounds in TR sediment, soils, and mink. In the first study, adult

7

female ranch mink were fed TCDF, PeCDF, or a mixture of TCDF and PeCDF for 180 d.
Doses were approximately eight times greater than doses in wild mink estimated in the TR
field study (Moore et al., 2009). This study was conducted to assess: (1) the dosages of
TCDF and PeCDF necessary to achieve liver concentrations bracketing those observed in
wild mink, (2) time to achieve steady-state concentrations of the two congeners, and (3)
effect of co-administration of TCDF and PeCDF on the toxicokinetics and distribution of
each congener (Zwiernik et al., 2008b). This study also evaluated dose- and time-dependent
effects of TCDF, PeCDF, or a mixture of these two congeners on hepatic P450 enzyme
activity and tissue morphology, including jaw histology (Moore et al., 2009 and Chapter 2).
Since TCDD, PeCDF and TCDF made up the majority of the calculated toxic potency based
on TEQ using current WHO TEFs for the TR, the second study (Moore et al., 2012 and
Chapter 3) assessed the reproductive performance of female mink fed diets containing
TCDD, PeCDF or TCDF and the growth and viability of their offspring. In addition to
bracketing field exposures, the dosing regime was expanded to cover a range of
concentrations including those expected to elicit effects previously reported in mink exposed
to TCDD-like compounds. Lesser doses were set to mimic nominal, environmentally
relevant concentrations and were expected to result in no effects except for the most sensitive
responses at the molecular level. In contrast, the highest dose for each congener expressed as
TEQ exceeded median predicted environmental exposures for the TR. This highest dose was
expected to cause reproductive effects based on results of laboratory studies in which mink
fed TEQ-normalized concentrations of PCBs (Beckett et al., 2008, Bursian et al., 2006a,b,c,
Heaton et al., 1995a, Heaton et al., 1995b, Tillitt et al., 1996) at similar levels experienced
decreased litter size and/or reduce offspring viability.

8

The two studies presented herein contribute to the current body of knowledge used by
risk managers to assess risk of harm to mink exposed to site-specific or environmentally
relevant concentrations of TCDD-like compounds. Exposures at TEQ-normalized
concentrations of TCDF and PeCDF in both studies resulted in adverse effects that were less
than expected based on the TEFs assigned by the WHO as well as TRVs from other
mammalian studies. In addition, the additive assumption of the TEQ method conflicts with
results from the first study where two PCDFs were coadministered. Finally, results from the
second study described herein and other reproductive feeding studies performed at the same
facility with similar methodology suggest that the TEF for PCB 126 is underestimated and
that PCB 126 should be evaluated relative to TCDD and TCDD-like compounds, or
consideration should be given to standardize the toxicity of PCB 126 outside the TCDDcentric TEF approach. It is recommended that interactions of TCDD-like congeners be
evaluated further while relative potency studies are necessary to compare single congener
exposures of TCDD-like compounds to TCDD as well as PCB 126 to derive species-specific
TEFs for a sensitive environmentally relevant wildlife receptor.

9

CHAPTER 2
HEPATIC P450 ENZYME ACTIVITY, TISSUE MORPHOLOGY AND HISTOLOGY OF
MINK (MUSTELA VISON) EXPOSED TO POLYCHLORINATED DIBENZOFURANS
(PCDFS)

ABSTRACT
Dose- and time-dependent effects of environmentally relevant concentrations of
2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ) of 2,3,7,8-tetrachlorodibenzofuran
(TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of these two congeners on
hepatic P450 enzyme activity and tissue morphology, including jaw histology, of adult ranch
mink were determined under controlled conditions. Adult female ranch mink were fed either
TCDF (0.98, 3.8, or 20 ng TEQTCDF/kg body wt/d) or PeCDF (0.62, 2.2, or 9.5 ng
TEQPeCDF/kg body wt/d) or a mixture of TCDF and PeCDF (4.1 ng TEQTCDF/kg body wt/d
and 2.8 ng TEQPeCDF/kg body wt/d, respectively) for 180 d. Doses used in this study were
approximately eight times greater than those reported in a parallel field study. Activities of
the cytochrome P450 1A enzymes, ethoxyresorufin O-deethylase (EROD) and
methoxyresorufin O-deethylase (MROD) were significantly greater in livers of mink exposed
to TCDF, PeCDF and a mixture of the two congeners, however, there were no significant
histological or morphological effects observed. It was determined that EROD and MROD
activity can be used as sensitive biomarkers of exposure to PeCDF and TCDF in adult female
mink, however, under the conditions of this study the response of EROD/MROD induction
occurred at doses that were less than those required to cause histological or morphological
changes.
10

INTRODUCTION
Recently, there has been concern about the concentrations of polychlorinated
dibenzofurans (PCDFs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated
biphenyls (PCBs) in floodplain soil and sediment from the Tittabawassee River (Hilscherova
et al., 2003). The Tittabawassee River flows into the Saginaw River and Saginaw Bay,
Michigan, USA, as part of the Lake Huron watershed. Both field and laboratory-based
studies have been conducted to assess the potential risks of these concentrations of PCDD,
PCDF and PCBs on terrestrial and aquatic organisms (Zwiernik et al., 2008a).
The mink (Mustela vison) has been utilized as a sentinel species for ecological risk
assessments at sites where contaminants of concern are chemicals that can bind to the
aromatic hydrocarbon receptor (AhR) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
and structurally similar compounds (Giesy et al., 1994; Tillitt et al., 1996; Blankenship et al.,
2008; Basu et al., 2007). The mink is considered to be among the more sensitive mammals
to TCDD and related compounds (Hochstein et al., 1988, 1998; Beckett et al., 2008). Mink
have a relatively great potential for exposure to these persistent, bioaccumulative chemicals
(Basu et al., 2007).
An ecological risk assessment using previously-established toxicity reference values
(TRVs) derived primarily from studies of the effects of TCDD and other AhR-active
compounds on mink (Blankenship et al., 2008) and concentrations of TCDD equivalents
(TEQ) in the dietary and tissues of mink inhabiting the Tittabawassee River has been
conducted (Zwiernik et al., 2008a). This study indicated that mink might be at risk of being
adversely affected by these compounds with hazard quotients between <1 to 10 being
11

calculated. However, despite accumulating relatively great concentrations of 2,3,4,7,8pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (TCDF) in their
livers the conditions of individual mink from the more highly contaminated areas of the
Tittabawassee River was comparable or superior to that of mink collected in reference areas
and that the population was robust (Zwiernik et al., 2008a). The inconsistency between the
apparent healthy population and the elevated hazard quotient (HQ) estimates may due to
several factors including: (1) World Health Organization (WHO) toxic equivalent factor
(TEF) values and resulting TEQ concentrations are conservative and may have overestimated
risk; (2) The toxicity reference values (TRVs) used to estimate the HQs may not have been
accurate for mink due to lack of toxicological information for the dominant PCDF congeners
identified in mink at the site relative to data available in the literature to derive TEFs: and (3)
Uptake rates, metabolism, excretion and disposition of TCDF and PeCDF may differ from
TCDD or PCBs that have been studied in mink (Beckett et al., 2008; Zwiernik et al., 2008b).
A 180-day dietary study was conducted to: (1) determine rates of assimilation and
distribution of environmentally relevant doses of TCDF, PeCDF or a combination of the two
congeners in liver tissue of mink (Zwiernik et al., 2008b); (2) examine the relationship
between chemical exposure and hepatic cytochrome P4501A enzyme activities, potential
functional indicators of exposure to AhR agonists (Hahn, 1998; Whitlock Jr., 1999; Kawajiri
et al., 2007). Ethoxyresorufin O-deethylase (EROD) activity is most directly associated with
the induction of hepatic activity of the cytochrome P4501A1 enzymes whereas
methoxyresorufin O-deethylase (MROD) activity is more associated with P4501A2 enzymes.
However, while both enzymes can metabolize either substrate to some extent, metabolism of
both substrates provides valuable information as to P4501A activity in an organism relative

12

to its exposure to xenobiotics; and (3) examine relationships between EROD and MROD
activity in liver to other morphological and histological changes in mink. This chapter
presents the results of the effects of TCDF and PeCDF on hepatic EROD and MROD
activities and selected morphological and histological parameters in mink.

MATERIALS & METHODS
Mink husbandry, exposure and necropsy
Adult, female, ranch mink were randomly assigned and housed individually in wire
mesh breeder cages (61 cm L x 76 cm W x 46 cm H) with wooden nest boxes (30 cm L x
22.5 cm W x 25 cm H) within an indoor facility at Michigan State University (MSU). A total
of 50 female mink were distributed among eight treatments with six individuals in each of
seven furan-dosed groups (three TCDF groups, three PeCDF groups and one TCDF plus
PeCDF group) and eight female mink in the control group. Doses were expressed as TEQ
(Table 2.1) calculated by use of toxic equivalency factors (TEFs) reported by Van den Berg
et al. (2006).

13

Table 2.1. Daily dose and concentrations of 2,3,7,8-tetrachlorodibenzofuran (TCDF)
and/or 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) in the liver of mink (Mustela
a
vison) .
Daily dose
Liver concentration (ng TEQ/kg,
b
ww)
(ng TEQ/kg body
0d
90 & 180 d
wt/d)
Treatment
Control
d
d
TCDF
<LOD
<LOD
0.79 ± 0.24
d

c

PeCDF

<LOD
0.98
3.8
20
0.62
2.2
9.5

<LOD
NA
NA
NA
NA
NA
NA

0.61 ± 0.46
1.2 ± 0.27
2.3 ± 0.22
7.1 ± 1.1
52 ± 18
270 ± 25
1600 ± 530

TCDF
PeCDF

4.1
2.8

NA
NA

1.4 ± 0.24
360 ± 80

TCDF

PeCDF

Mixture

a

Each treatment group had six mink while the control group had eight mink.
Control animals were sampled at 0, 90 and 180 d; three treated animals per dose
group were sampled at 90 and 180 d. All concentrations were converted to 2,3,7,8TCDD equivalent
b
Liver concentrations are presented as mean ±1 SD.
LOD = Limit of detection
c
d

LOD = 0.1 ng TEQ/kg, ww
LOD = 0.01 ng TEQ/kg, ww

e

n=2, so no SD was calculated; one mink was euthanized because of kidney failure
that was not treatment related.
NA indicates that samples were not collected.

The test chemical for each treatment was dissolved in hexane to produce a stock
solution and aliquots of the stock were then diluted appropriately with 100 ml corn oil. The
corn oil containing test chemical was added to the water component of the mink diet and
mixed well in a paddle mixer prior to addition of the other feed ingredients. After addition of
all of the dietary ingredients, the feed was mixed for an additional 20 minutes. Each morning
14

for 180 d, 25 g of feed containing the furan congener(s) was given to each animal. This
procedure ensured complete ingestion of the contaminated feed, eliminating the need to
measure daily feed consumption in order to estimate doses. After this feed was consumed,
an additional 100 g of uncontaminated feed was given to each animal. Water was provided
ad libitum. Full-spectrum lighting controlled by a timer simulated the natural light/dark
cycle for the Eastern Standard Time Zone. Temperature was maintained between 13º C and
28º C and humidity ranged from 26% to 91%. Mink were observed daily for signs of toxicity
including a decrease in feed consumption and lethargy. Individual body masses (g) were
measured at the beginning of the study (January 31, 2006) and every 30 d thereafter.
Three animals from the control group were euthanized by asphyxiation with carbon
dioxide at initiation of the exposure (0 d) and three animals from each of the eight treatment
groups were euthanized at 90 and 180 d of exposure for subsequent necropsy. Body mass (g)
and length (cm) including and excluding the tail were recorded for each female mink. Mink
were examined externally and internally for overall condition, nutritional status and the
presence of gross abnormalities. Livers were removed and weighed. Sub-samples of liver
were frozen in liquid nitrogen for subsequent measurement of EROD and MROD activities.
Approximately 2.0 g of liver tissue was placed in a 10% formalin-saline solution (10%
formalin in 0.9% sodium chloride) for histological examination. The remaining liver was
placed in I-Chem® jars (I-Chem, New Castle, DE, USA) and frozen at -20˚ C for subsequent
determination of TCDF and PeCDF concentrations using High Resolution-Gas
Chromatography/Mass Spectrometry (HR-GC/MS). In addition, the spleen, kidney, thymus,
mesenteric lymph node, and brain were removed and preserved for subsequent histological
examination. The head was placed in formalin-saline solution for subsequent histological

15

examination of mandibular and maxillary squamous epithelial cell proliferation as described
by Beckett et al. (2005). The lesion was graded as mild, moderate, or severe based on the
number and size of foci of squamous cell proliferation in the maxilla and mandible (Beckett
et al., 2005). The MSU Institutional Animal Care and Use Committee approved this study
(AUF 12/05 – 165 – 00).

Chemicals and reagents
PeCDF and TCDF were obtained from Accustandard Inc. (New Haven, CT, USA)
and dissolved in hexane to produce a stock solution. Working solutions and dilutions of
PeCDF and TCDF were prepared in pesticide residue analysis grade OmniSolv n-hexane
(EM Science, Lawrence, KS, USA). For biochemical analyses, 7-ethoxyresorufin (7-ER)
was obtained from Molecular Probes (Eugene, OR, USA) while 7-methoxyresorufin (7-MR)
and resorufin were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other
biochemical reagents including NADPH were obtained from Sigma-Aldrich and were
reagent grade or better unless stated otherwise.

EROD and MROD quantification
Liver microsomes were prepared by homogenizing 0.5 g of liver in Tris buffer (0.05
M Tris and 1.15% KCl, pH 7.5) and centrifuged to obtain the microsomal fraction. The
microsomal pellet was resuspended in microsomal stabilization buffer (20% glycerol, 0.1 M
KH2PO4, 1mM EDTA, and 1mM dithiothreitol, pH 6.25) and aliquots were stored at -80o C.
EROD and MROD activities measured using a modification of methods described by
Kennedy and Jones (1994). The assays were optimized and conducted in 96-well plates

16

(Corning Costar Corp., Corning, NY, USA) where both microsomal cytochrome P450
activity and protein concentration were measured simultaneously using a Fluoroscan Ascent
microplate fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). For EROD
assays, the range of the working resorufin standards was 0 to 210 pmol/well. The reaction
mixture included 3.0 µl of microsome preparation in 0.05 M HEPES buffer (pH 7.8), 0.3 mM
NADPH and 5mM ethoxyresorufin (7-ER) per well. For MROD assays, the working
resorufin standard range was 0 to 180 pmol/well. The reaction mixture included 8 µl of
microsome preparation in 0.05 HEPES buffer (pH 7.8), 0.3 mM NADPH and 2.5 mM
methoxyresorufin (7-MR) per well. Following the addition of the substrates (7-ER or 7MR), all assay plates were pre-incubated for 10 min at 37°C prior to the addition of NADPH
to initiate the reaction. EROD and MROD activities were determined kinetically by
measuring the formation of resorufin every 2 min for 30 min. The reaction was terminated
by adding 60 ml acetonitrile (Burdick and Jackson, Muskegon, MI, USA) containing 0.4 mM
fluorescamine (Sigma-Aldrich, St. Louis, MO, USA) to each well followed by the
determination of protein concentrations (Kennedy and Jones, 1994). EROD and MROD
activities were determined from the linear range of the time-curves for each well and the
results were expressed as pmol substrate converted per min per mg protein (pmol/min/mg).

Quantification of PCDD, PCDF and TEQ
To insure that co-contaminants were not a factor in the study, the concentrations of 17
individual 2,3,7,8-substituted PCDF and PCDD congeners and 12 individual PCB congeners
were measured in the dietary items and mink tissues as described in Zwiernik et al. (2008b).
Concentrations of TEQ were calculated as the sum of the products of the concentrations of

17

congeners multiplied by their respective TEF (Van den Berg et al., 2006). A surrogate value
of one-half the method detection limit (MDL) was used for concentrations less than the MDL

Data analysis
All statistical analyses were performed with SAS (SAS, Ver. 9.1; Cary, NC, USA).
Because of the nature of the parameters, several statistical models were used for data
analyses. The study was designed for the application of both fixed effects models (test for
differences among exposure groups) and regression analysis (correlation of liver PeCDF and
TCDF concentrations and EROD and MROD enzyme activities). Prior to conducting
statistical comparisons, data were tested for normality using the Shapiro-Wilkes test and
probability plots. If necessary, values were log-transformed to approximate normality.
Differences among exposure groups were tested using a one-way ANOVA followed by
Dunnett’s test (PROC ANOVA). A sensitivity analysis was conducted to determine the bias
introduced by assuming a value of half the limit of quantification (LOQ) for censured data
sets.

RESULTS
PCDF concentration in liver
Concentrations of TCDF and PeCDF in livers of mink fed daily doses of TCDF,
PeCDF or a mixture of the two congeners did not differ between 90 or 180 d, thus a single
mean concentration is presented (Table 2.1).
Concentrations of TCDF in the liver varied among doses dose, ranging from 30%
greater than the daily dose (0.98 TEQTCDF/kg body wt/d) to 65% less than the daily dose (20
18

TEQTCDF/kg body wt/d). Concentrations of PeCDF in mink liver increased significantly
with dose, with bioaccumulation factors (BAFs) of 9.5 and 17 for the two doses 0.62 and 9.5
ng TEQPeCDF/kg body wt/d, respectively. Concentrations of TCDF and PeCDF in livers of
mink fed the TCDF/PeCDF mixture were similar to concentrations in the livers of mink
receiving a similar dose of the individual congeners. Hepatic BAFs based on TEQ
concentration were 0.032 for TCDF (4.1 ng TEQTCDF/kg body wt/d) and 12 for PeCDF (2.8
ng TEQPeCDF/kg body wt/d).

Gross morphology and histology
There were no treatment-related changes in gross morphology or histology. No
external lesions or abnormalities that were attributable to treatment were observed and the
nutritional status of all mink, except for one individual was classified as “good” to “very
good”. There were no significant changes in body mass or liver mass over the course of the
study (data not presented). The most frequent histological alteration was hepatocellular
vacuolation that occurred in all groups, and thus, was not considered to be treatment-related
(Table 2.2).

19

Table 2.2. Incidence of gross and histological effects in female mink exposed to either TCDF, PeCDF singly or as a
a
mixture through the diet for up to 180 d .
TCDF (ng TEQ/kg body
PeCDF (ng TEQ/kg body
TCDF/
wt/d)
wt/d)
PeCDF
b
c
Control
0.98
3.8
20
0.62
2.2
9.5
Pathological Endpoints
Mixture
Oral Lesions
Squamous epithelial osteoinvasion
Osetoclasts and bone resorption
Periodontitis
Liver
Hepatocellular vacoulation
Periportal lymphocytic/plasmytic
Fatty liver
Bile duct hyperplasia
Kidney
Medullary tubules or uroliths
Infection
Nephritis
Lymphoid aggregates
Spleen
Hemorrhage
Scar fissure

0
0
1

0
0
0

0
0
0

0
0
0

0
0
0

0
0
0

1
1
0

0
0
0

5
0
0
0

6
0
1
0

6
1
0
1

6
0
1
0

6
0
1
1

6
0
3
1

6
0
2
0

6
0
0
0

1
0
0
0

1
0
1
0

1
1
0
0

1
0
0
0

0
0
0
0

1
0
0
2

1
0
0
0

2
1
0
0

0
1

0
0

0
0

0
0

0
0

0
0

0
0

1
0

a

Treatment concentrations are estimated daily doses reported as TEQ values. Mammalian TEF used were 0.3 for PeCDF
and 0.1 for TCDF (Van den Berg et al. 2006). Mixture consisted of 4.1 and 2.8 ng TEQ/kg bw/d for TCDF and PeCDF,
respectively.
b
Values given for each endpoint represent the number findings (mink) associated with each treatment group (n=6 mink
per treatment).
c
Mixture consisted of 4.1 and 2.8 ng TEQ/kg body wt/d for TCDF and PeCDF, respectively.
20

There were a few cases of bile duct hyperplasia and minimal to mild mineralization
of renal medullar tubules that occurred across all treatments. There was a numerically
greater incidence of fatty liver in mink fed only PeCDF, compared to the other groups (Table
2.2). Periodontitis was observed in one mink from the control group, but this was considered
incidental and not treatment-related. Jaw lesions classified as mild were observed at the
termination of the study in two mink from the 9.5 ng TEQPeCDF/kg body wt/d treatment
group (Table 2.2). One of these mink exhibited a single cyst consisting of squamous
epithelial cells (Figure 2.1). However, the presence and severity of this lesion was not dosedependent, and therefore, was considered incidental.

A

B

For interpretation of the references to color in this and all other figures, the reader
is referred to the electronic version of this thesis.

Figure 2.1. Single cyst consisting of squamous epithelial cells

21

EROD and MROD activities
Mink fed TCDF alone had significantly greater EROD and MROD activities in the
liver compared to controls. Because there were no significant treatment by time interactions,
enzyme activities measured after 90 and 180 d of exposure were averaged. Exposure to
TCDF resulted in significantly greater activities of both EROD and MROD in mink at doses
of 3.8 and 20 ng TEQTCDF/kg body wt/d (Figure 2.2).

22

A

EROD (pmol/min/mg protein)

1000
c
800
b
600
a
400
a
200
0
Control

0.98

3.8

20

TCDF Dose (ng TEQ/kg body wt/d)
B

MROD (pmol/min/mg protein)

160
c

140
120

b
*

100
a

80
a
60
40
20
0
Control

0.98

3.8

20

TCDF Dose (ng TEQ/kg body wt/d)

Figure 2.2. TCDF exposure and EROD and MROD activity in mink

23

Additionally, EROD and MROD activities in mink fed 20 ng TEQTCDF/kg body wt/d were
significantly greater than activities of those fed 3.8 ng TEQTCDF/kg body wt/d group. Both
EROD (Figure 2.3A) and MROD (Figure 2.3B) activities were positively correlated with
concentrations of TCDF expressed as TEQ in the liver.

24

EROD (pmol/min/mg protein)

1200
Control

1000

Day 90
Day 180

800
600
400
y = 221.44Ln(x) + 261.51
R2 = 0.8288

200
0
0

1

2

3

4

5

6

7

8

9

10

Liver TCDF Conc. (ng TEQ/kg, ww)

MROD (pmol/min/mg protein)

160
Control

140

Day 90
120

Day 180

100
80
60

y = 32.099Ln(x) + 50.969

40

2

R = 0.8326

20
0
0.0

2.0

4.0

6.0

8.0

10.0

Liver TCDF Conc. (ng TEQ/kg, ww)

Figure 2.3. TCDF liver concentration and EROD and MROD activity in mink (Moore et al..
2009)

25

Exposure to PeCDF resulted in statistically significant greater EROD and MROD activities
relative to controls (Figure 2.4). Because there were no statistically significant differences in
either EROD or MROD enzyme activities at 90 and 180 d and there were no interactions
between treatment and time the values of each of these enzyme activities at the two times
were averaged. EROD activities in all PeCDF-dosed groups were significantly greater than
control activity (Figure 2.4A).

26

A
1200
EROD (pmol/min/mg protein)

c
1000

b, c

800
b

600
400
a
200
0
Control

0.62

2.2

9.5

PeCDF Dose (ng TEQ/kg body wt/d)

MROD (pmol/min/mg protein)

B
200
180
160
140
120
100
80
60
40
20
0

c
c
b
a

Control

0.62

2.2

9.5

PeCDF Dose (ng TEQ/kg body wt/d)

Figure 2.4. PeCDF exposure and EROD and MROD activity in mink

27

EROD activity in the 9.5 ng TEQPeCDF/kg body wt/d group was significantly greater than
enzyme activities in the 0.62 and 2.3 ng TEQPeCDF/kg body wt/d dose groups. MROD
activities were also significantly greater than control activities at all PeCDF doses with
activities in livers of mink fed 2.2 or 9.5 ng TEQPeCDF/kg body wt/d being significantly
greater than activities in livers of mink fed 0.62 ng TEQPeCDF/kg body wt/d. Both EROD
and MROD activities were positively correlated with concentrations of PeCDF expressed as
TEQ in the liver (Figure 2.5A, B).

28

EROD (pmol/min/mg protein)

1200
1000
800
600
400

Control
y = 79.822Ln(x) + 194.71
R2 = 0.6976

Day 90

200

Day 180

0
0

500

1000

1500

2000

2500

3000

Liver PeCDF Conc. (ng TEQ/kg, ww)

MROD (pmol/min/mg protein)

180
160
140
120
100
80
60

Control
Day 90
Day 180

40
20

y = 12.436Ln(x) + 44.211
2
R = 0.7852

0
0

500

1000

1500

2000

2500

3000

Liver PeCDF Conc. (ng TEQ/kg ww)

Figure 2.5. PeCDF liver concentrations and EROD and MROD activity in mink (Moore et
al., 2009)

29

EROD and MROD activities in livers of mink fed a mixture of TCDF (4.1 ng TEQTCDF/kg
body wt/d) and PeCDF (2.8 TEQPeCDF/kg body wt/d) were significantly greater than
activities in livers of control mink (Figure 2.6). EROD activity in the livers of mink fed the
mixture of TCDF and PeCDF were similar to the activities in mink fed 3.8 TEQTCDF/kg
body wt/d and those fed 2.2 ng TEQPeCDF/kg body wt/d (Figure 2.6A). MROD activity in
livers of mink fed the mixture was significantly greater than enzyme activity in livers of
mink fed 3.8 ng TEQTCDF/kg body wt/d, but did not differ from activity in livers of mink fed
2.2 ng TEQPeCDF/kg body wt/d PeCDF (Figure 2.6B).

30

EROD (pmol/min/mg protein)

A
1000
b
b

800
b

600
400
a
200
0
Control

TCDF

PeCDF

Mixture

Doses (ng TEQ/kg body wt/d)

MROD (pmol/min/mg protein)

B
200
180

c

160
140
120
100
80
60
40
20
0

c
b
a

Control

TCDF

PeCDF

Mixture

Doses (ng TEQ/kg body wt/d)

Figure 2.6. TCDF and PeCDF exposure and EROD and MROD activity in mink

31

DISCUSSION
PCDF concentrations in liver
2,3,4,7,8-Pentachlorodibenzofuran accumulated in the liver of the mink to a much
greater extent than did TCDF when administered as a single congener or in combination with
TCDF (Table 2.1). Hepatic sequestration of PeCDF relative to that of PCDDs and other
PCDFs including TCDF is consistent with what has been reported in other studies with
mammals (Brewster and Birnbaum 1987, 1988; Devito et al., 1997). These studies have
shown that PeCDF accumulates in the liver of rodents by binding to hepatic CYP1A2 protein
(Dilberto et al., 1999) and presumably, PeCDF could be sequestered in livers of mink by the
same mechanism (Zwiernik et al., 2008b). The lesser concentrations of TCDF accumulated
in livers of the mink suggest an efficient elimination and/or metabolism of the congener. The
BAF of this congener has been reported to be inversely proportional to dose (Zwiernik et al.,
2008b), which suggests inducible metabolism of TCDF. This is similar to what has been
reported in rodents (Tai et al., 1993). The fact that the presence of PeCDF reduced the
accumulation of TCDF in the liver of the mink to an even greater extent strengthens the
argument that induction of CYP1A1 reduced accumulation of TCDF (Zwiernik et al.,
2008b).
The whole-body half-time for elimination of PeCDF observed for mink in this study
was estimated to be approximately 8 d while the half-time for elimination of TCDF was less
than half a day in mink (Zwiernik et al., 2008b). The half-time for elimination for TCDF and
PeCDF in the mink are less than those reported for rodents. The half-time of TCDF is
approximately 2 d in mice (Devito et al., 1997) and the half-time of PeCDF in the rat is more
than 60 d (Brewster and Birnbaum, 1987).

32

Histology
In this study, TCDF and PeCDF, administered singly or in combination, at
environmentally relevant doses for 180 d did not result in changes in gross morphological or
histological endpoints (Table 2.2) that have been reported for other studies in which mink
were exposed to dioxin or dioxin-like compounds (Hochstein et al., 1988, 1998; Render et
al., 2000a,b, 2001). Recent studies (Bursian et al., 2006b,c) suggest that a very sensitive
indicator of exposure of mink to environmentally relevant concentrations of TCDD-like
compounds is proliferation of mandibular and maxillary squamous epithelia. Previous
studies have indicated that ranch mink exposed to 24.0 µg 3,3’,4,4’,5-pentachlorobiphenyl
(PCB 126)/kg or 2.4 µg TCDD/kg feed (2.4 µg TEQ/kg feed or approximately 300 ng
TEQ/kg body wt/d) developed clinical signs of mandibular and maxillary squamous
epithelial hyperplasia that in severe cases resulted in the loss of teeth (Render et al., 2000a;
2001). Mink fed diets containing concentrations as little as 0.24 µg PCB 126/kg feed (0.024
µg TEQ/kg feed or 3 ng TEQ/kg body wt/d) (Beckett et al., 2008) exhibited the lesion (K.
Beckett, personal communication) as did mink fed a diet containing fish containing PCBs,
PCDDs, and PCDFs that provided an estimated daily dose of 1 ng TEQ/kg body wt/d
(Bursian et al., 2006b). In the present study, only one animal, which had been fed 9.5 ng
TEQPeCDF/kg body wt/d had a single cyst of squamous epithelial cells at 180 d. The
concentration of PeCDF in the liver of that mink was 1.3 ng TEQ/g, ww. In those mink
studies where jaw lesion incidence and liver TEQ concentrations were assessed, results
indicated that histological lesions were evident in animals with hepatic TEQ concentrations
ranging from 40 to 75 ng/kg, ww in the liver (Bursian et al., 2006b,c). Wild mink with
33

histological evidence of proliferation of mandibular and maxillary squamous epithelia had an
average concentration of 610 ng TEQ/kg, ww (Beckett et al., 2005). There are two possible
explanations for the scarcity of the jaw lesions in the present study. One possibility is that
the age at which exposure was initiated was too late and/or the duration of exposure was not
sufficient. In the studies with ranch mink in which effects were observed at concentrations
similar or less than those tested in this study, exposure began in utero and continued until
mink were approximately 7 mo old (Bursian et al., 2006b,c). In those studies where
exposure periods ranged from 30 to 60 d (Render et al., 2000a, 2001), the mink were
approximately 6 wk old and the dose was approximately 30-fold greater than the dose in the
present study (300 ng TEQ/kg body wt/d versus 9.5 ng TEQ/kg body wt/d). A second
possibility is related to the specific PCB/PCDD/PCDF congeners contributing to the TEQs.
In studies of ranch mink utilizing individual congeners, TEQs were provided by either TCDD
or PCB 126 (Render et al., 2000a,b, 2001). In those studies of mink fed diets containing
contaminated fish, the majority of TEQs were contributed by congeners other than furans.
For example, in a study that assessed the effects of feeding diets containing fish from the
Housatonic River, PCB 126 and TCDD contributed 61% of the total TEQs while TCDF and
PeCDF contributed 4% (Bursian et al., 2006a,b). In a similar study utilizing fish from the
Saginaw River, PCB 126 and TCDD contributed 39% of the total while TCDF and PeCDF
accounted for 25% of the total. It is possible that TCDF and PeCDF are less effective than
PCB 126 and TCDD in inducing proliferation of mandibular and maxillary squamous
epithelia. Furthermore, it has been determined that the effects of PCDFs can not be
accurately predicted from the use of TEQ-based TRVs developed from studies of PCDDs
and PCBs (Blankenship et al., 2008). This suggests that there are differences in the

34

sensitivity of mink to PCBs and PCDFs that are not appropriately reflected by the currently
utilized TEQ approach (Van den Berg et al., 2006).

Enzyme Induction
Basal EROD activities measured in livers of mink during this study fell within a
range of control activities that have been reported in other studies with mink (Smits et al.,
1995; Shipp et al., 1998; Brunström et al., 2001; Käkelä et al., 2001; Martin et al., 2007).
Values in this study were similar to those reported by Smits et al. (1995), Kakela et al. (2001)
and Martin et al. (2007), but were less than those values reported by Brunström et al. (2001).
However, given the inconsistencies between all of these studies relative to experimental
design, age, and sex of animals, as well as potential contaminants associated with their feed,
a direct comparison between these studies is not possible. Given that the basal EROD
activities in our study are similar to those enzyme activities measured in other studies, it can
be assumed that the cytochrome P4501A1 system was functioning properly.
To our knowledge, there have been no reports of MROD enzyme activities in mink to
date. Basal MROD activity was less than that reported for EROD, which is in accordance
with studies in other mammals such as rats or monkeys (Lubet et al., 1990; Weaver et al.,
1994; Suzuki et al., 2001) but opposite to reports on other species such as various mice
strains, hamster, or humans (Weaver et al., 1994; Hamm et al., 1998). The relative difference
between EROD and MROD activities was greater (~5-fold) when compared to that reported
for rats or monkey (<2- to 3-fold). It has been previously reported that the specificities of
orthologous forms of P450s are expressed differently among mammalian species. In rats
CYP1A1 and CYP1A2 selectively catalyze EROD and MROD, respectively, while in humans

35

CYP1A2 has similar activities for both EROD and MROD. From the data presented here it
appears that mink are associated more closely with rat or monkey regarding their basal
EROD/MROD profiles. However, further elucidation of the specificities of different forms
of P450s for the different alkylresorufin O-dealkylases (AROD) is necessary to be able to
assign mink to a certain mammalian metabolism type.
There were no significant differences in enzyme activity in mink receiving daily
doses of TCDF and/or PeCDF between 90 and 180 d. This suggests that maximum induction
of CYP1As in livers of mink as a function of time in response to the exposure with TCDF
and PeCDF occurs earlier than the first sampling time point at 90 d.
The EROD activity in mink dosed with the mixture of TCDF and PeCDF was similar
to enzyme activity in those mink dosed with either TCDF or PeCDF while MROD activity
was similar to activity in those mink dosed with PeCDF. This suggests that induction
resulting from the combination of the two furan congeners may not have been additive and
perhaps was due primarily to the action of only one of the congeners. Based on liver
concentration data indicating greater concentration of PeCDF compared to TCDF, it is
possible that enzyme induction in those animals receiving the mixture was due primarily to
PeCDF. Alternatively, TCDF may also have contributed to the increase in enzyme activities,
but due to metabolism, its concentration in the liver was less than that of PeCDF.

ACKNOWLEDGEMENTS
This research was supported by an unrestricted grant from ENTRIX Inc. to Steven J.
Bursian, John P. Giesy and Matthew J. Zwiernik. Special thanks go to Angelo Napolitano,
Jeff Greenlee, C.P. Napolitano, David Hamman, Patrick Bradley, Michael Kramer, Nozomi
36

Ikeda, Molly Wiersema, and Melissa Shotwell for the expertise and support they provided
during the study.

37

CHAPTER 3
EFFECTS OF DIETARY EXPOSURE OF MINK (MUSTELA VISON) TO 2,3,7,8 –
TETRACHLORODIBENZO-P-DIOXIN (TCDD), 2,3,4,7,8PENTACHLORODIBENZOFURAN (PECDF) AND 2,3,7,8TETRACHLORODIBENZOFURAN (TCDF) ON REPRODUCTON AND OFFSPRING
VIABILITY AND GROWTH
ABSTRACT
This study assessed the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8 tetrachlorodibenzofuran (TCDF) on
the reproductive performance of female mink (Mustela vison) and the viability and growth of
their offspring. Nine adult female mink each were randomly assigned to one of 13 dietary
treatments (one control and four doses each of TCDD, PeCDF and TCDF [2.1-8.4, 4.0-15
and 5.2-25 ng TCDD toxic equivalents (TEQ)/kg body wt/d]. Diets were fed from two
months prior to breeding through weaning of offspring at six weeks of age. At least nine kits
per treatment group were maintained on their diets through 27 weeks of age. There were no
effects on litter size or viability of offspring. No consistent effects were observed on body
mass or relative organ masses of animals at any age. 2,3,7,8-Tetrachlorodibenzo-p-dioxin
and PeCDF accumulated in the liver and adipose tissue, but TCDF was rapidly cleared. The
lack of significant effects on reproduction and offspring viability contrasts with effects
reported for mink exposed to environmentally derived PCB mixtures with equivalent TCDD
potencies. This suggests that it may be inappropriate to apply toxicity reference values
associated with PCB mixtures to animals also exposed to TCDD, PeCDF or TCDF and the
World Heath Organization TCDD toxic equivalency factors for some congeners may not be
appropriate for mink.

38

INTRODUCTION
Elevated concentrations of polychlorinated dibenzo-p-dioxins (PCDDs) and
polychlorinated dibenzofurans (PCDFs) have been detected in sediments, floodplain soils,
and fish of the Tittabawassee River (MI, USA) (Hilsherova et al., 2003). Polychlorinated
dibenzo-p-dioxins and PCDFs are persistent, bioaccumulative compounds; therefore, top
trophic level predators have the greatest potential for exposure. Mink (Mustela vison) are a
species of special interest because they forage within the riparian zone and have a prey base
consisting of both terrestrial and aquatic organisms. The home range of an adult male is
estimated to average 2.6 km in stream length and that of an adult female averages 1.9 km
(Linscombe et al., 1982). In addition, laboratory studies have shown that mink are among
the most sensitive species to the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and
TCDD-like compounds (Hochstein et al., 1988, Hochstein et al., 1998, Beckett et al., 2008).
The combination of exposure potential and sensitivity to the site-specific contaminants of
concern make the mink a good species for interpreting risk of harm to piscivorous
mammalian wildlife species, as discussed by Basu et al. (2007) residing within the
Tittabawassee River floodplain.
Concentrations of PCDDs and PCDFs in tissues of mammals residing within the
Tittabawassee River basin are among the highest ever reported (Zwiernik et al., 2008a).
When concentrations are expressed as TCDD toxic equivalents (TEQ) using World Health
Organization toxic equivalency factors (TEFs) (Van den Berg et al., 2006), livers from 22
wild mink, collected downstream of Midland, MI, USA, had an average of 400 ng TEQ/kg
(wet wt), of which 290 ng TEQ/kg (wet wt) was contributed by PCDFs and 21 ng TEQ/kg
(wet wt) was contributed by PCDDs. Mink collected upstream of the study area had a

39

concentration of 20 ng TEQ/kg (wet wt) in liver tissue, which was distributed more evenly
among the PCDDs, PCDFs, and TCDD-like polychlorinated biphenyls (PCBs) (Zwiernik et
al., 2008a). Based on the present understanding of the toxicological potency of these
mixtures, dietary- and tissue-based exposure data suggest that mink, as one of the most
highly exposed and most sensitive species, should be experiencing adverse effects (Bursian
et al., 2006a,b,c) along the Tittabawassee River. Conversely, selected measures of individual
health, including histological and morphological measures, as well as measures of population
conditions such as abundance and demographics, indicate that mink appear to be healthy and
populations are stable and at or close to carrying capacity for the Tittabawassee River
(Zwiernik et al., 2009). From this apparent disparity between the predicted and observed
condition of resident mink, it was concluded that additional information on the potency of the
toxic mixture of compounds found in the Tittabawassee River soils, sediments, and wildlife
was needed.
To provide risk managers with the best possible information pertaining to the potency
of the site-specific contaminant mixture, a controlled feeding study was conducted in which
ranch mink were exposed to relevant PCDD and PCDF congeners at concentrations
bracketing those observed in the field. These included TCDD, 2,3,4,7,8pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (TCDF), which were
the three compounds that made up the majority of calculated toxic potency based on TEQ
using current World Health Organization TEFs (Van den Berg et al., 2006). Because the
present study design included TCDD in a side-by-side comparison of toxicity to the two
furans, the results also provide animal-based relative potency data that can be used by the
World Health Organization for calculating the mammalian TEFs for PeCDF and TCDF. In

40

addition to bracketing field exposures, the dosing regime was expanded to cover a range of
concentrations including those expected to elicit effects previously reported for mink
exposed to TCDD-like compounds. Lesser doses were set to mimic nominal
environmentally relevant concentrations and were expected to result in no effects except for
the most sensitive responses at the molecular level. In contrast, the highest dose for each
congener expressed as TEQ using the current World Health Organization TEFs (Bursian et
al., 2006a) (TCDD = 8.4 ng TEQTCDD/kg body wt/d, PeCDF = 15 ng TEQPeCDF/kg body
wt/d, and TCDF = 25 ng TEQTCDF/kg body wt/d) exceeded the median predicted
environmental exposures for the Tittabawassee River of 3.9 ng TEQ/kg body wt/d. This
highest dose was expected to cause reproductive effects based on the results of laboratory
studies where mink fed TEQ-normalized concentrations of PCBs (Beckett et al., 2008,
Bursian et al., 2006a,b,c, Heaton et al., 1995a, 1995b, Tillitt et al., 1996) at similar levels
experienced decreased litter size and/or reduced offspring viability.
The present report describes the effects of consumption of diets containing various
concentrations of TCDD, PeCDF or TCDF on adult female reproductive performance and
offspring viability and growth through 27 weeks of age.

MATERIALS and METHODS
Chemicals and reagents
2,3,7,8-Tetrachlorodibenzo-p-dioxin, PeCDF and TCDF were obtained from
AccuStandard and dissolved in hexane (OmniSolv, EMD Chemicals) to produce a stock
solution for each congener. Working solutions of TCDD, PeCDF and TCDF were then
41

prepared by serial dilution in hexane. One ml of each working solution was added to 100 ml
corn oil for incorporation into the feed.

Dietary treatments
The treatment diets were based on the Michigan State University (MSU)
Experimental Fur Farm ranch diet formulated to meet the nutritional requirements of mink
(Table 3.1) (National Research Council, 1982). The treatment diets were prepared by adding
water to a 500-kg-capacity paddle mixer, followed by fishmeal, wheat middlings, and
soybean oil. These ingredients were thoroughly mixed prior to addition of the working
solutions, which had been diluted 1:100 with corn oil. A solution of 1 ml hexane and 100 ml
corn oil was added to the control feed. After an additional period of mixing to allow the
hexane to evaporate, the remaining ingredients were added and mixed thoroughly. Three
grab samples consisting of five subsamples per grab sample were collected for each diet for
congener analysis (Vista Laboratories), as well as a sample for nutrient analysis (Litchfield
Analytical Services). The treatment diets were packaged in labeled, one-gallon aluminum
containers that were stored in a walk-in freezer (-20º C) at the MSU Experimental Fur Farm.
Twenty-fours hours prior to use, containers were transferred from the walk-in freezer to a
walk-in cooler (4º C) to allow the feed to thaw. One container was sufficient to feed a group
of nine mink for approximately 3 d. Feed was mixed and sampled a second time halfway
through the trial as described above.

42

Table 3.1. Composition and nutrient analysis of basal
experimental diets (as fed basis).
Ingredient
Composition (%)
Water
34.0
a
Soybean oil
6.0
Spray-dried poultry liver
Spray-dried eggs

4.0

b

Spray-dried blood cells
Chicken

b

5.0
c

4.0

d

26.0

Wheat middlings
Fishmeal

a

15.0

a

4.0

Vitamin premix
Mineral premix

e

0.5

f

Phosphoric acid

0.5

g

1.0

h

Larvacide (ml/kg feed)

0.2

i

d-biotin (mg/kg feed)
Nutrient analysis (%)
Moisture
Protein
Fat
Ash
Crude fiber
Total digestible nutrients
a

53.8
17.6
11.0
4.7
1.7
43.9

North American Nutrition, Lewisburg, OH, USA.

b
c

2.4

VanElderen, Martin, MI, USA.

California Spray Dry, Stockton, CA, USA.

d

Whole ground chicken, Whalen Foods, Chaska, MN, USA.

e

Calcium, 13.40%; copper, 2000 mg/kg; iodine, 30 mg/kg;
iron, 2.0 %; manganese, 2000 mg/kg; selenium, 60 mg/kg;
zinc, 2.0 %; Akey, Louisburg, OH, USA.

43

Table 3.1. Cont’d.
f

Vitamin A, 916,652 IU/kg; vitamin D3, 91,674 IU/kg; activity,
vitamin E, 11,000 IU/kg; vitamin K 2200 mg/kg; menadione,
733 mg/kg; vitamin B12, 5.5 mg/kg; riboflavin, 733 mg/kg; dpantothenic acid, 2935 mg/kg; niacin, 4400 mg/kg; thiamine,
183 mg/kg; pyridoxine, 33 mg/kg; Akey, Louisburg, OH.
g
Astaris, St. Louis, MO, USA.
h

Active ingredient: cyromazine (N-cyclopropyl-1,3,5- triazine2,4,6-triamine, 2%), Novartis Animal Health, Greensboro, NC,
USA.
i
Biotin 100 (100 mg/lb), ADM, Des Moines, IA, USA.

Targeted dietary concentrations were 21, 42, 73 and 104 ng TCDD/kg feed; 139, 243,
347 and 533 ng PeCDF/kg feed; 728, 1600, 2560 and 3120 ng TCDF/kg feed. Actual dietary
concentrations reflecting both mixes, as determined by high resolution gas
chromatography/high resolution mass spectrometry (HRGC/HRMS), and daily doses of
TCDD, PeCDF, and TCDF as well as the corresponding TEQs (based on TEFs reported by
Van den Berg et al., 2006) are presented in Table 3.2. The TEQ concentration for each of the
TCDD, PeCDF, and TCDF groups reflects the concentration provided by that congener only
because the TEQs contributed by other congeners were less than 1% of the total. Dose
calculations were based on the average estimated feed consumption and body mass of adult
females in each treatment group through the first 15 weeks of the trial. Feed consumption
was estimated by providing each animal with a daily allotment of 125 g of feed, which was
slightly greater than the consumption of 115 g/d previously reported for adult female ranch
mink (Bleavins et al., 1981), and determining the amount feed remaining at the time of next
feeding.

44

Table 3.2. Dietary concentrations and corresponding doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF).
a
Estimated dose
Dietary concentration

Treatment
TCDD

PeCDF

TCDF

ng/kg
feed
23
53
77
101
166
288
363
619
679
1464
2402
2866

b

SE
0.6
1.6
2.6
3.9
3.1
4.7
23.2
12.2
21.3
35.3
150.5
163.6

ng
c
TEQ /kg
feed
23
53
77
101
50
86
109
186
68
146
240
287

Mean
estimated
daily feed
intake
wk 1-15 (g)
99.5
103.2
100.3
102.3
99.1
102.8
99.3
96.8
100.0
99.3
99.1
93.5

SE
1.1
0.5
0.9
0.8
0.9
0.6
0.9
0.9
1.0
1.0
1.0
1.2

Mean body
weight
wk 1-15
(g)
SE
1088
27
1187
31
1286
21
1226
23
1241
14
1172
23
1207
29
1215
20
1318
26
1254
26
1110
19
1091
30

ng/kg
body
wt/d
2.1
4.6
6.0
8.4
13
25
30
49
52
116
214
246

ng TEQ/kg
body wt/d
2.1
4.6
6.0
8.4
4.0
7.6
9.0
15
5.2
12
21
25

a

Dose based on estimated feed consumed from a daily allotment of 125 g feed and mean body weights of
adult female mink through week 15 of the study.
b
SE refers to standard error.
c

TEQ refers to toxic equivalents that are based on toxic equivalency factors of 1.0, 0.3 and 0.1 for TCDD,
PeCDF and TCDF, respectively [Van den Berg et al., 2006].

45

Animals
One hundred seventeen first-year (virgin) and second-year (proven breeder), natural
dark, female mink from the MSU Experimental Fur Farm herd were assigned randomly on
November 20, 2006 to 13 dietary treatment groups (nine mink per group) with the exception
that littermates were not placed in the same treatment group to minimize genetic
predisposition to compound toxicity. Untreated, natural dark, male mink were used for
breeding purposes only. The MSU Institutional Animal Care and Use Committee approved
the use of animals for this trial.

Housing
Female mink were housed individually in wire breeder cages (76 cm L x 46 cm W x
38 cm H) suspended above the ground in an open-sided mink shed. Nine animals per
treatment group were assigned randomly to a bank of nine cages separated from the next
bank of nine cages by an empty cage. Assignment of treatments to banks of cages was done
to minimize the potential for cross-contamination between groups. A wooden nest box (38
cm L x 25 cm W x 29 cm H) bedded with excelsior (wood wool) prebreeding or aspen
shavings postbreeding was attached to the outside of each cage. The standard guidelines for
the operation of mink farms in the United States (Fur Commission U.S.A., 2003) were
followed to house and maintain the animals.

Exposure period
Mink were started on their treatment diets on December 30, 2006, after a one-week
acclimation period. The daily allotment of feed (125 g) was placed on a cleaned grid on the

46

top of the cage. Water was available ad libitum. Animals were weighed every four weeks
until the initiation of breeding (March 1, 2007).
Adult females were mated to untreated males between March 1 and March 26, 2007.
Each female was given an opportunity to mate every fourth day until a successful mating was
obtained. Females were assumed to have bred successfully if evidence of vulvar swelling
appeared following a copulation period of at least 10 minutes. Mated females were given an
opportunity to breed with a different male the day following a successful mating and on the
eighth and ninth days after the first successful mating (a common commercial mink breeding
practice).
Whelping began on April 15, 2007 and ended on May 15, 2007. Nest boxes were
checked on a daily basis for the presence of mink kits. Live kits were enumerated, and body
masses were recorded at birth and at three and six weeks of age. Body masses of adult
females were recorded at the time their litters were weighed.
All surviving adults and a representative number of kits from each treatment group
were euthanized (CO2) when kits were six weeks old (May 22 to June 25, 2007). These
individuals were necropsied and samples of selected tissues were taken for analytical and
histological assessment. At least nine kits per treatment were maintained on their diets until
they were 27 weeks old (October 22 to November 2, 2007), at which time they were
euthanized and processed as above. At least four males and four females, but no more than
nine mink, were selected randomly from each treatment for the final necropsy and tissue
analysis. The thyroid gland, thymus, heart, adrenal glands, kidneys, spleen, reproductive
organs (uterus with ovaries/testes), liver, and brain were removed, weighed, and placed in
10% neutral buffered formalin for subsequent histological assessment.
47

Chemical analysis
To ensure that cocontaminants were not a factor in the present study, concentrations
of 17 2,3,7,8-substituted PCDF and PCDD congeners and 12 TCDD-like PCB congeners
were measured in the dietary items, feed samples, and liver tissue as described by Zwiernik
et al. (2008b). Cocontainments accounted for less than 1% of the TEQ contributed by
TCDD, PeCDF, or TCDF. Thus, concentrations of dietary and hepatic TEQ for each of the
TCDD, PeCDF, and TCDF treatment groups were calculated as the product of the
concentration of that congener only multiplied by its respective TEF (Van den Berg et al.,
2006). A surrogate value of one-half the method detection limit (MDL) was used for
concentrations less than the MDL. Liver tissues were extracted following a modification of
U.S. Environmental Protection Agency (U.S. EPA) Method 1613B (Telliard, 1994)). Liver
tissue extracts were shipped on dry ice to Vista Laboratories for congener analysis by highresolution gas chromatography/high-resolution mass spectrometry according to U.S. EPA
Method 1613B (Telliard, 1994).

Histological analysis
Histological examination of tissues was performed at MSU’s Diagnostic Center for
Population and Animal Health. Tissues were embedded in paraffin, sectioned at 5 µm, and
stained with hematoxylin and eosin. A board-certified veterinary pathologist examined slides
of the thyroid gland, thymus, heart, adrenal glands, kidneys, spleen, reproductive organs
(uterus with ovaries/testes), liver, brain, and maxilla and mandible of each mink sampled at
necropsy.

48

Statistical analysis
All statistical analyses were performed with SAS Version 9.1. Because of the nature
of the parameters, several statistical models were used for data analyses. The present study
was designed for the application of fixed effects models to test for differences among
exposure groups. Prior to conducting statistical comparisons, data were tested for normality
using the Shapiro-Wilkes test and probability plots. If necessary, values were log
transformed to approximate normality. Differences among treatment groups were evaluated
by analysis of variance using SAS PROC Mixed. Because of the unbalanced experimental
design (unequal sample sizes), least square means were used in the analyses. When group
effects were statistically significant, differences among treatment groups were tested with
Tukey-Kramer test to account for differences in sample size among the groups. Differences
among groups were considered significant at p < 0.05.

RESULTS
Reproductive performance and offspring viability
All females bred at least once. The percent of bred females whelping ranged from
78% to 100% with the exception of the greatest PeCDF treatment group (49 ng PeCDF/kg
body wt/d or 15 ng TEQPeCDF/kg body wt/d), which had a 56% whelping rate. Mean litter
sizes at birth for females that whelped were not significantly different from the control group
irrespective of the treatment compound or dose. Similarly, no significant differences were
observed in kit viability among treatment groups compared with controls through six weeks
of age (Table 3.3). Although differences in kit viability among dose groups were not
statistically significant because of sample size and variability, the percentages of viable kits

49

in the control and low-dose PeCDF groups were numerically greater compared to the other
groups.

50

Table 3.3. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran
(PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) on reproduction and kit growth and survivability
a
through six weeks of age .

7 of 9
8 of 9

8.4
PeCDF

7 of 9

6.0

TCDD

2.1
4.6

Treatment
Control

Dose (ng/kg
body wt/d)
0

Number of
females
whelping/total
number of
females
9 of 9

8 of 9

13

8 of 9

25

8 of 9

30

8 of 9

49

5 of 9

b

c

d

Litter size
(live kits)
6.1
(0.70)

Mean body
mass (g) at
birth
9.91
(0.60)

Mean body
mass (g) at
six weeks
250.13
(23.50)

Survivability
through six
weeks of age
(%)
80.3
(0.5 - 1.1)

5.6
(0.80)
4.9
(0.80)

7.57
(0.70)
8.39
(0.70)

215.6
(33.30)
182.06
(33.30)

46.3
(0.0 – 0.9)
53.1
(0.1 - 1.0)

5.7
(0.70)
4.6
(0.70)
5.4
(0.80)
4.8
(0.80)
5.4
(0.80)

9.3
(0.60)
9.95
(0.60)
8.65
(0.60)
9.53
(0.60)
9.9
(0.70)

255.27
(27.27)
181.12
(27.20)
275.39
(23.50)
184.04
(29.80)
172.78
(27.20)

51.6
(0.2 – 0.8)
67.7
(0.3 – 1.0)
81.3
(0.6 – 1.0)
36.3
(0.0 – 0.7)
51.5
(0.2 – 0.9)

4.4
(1.00)

9.68
(0.80)

180.5
(33.30)

65.0
(0.1 – 1.2)

51

Table 3.3. Cont’d.

TCDF

52

9 of 9

116

9 of 9

214

8 of 9

246

7 of 9

4.4
(0.70)
5.9
(0.70)
4.6
(0.80)
5.1
(0.80)

10.19
(0.60)
9.22
(0.60)
9.04
(0.70)
7.7
(0.70)

a

228.73
(25.10)
238.13
(22.20)
228.08
(29.80)
181.75
(27.20)

51.8
(0.2 – 0.9)
61.1
(0.4 – 0.9)
57.1
(0.2 – 1.0)
66.7
(0.3 – 1.0)

Data are presented as means with (standard error) or (95% confidence interval)
beneath.
b
One female died due to renal failure caused by bacterial pyelonephritits. Uterus contained six fetuses.
c

One female died due to bacterial pneumonia. Uterus did not contain fetuses.

d

One female died due to ruptured uterus. Uterus contained five fetuses.

52

Body mass
Mean body masses of adult females prior to the whelping period were not
significantly different compared to controls (Table 3.4). Similarly, no significant differences
were noted in mean body masses of kits at birth and six weeks of age compared with controls
(Table 3.3). Conversely, some significant treatment-related differences were observed in
juvenile male mean body masses compared to controls at week 14 and 27. Treatment
differences were generally not consistent in terms of age and/or dose (Table 3.4). The mean
body mass of male juveniles exposed to the highest dose of TCDD (8.4 ng/kg body wt/d; 8.4
ng TEQTCDD/kg body wt/d) was significantly less than the mean body mass of the control
group counterparts at week 14 of the trial; however, by week 27, masses were no longer
different. For the next-lesser-dose TCDD group (6.0 ng TCDD/kg body wt/d; 6.0 ng
TEQTCDD/kg body wt/d), male mean body mass did not differ from the control group
counterparts at week 14 but did differ at week 27. In males exposed to PeCDF, mean body
masses were significantly less compared with controls at weeks 14 and 27 in the 25 and 49
ng PeCDF/kg body wt/d (7.6 and 15 ng TEQPeCDF/kg body wt/d) treatment groups but not
in the 30 ng PeCDF/kg body wt/d (9.0 ng TEQTCDD/kg body wt/d) group. Only at the
highest dose of TCDF (246 ng TCDF/kg body wt/d; 25 ng TEQTCDF/kg body wt/d) was
mean body mass of juvenile males significantly different compared with controls at 14 weeks
of age but not at 27 weeks of age. Regardless of congener or dose, mean body mass in
juvenile females did not differ significantly from controls (Table 3.4).

53

Table 3.4. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8tetrachlorodibenzofuran (TCDF) on adult female pre-whelping mass (g) and juvenile male and female mass (g) from 14 to 27
a,b
weeks of age .
Adult Females

8
9
8

8.4
PeCDF

2.1

6.0

TCDD

n
9

4.6

Treatment
Control

Dose
(ng/kg
body wt/d)
0

9

13

9

25

9

30

9

49

8

Wk 0 prewhelping
1275
(62)
1274
(62)
1311
(62)
1326
(62)
1264
(62)
1250
(62)
1303
(62)
1244
(62)
1272
(62)

Wk 15 prewhelping
1290
(49)
1018
(49)
1225
(49)
1362
(49)
1285
(49)
1284
(49)
1254
(49)
1270
(49)
1215
(49)

Juvenile males

Wk 14 of
age
1164
(58)
972
(72)
1066
(72)
950
(75)
841 A
(58)
1037
(51)
866 A
(75)
1066
(56)
805 A
(70)

n
12
7
7
6
9
10
3
7
7

54

Wk 27 of
age
1537
(79)
1355
(96)
1285
(96)
1073 A
(102)
1226
(81)
1307
(70)
1093 A
(123)
1494
(83)
1119 A
(88)

Juvenile females

n
17
7
7
16
7
13
7
8
2

Wk 14 of
age
802
(41)
836
(66)
765
(60)
815
(76)
689
(60)
795
(44)
733
(63)
762
(55)
651
(109)

Wk 27 of
age
1164
(53)
999
(86)
983
(78)
1013
(60)
952
(78)
1123
(63)
938
(90)
1043
(79)
821
(155)

Table 3.4. Cont’d.

TCDF

52

9

116

9

214

9

246

9

1387
(62)
1350
(62)
1256
(62)
1248
(62)

1373
(49)
1249
(49)
1096
(49)
1095
(49)

9

1102
(58)
1055
(52)
926
(61)
911 A
(56)

12
9
9

1354
(89)
1572
(79)
1357
(92)
1304
(86)

a

Data are presented as means with (standard error) beneath.

b

Means that are significantly different then the control mean at p < 0.05 are designated with an A.

55

4
12
9
9

869
(61)
816
(45)
712
(46)
737
(49)

1026
(79)
1144
(57)
1008
(59)
1035
(63)

Organ mass
Relative masses (percent of body mass) of the spleen and liver were greater compared
to controls at the highest doses of the three congeners, depending on age, whereas changes in
relative masses of other organs were inconsistent across doses (Table 3.5). Mean relative
spleen mass in the adult females receiving the greatest dose of TCDD (8.4 ng TCDD/kg body
wt/d; 8.4 ng TEQ/kg body wt/d) was significantly greater compared to controls (mean [95%
confidence interval]; 0.34 [0.30-0.42] vs 0.25 [0.22-0.28]) as was mean relative spleen mass
in the juvenile males dosed with 8.4 ng TCDD/kg body wt/d (8.4 ng TEQTCDD/kg body
wt/d). Mean relative liver masses of juvenile males at the highest PeCDF (49 ng PeCDF/kg
body wt/d; 15 ng TEQPeCDF/kg body wt/d) and TCDF (246 ng TCDF/kg body wt/d; 25 ng
TEQTCDF/kg body wt/d) doses were significantly greater compared to controls. There
appeared to be a dose-related trend of increasing relative liver masses in the TCDD and
PeCDF groups. Other significant changes in relative organ masses included increased mean
relative kidney masses at 4.6 ng TCDD/kg body wt/d (4.6 ng TEQTCDD/kg body wt/d) and
25 and 49 ng PeCDF/kg body wt/d (7.6 and 15 ng TEQPeCDF/kg body wt/d) in juvenile
males. In juvenile females, mean relative thymus masses were significantly decreased at 6.0
ng TCDD/kg body wt/d (6.0ng TEQTCDD/kg body wt/d) and 52 ng TCDF/kg body wt/d (5.2
ng TEQTCDF/kg body wt/d), mean relative heart masses were significantly increased at 6.0
ng TCDD/kg body wt/d (6.0 ng TEQTCDD/kg body wt/d) and 25 ng PeCDF/kg body wt/d
(7.6 ng TEQPeCDF/kg body wt/d) and mean relative adrenal gland mass was significantly

56

increased at 8.4 ng TCDD/kg body wt/d (8.4 ng TEQTCDD/kg body wt/d) compared with
controls. Absolute and relative masses for all adult female, kit, and juvenile organs are
presented in Supplemental Data (Appendix).

57

Table 3.5. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran
(PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) on juvenile male and female relative organ mass (%
a,b,c
.
of body mass) at 27 weeks of age
Males

7
6
6

8.4
PeCDF

2.1

6.0

TCDD

n
12

4.6

Treatment
Control

Dose
(ng/kg
body
wt/d)
0

9

13

10

25

3

30

7

49

8

Body massnecropsy (g)
1536
(79)
1355
(96)
1295
(103)
1072 A
(103)
1225
(81)
1307
(71)
1093 A
(128)
1497
(85)
1057 A
(82)

Liver
5.1
4.79-5.42
5.19
4.28-6.11
5.54
4.73-6.36
5.99
5.23-6.75
5.97
5.50-6.45
5.38
4.62-6.15
6.67
1.78-11.56
5.70
5.35-6.06
6.83 A
6.01-7.64

58

Spleen
0.19
0.16-0.21
0.18
0.16-0.20
0.22
0.20-0.25
0.18
0.16-0.20
0.26 A
0.21-0.31
0.19
0.17-0.22
0.78
0.00-3.25
0.27
0.23-0.32
0.31
0.25-0.38

Kidneys
0.66
0.60-0.72
0.75
0.07-0.84
0.83 A
0.70-0.95
0.83
0.74-0.92
0.76
0.70-0.82
0.72
0.65-0.80
0.86 A
0.56-1.17
0.68
0.62-0.74
0.91 A
0.82-0.10

Table 3.5. Cont’d.

TCDF

12

214

10

246

TCDD

9

116

Treatment
Control

52

9

Dose
(ng/kg
body
wt/d)
0

n
17

2.1

7

4.6

8

6.0

16

8.4

7

1357
(90)
1574
(79)
1323
(90)
1302
(87)

4.99
4.50-5.48
4.98
1.77-5.19
5.31
4.87-5.75
5.85 A
5.48-6.22
Females

59

0.73
0.65-0.80
0.68
0.65-0.70
0.75
0.67-0.82
0.74
0.67-1.80

Thymus
0.11
0.09-0.12
0.08
0.06-0.11
0.09
0.06-0.11
0.07 A
0.06-0.08
0.08
0.06-0.10

Body massnecropsy (g)
1164
(54)
999
(87)
990
(78)
1013
(60)
952
(79)

0.21
0.14-0.28
0.20
0.17-0.24
0.24
0.19-0.28
0.27
0.24-0.29

Heart
0.71
0.65-0.77
0.83
0.70-0.97
0.85
0.77-0.93
0.92 A
0.87-0.96
0.86
0.72-0.99

Adrenal glands
0.026
0.021-0.030
0.035
0.024-0.047
0.028
0.020-0.036
0.029
0.025-0.032
0.038 A
0.033-0.043

Table 3.5. Cont’d.

PeCDF

13

25

7

30

8

49
52

1
4

116

12

214

8

246

TCDF

13

9

1123
(63)
938
(90)
1043
(79)
967
1025
(80)
1144
(58)
1019
(61)
1035
(64)

0.09
0.08-0.11
0.07
0.04-0.09
0.07
0.05-0.09
0.06
0.06 A
0.01-0.11
0.08
0.06-0.09
0.07
0.06-0.09
0.07
0.06-0.09

0.78
0.72-0.84
0.96 A
0.74-1.18
0.76
0.68-0.84
0.76
0.89
0.63-1.15
0.70
0.62-0.78
0.84
0.72-0.96
0.80
0.70-0.89

0.027
0.020-0.034
0.039
0.025-0.053
0.032
0.022-0.042
0.028
0.027
0.018-0.037
0.031
0.026-0.037
0.030
0.021-0.039
0.027
0.021-0.032

a

Body mass data are presented as the least squares mean with (standard error) beneath.

b

Relative organ relative mass data are presented as the mean with 95% confidence interval beneath.

c

Means that are significantly different than the control mean at p < 0.05 are designated with an A.

60

Pathology
Exposure to TCDD, PeCDF, and TCDF did not induce any consistent treatmentrelated histological changes in the tissues examined, with the exception of mandibular and
maxillary squamous epithelial proliferation in six-week-old kits and 27-week-old juveniles
(Bursian et al., 2012) and significant mineralization of the liver, heart, and thyroid gland in
juveniles exposed to the greatest dose of TCDF (246 ng TCDF/kg body wt/d; 25 ng
TEQTCDF/kg body wt/d) (Table 3.6). Additionally, in both six-week-old kits (data not
shown) and 27-week-old juveniles, evidence of mild renal mineralization and hepatic
vacuolation was observed in all dose groups, including the controls.

61

Table 3.6. Effects of dietary 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran
(PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) on organ histology of juvenile mink.

Treatment

Control
TCDD

PeCDF

TCDF

Dose
(ng/kg
body
wt/d)
2.1
4.6
6.0
8.4
13
25
30
49
52
116
214
246

n

Kidney
a
mineralization

Hepatic
b
vacuolation

14
9
9
9
9
10
7
9
9
9
10
9
10

1.07
1.11
1.00
1.11
1.11
1.00
1.00
1.00
1.00
1.00
0.91
1.00
1.00

Hepatic
Cardiac
Thyroid
a,c
a,c
a,c
mineralization
mineralization
mineralization

1.86
1.89
1.75
1.67
2.00
1.70
2.00
1.89
1.89
2.00
1.89
2.00
2.00

0
0
0
0
0
0
0
0
0
0
0
0.11
0.60 A

0
0
0
0
0.11
0.10
0.14
0
0
0.22
0.20
0.56
0.90 A

a

A value of 1 = mild mineralization; 2 = moderate mineralization

b

A value of 1 = mild fatty vacuolation; 2 = moderate fatty vacuolation.

c

Means that are significantly different than the control mean at P < 0.05 are designated with an A.

62

0
0
0
0
0
0
0
0
0
0
0
0.11
0.60 A

Hepatic and adipose TCDD/PeCDF/TCDF concentrations
Concentrations of TCDD, PeCDF, and TCDF in liver and adipose of adult females
and 27-week-old juveniles generally increased with dose (Table 3.7). Concentrations of the
three congeners in livers of adults were significantly different from those in livers of controls
at the two highest doses of TCDF, all doses of PeCDF, and the two highest doses of TCDF.
In adult female adipose tissue, concentrations of TCDD, PeCDF, and TCDF were
significantly greater than control concentrations at all doses. Congener concentrations in
livers of juvenile mink fed TCDD, PeCDF, and TCDF were significantly greater than those
in livers of controls at all doses except the lowest doses of TCDD and TCDF.
Concentrations of TCDD, PeCDF, and TCDF in adipose tissue of juveniles were
significantly greater at all doses than those in adipose tissue of unexposed juvenile mink.
Concentrations were generally similar between adults and juveniles.
Bioaccumulation factors were generally consistent across treatment groups for each
congener (Table 3.7). Bioaccumulation factors were greater than one for TCDD and PeCDF
in both liver and adipose tissue of adults and juveniles but less than one for TCDF in all
treatment groups. The bioaccumulation factors indicated that TCDD bioaccumulated to a
greater extent in adipose tissue than in the liver, whereas PeCDF bioaccumluated to a greater
extent in liver than in adipose tissue. TCDF did not bioaccumulate in either tissue relative to
the diet being fed.

63

Table 3.7. Hepatic and adipose concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) and
bioaccumulation factors in adult female mink and their juvenile offspring.
Adults
Liver
Dietary
treatment
Control
TCDD

Control
PeCDF

Control
TCDF

Dose
(ng/kg
body
wt/d)
2.1
4.6
6.0
8.4
13
25
30
49
52
116
214
246

n
9
9
7
7
7
9
9
7
8
7
9
3
5
4
5

Concentration
(ng/kg ww)
0.14
56

a

SE

23.6
23.6
26.7
26.7
26.7
336
336
381
356
381
13.2
22.9
17.7
19.8
17.7

c

157 A
250 A
364 A
0.53
1851 A
3066 A
4078 A
7209 A
0.66
46
58
109 A
125 A

64

p value
dose vs.
control
0.4660
0.0009
< 0.0001
< 0.0001
0.0036
< 0.0001
< 0.0001
< 0.0001
0.4560
0.1080
0.0014
< 0.0001

Bioaccumulation
b
factor
2.33
2.46
2.93
3.23
3.61
4.08
11.2
10.7
11.2
11.7
0.250
0.068
0.040
0.045
0.044

Table 3.7. Cont’d.

Adipose
Dietary
treatment
Control
TCDD

Control
PeCDF

Control
TCDF

Dose
(ng/kg
body
wt/d)
2.1
4.6
6.0
8.4
13
25
30
49
52
116
214
246

n

Concentration
(ng/kg ww)

SE

6
7
7
7
5
6
6
5
7
7
6
3
3
4
3

0.89
344 A
540 A
969 A
1418 A
3.7
1314 A
1704 A
2396 A
3008 A
0.44
289 A
318 A
568 A
791 A

51.9
48.0
48.0
48.0
56.8
190
190
208
176
176
44.1
62.4
62.4
54.0
62.4

65

p value
dose vs.
control
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.0004
0.0002
< 0.0001
< 0.0001

Bioaccumulation
b
factor
14.8
15.1
10.1
12.5
14.1
26.2
7.92
5.93
6.59
4.86
0.170
0.425
0.217
0.237
0.276

Table 3.7. Cont’d.

Juveniles
Liver
Dietary
treatment
Control
TCDD

Control
PeCDF

Control
TCDF

Dose
(ng/kg
body
wt/d)
2.1
4.6
6.0
8.4
13
25
30
49
52
116
214
246

n

Concentration
(ng/kg ww)

SE

13
11
9
9
9
13
9
11
9
10
13
9
11
9
10

0.18
59
165 A
289 A
338 A
0.94
1662 A
3161 A
5261 A
8167 A
0.17
39
125 A
207 A
207 A

21.9
32.1
25.8
22.4
23.9
341
364
431
381
457
18.0
21.6
19.6
21.6
20.5

66

a

p value
dose vs.
control
0.0788
< 0.0001
< 0.0001
< 0.0001
0.0002
< 0.0001
< 0.0001
< 0.0001
0.0824
< 0.0001
< 0.0001
< 0.0001

Bioaccumulation
b
factor
3.00
2.60
3.08
3.74
3.34
7.28
10.0
11.0
14.5
13.2
0.064
0.057
0.085
0.086
0.072

Table 3.7. Cont’d.

Adipose
Dietary
treatment
Control
TCDD

Control
PeCDF

Control
TCDF

Dose
(ng/kg
body
wt/d)
2.1
4.6
6.0
8.4
13
25
30
49
52
116
214
246

n

Concentration
(ng/kg ww)

SE

7
3
4
3
3
7
6
5
3
3
7
3
3
3
3

0.09
371 A
825 A
1105 A
1560 A
0.75
966 A
1536 A
1918 A
2903 A
0.53
323 A
544 A
633 A
756 A

86.5
113
112
113
113
141
152
166
214
214
23.2
29.9
29.9
29.9
29.9

p value
dose vs.
control
0.0065
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001

Bioaccumulation
b
factor
15.5
16.4
15.5
14.3
15.5
5.36
5.82
5.34
5.28
4.69
0.200
0.475
0.372
0.264
0.264

a

SE refers to standard error.

b

Bioaccumulation factor = (liver or adipose concentration/feed concentration).

c

Means that are significantly different than the control mean at p < 0.05 are designated with an A.

67

DISCUSSION
Reproductive performance and offspring viability
The reproductive performance of the control mink and viability of their offspring in
the present study were comparable to those of control mink in two other reproduction trials
(Bursian et al., 2006a,c) conducted at the MSU Experimental Farm using similar
methodology. Average litter size at birth in the present study was 6.1 kits per litter compared
to 5.7 (Bursian et al., 2006c) and 4.6 (Bursian et al., 2006a) kits per litter. Kit survivability
through six weeks of age was 80.3% in the present study compared to 88.9% (Bursian et al.,
2006c) and 85.0% (Bursian et al., 2006a).
Toxic equivalent doses of TCDD, PeCDF and TCDF as great as 8.4 ng TEQTCDD/kg
body wt/d, 15 ng TEQPeCDF/kg body wt/d and 25 ng TEQTCDF/kg body wt/d, respectively,
had no significant effect on reproductive performance of mink and viability of their
offspring. These doses corresponded to maternal hepatic TEQ concentrations of 364, 2163
and 13 ng /kg ww, respectively. However, only 56% of the bred females in the highest
PeCDF dose group whelped compared to 100% in the control group. It is possible that the
dose of 15 ng TEQPeCDF/kg body wt/d affected the whelping rate, although one of the four
females not whelping died of a ruptured uterus that contained five fetuses, which was not
considered treatment related. The females that did whelp had a mean litter size that did not
differ from the mean litter size of control females. In a mink feeding study utilizing
3,3’,4,4’5-pentachlorobiphenyl (PCB 126), females fed diets containing 240 ng TEQPCB
126/kg feed (30 ng TEQPCB 126/kg body wt/d) and higher experienced complete reproductive

68

failure, whereas animals fed diet containing 24 ng TEQPCB 126/kg feed (3.0 ng TEQPCB
126/kg body wt/d) were not affected (Beckett et al., 2008).

The general lack of an effect on reproductive performance and offspring viability was
unexpected in that reproductive impairment and reduced offspring viability have been
associated with similar or lesser TEQ doses in other mink feeding studies using a similar
exposure scenario (Beckett et al., 2008, Bursian et al., 2006a,b, Zwiernik et al., 2009, Heaton
et al., 1995a, Tillitt et al., 1996, Hochstein et al., 2001). From studies utilzing single
congeners, Hochstein et al. (1998) reported a 125 d LC50 value for TCDD in mink of 47 ng
TEQTCDD/kg body wt/d, which is less than twice the highest TEQ dose provided by TCDF.
Hochstein et al. (2001) also attempted a mink reproduction study utilizing TCDD at
estimated daily doses of 2.0, 6.6, 22.5 and 175 ng TEQTCDD/kg body wt/d (16, 53, 180 and
1,400 ng TEQTCDD/kg feed, respectively). An effect on reproduction could not be clearly
determined because of subnormal reproductive performance of the control group, which was
attributed to the fact that the trial was conducted indoors. The highest dose resulted in 17%
adult mortality and a 26% decrease in body weight. Significant dose-dependent decreases
were noted in kit birth mass and survival from birth to three weeks of age in the groups that
had reproduction (animals in the 16 ng TEQTCDD/kg feed [2.0 ng TEQTCDD/kg body wt/d]
did not reproduce). Zwiernik et al. (2009) reported that dietary concentrations of 240 and
2,400 ng TCDF/kg feed (26 and 240 ng TEQTCDF/kg feed or estimated doses of 3.3 and 30
ng TEQTCDF/kg body wt/d) did not affect reproduction and kit viability, but body masses of

69

offspring through 36 weeks of age were decreased compared with controls at various time
points.
Several mink feeding studies have been conducted using contaminated fish collected
from specific bodies of water. In one such study, mink were fed diets containing fish
collected from Saginaw Bay (MI, USA) that were contaminated with a mixture of PCB,
PCDF and PCDD congeners (Heaton et al., 1995a, Tillitt et al., 1996). Mated females
exposed to a dose of 8.3 ng TEQPCBs/PCDDs/PCDFs/kg body wt/d (based on a dietary
concentration of 66 ng TEQPCBs/PCDDs/PCDFs/kg feed with TEQ recalculated using TEFs
presented by Van den Berg et al. (2006) produced fewer live kits compared to controls,
while a dose of 2.1 ng TEQPCBs/PCDDs/PCDFs/kg body wt/d (dietary concentration of 17 ng
TEQPCBs/PCDDs/PCDFs/kg feed) significantly reduced kit viability through six weeks of age
compared with controls. In another study of similar design, PCB/PCDF/PCDD-contaminated
fish collected from the Housatonic River (MA, USA) resulting in a dose of 6.4 ng
TEQPCBs/PCDDs/PCDFs/kg body wt/d (dietary concentration of 51 ng
TEQPCBs/PCDDs/PCDFs/kg feed; TEQ recalculated using TEFs presented by Van den berg et
al. (2006)) also had reduced kit viability at six weeks of age (Bursian et al., 2006a). The
corresponding maternal hepatic concentrations were 226 ng TEQPCBs/PCDDs/PCDFs/kg and
189 ng TEQPCBs/PCDDs/PCDFs/kg for the Saginaw Bay (Heaton et al., 1995a, Tillitt et al.,
1996) and Housatonic River (Bursian et al., 2006b) studies. In contrast to the Saginaw Bay
(Heaton et al., 1995a, Tillitt et al., 1996) and Housatonic River (Bursian et al., 2006b) studies

70

and similar to the results in the present study, mink fed diets containing fish collected from
the Saginaw River (MI, USA) at dietary concentrations of 22, 36 and 57 ng
TEQPCBs/PCDDs/PCDFs/kg feed (TEQs were recalculated using TEFs presented by Van den
berg et al., 2006), which correspond to estimated doses of 2.8, 4.5 and 7.1 ng
TEQPCBs/PCDDs/PCDFs/kg body wt/d, experienced no effects on reproduction or kit
viability. The doses of TEQs in the present study at which no effects on reproduction and
survival were noted were up to fourfold greater than doses of TEQ in those fish feeding
studies that reported such effects.
This apparent difference in toxicity between studies could be a reflection of the
source of TEQ. Environmentally derived mixtures contain quantifiable PCBs and other
identified TCDD-like contaminants that contribute the calculated sum TEQ value, whereas
single congener studies provide a single congener source and subsequent TEQ value. In the
Saginaw Bay study (Heaton et al., 1995a, Tillitt et al., 1996), PCB 126 contributed 62 and
53%, TCDD contributed 11 and 8% and PeCDF contributed 6 and 24% of the dietary and
hepatic TEQs, respectively. In the Saginaw River study (Bursian et al., 2006c), PCB 126
contributed 33 and 34%, TCDD contributed 16 and 8%, and PeCDF contributed 17 and 44%
of the dietary and hepatic TEQ, respectively. In the Housatonic River study (Bursian et al.,
2006a,b), PCB 126 contributed 81 and 85%, TCDD contributed less than 1%, and PeCDF
contributed 7 and 6% of the dietary and hepatic TEQs, respectively. In mink feeding studies
using dietary TEQ provided exclusively by PCB 126 (Beckett et al., 2008) or TCDF (Bursian
et al., 2006b), PCB 126 caused complete reproductive failure at a concentration that was one
order of magnitude less than the greatest concentration of TCDF that resulted in no

71

reproductive effects. Thus, despite the fact that PCB 126 and TCDF have an identical TEF
of 0.1, it is apparent that when provided individually, PCB 126 is considerably more toxic to
mink than is TCDF. The same relationship could also be true for PCB 126 compared to
TCDD and PeCDF, explaining reproductive effects at lesser doses of TEQ that are provided
primarily by PCB 126, as in the Saginaw Bay (Heaton et al., 1995a, Tillitt et al., 1996), and
Housatonic River (Bursian et al., 2006a) studies when compared to the present study.

Body mass
Exposure of mink to TCDD, PeCDF, and TCDF did not have a significant effect on
body masses of adult females or male and female kits through six weeks of age and juvenile
females through 27 weeks of age and had an inconsistent effect on juvenile male body mass
at dietary TEQ concentrations as great as 287 ng/kg feed and TEQ doses up to 25 ng/kg body
wt/d. The results of other mink feeding studies have indicated variable effects of TCDD-like
chemicals on body mass. Adult female mink fed diets containing fish collected from
Saginaw Bay that provided TEQPCBs/PCDDs/PCDFs concentrations as little as 17 ng/kg feed
(2.1 ng/kg body wt/d) produced kits of significantly lesser body mass at three and six weeks
of age compared with control animals (Heaton et al., 1995a). Similarly, feeding mink diets
containing 51 ng TEQPCBs/PCDDs/PCDFs/kg feed (6.4 ng TEQPCBs/PCDDs/PCDFs /kg body
wt/d) derived from fish collected from the Housatonic River resulted in a transient decrease
in kit body masses at three weeks of age, but body masses of adult females and juveniles
were not affected (Bursian et al., 2006a). Body masses of adult female mink and their
offspring that were fed diets containing fish collected from the Saginaw River that provided
up to 57 ng TEQPCBs/PCDDs/PCDFs/kg feed (7.1 ng TEQPCBs/PCDDs/PCDFs/kg body wt/d)
72

were not adversely affected (Bursian et al., 2006c). Body masses of offspring of mink fed a
diet containing 24 ng TEQPCB 126/kg feed (3.0 ng TEQPCB 126/kg body wt/d) were not
significantly different compared to controls (Beckett et al., 2008). Body masses of male
mink kits exposed to TCDF in utero and during lactation at dietary concentrations of 24 and
240 ng TEQ/g feed (3.0 and 30 ng TEQTCDF/kg body wt/d) were less than those of controls
at three weeks of age, and body masses of female offspring were less compared with those of
controls from six to 36 weeks of age (Zwiernik et al., 2009).

Organ mass
Mink exposed to TCDD, PeCDF or TCDF had relative organ masses that were
different compared to controls in some cases, and, although the differences were not strictly
dose dependent, with the exception of what appeared to be a trend of increasing relative liver
masses with dose in juvenile males exposed to TCDD and TCDF, the changes were
comparable to those reported in other mink feeding studies involving TCDD-like chemicals.
In the present study, mean relative liver, heart, spleen, kidney and adrenal gland masses were
greater compared to controls for various doses of the three chemicals in the three age groups,
and mean relative thymus masses were reduced. Adult female mink fed diets containing fish
collected from Saginaw Bay that provided from 17 to 66 ng TEQPCBs/PCDDs/PCDFs/kg feed
(2.1 to 8.3 ng TEQPCBs/PCDDs/PCDFs/kg body wt/d, respectively) exhibited greater mean
relative spleen and liver masses at all doses, greater mean relative adrenal gland masses at 33
and 66 ng TEQPCBs/PCDDs/PCDFs/kg feed (4.1 and 8.3 ng TEQPCBs/PCDDs/PCDFs/kg body
wt/day, respectively) and greater mean relative kidney mass at 66 ng
73

TEQPCBs/PCDDs/PCDFs/kg feed (8.3 ng TEQPCBs/PCDDs/PCDFs/kg body wt/d) compared
with controls (Heaton et al., 1995a). Conversely, six-week-old kits generally had reduced
mean relative organ masses at 17 and 33 ng TEQPCBs/PCDDs/PCDFs/kg feed (2.1 and 4.1 ng
TEQPCBs/PCDDs/PCDFs/kg body wt/day, respectively). No kits survived in the 66 ng
TEQPCBs/PCDDs/PCDFs/kg feed (8.3 ng TEQPCBs/PCDDs/PCDFs/kg body wt/d) treatment
group (Heaton et al., 1995a). The mean relative liver mass in six-week-old kits whelped by
dams exposed to 36 and 57 ng TEQPCBs/PCDDs/PCDFs/kg feed (4.5 and 7.1 ng
TEQPCBs/PCDDs/PCDFs/kg body wt/d) provided by fish collected from the Saginaw River
were greater than those of individuals fed a control diet (Zwiernik et al., 2008). No
differences were observed in mean organ masses of adult female mink fed diets that provided
up to 51 ng TEQPCBs/PCDDs/PCDFs/kg feed (6.4 ng TEQPCBs/PCDDs/PCDFs/kg body wt/d)
derived from fish collected from the Housatonic River relative to those of controls, but sixweek-old female kits in the 51 ng TEQPCBs/PCDDs/PCDFs/kg feed (6.4 ng
TEQPCBs/PCDDs/PCDFs/kg body wt/d) treatment group had greater mean relative brain,
kidney, and liver masses, and 31-week-old male and female juveniles from the same
treatment group had increased relative spleen masses compared to controls (Bursian et al.,
2006b). Zwiernik et al. (2009) reported no effects on organ masses in mink that had been
exposed from conception through 72 weeks of age to 24 and 240 ng TEQTCDF/kg feed (3.0
and 30 ng TEQTCDF/kg body wt/d) provided by TCDF.

74

Pathology
Other than mandibular and maxillary squamous epithelial proliferation (Bursian et al.
2012) hepatic vacuolation and mineralization of the kidney, liver, heart and thyroid gland
were the only pathological effects noted in the present study. Hepatic vacuolation and renal
mineralization occurred in juveniles in all treatment groups, including the control group, and
thus, were not considered to be treatment related. These results are similar to those reported
by Bursian et al. (2006b). In contrast, Heaton et al. (1995b) reported that adult female mink
exposed to PCBs/PCDDs/PCDFs at dietary concentrations ranging from 17 to 66 ng
TEQPCBs/PCDDs/PCDFs/kg feed (2.1 to 8.3 ng TEQPCBs/PCDDs/PCDFs/kg body wt/d)
through dietary inclusion of fish collected from Saginaw Bay for a time period equivalent to
that in the present study had enlarged and diffusely yellow livers. Histologically, the livers
had various degrees of congestion, hepatocellular fatty change, and scattered aggregates of
lymphocytes. Mineralization of the liver, thyroid gland, and heart in animals exposed to
TCDF in the present study appeared to be related to dose. There are no reports in the
literature of soft tissue mineralization induced by TCDD-like chemicals.

Hepatic and adipose TCDD/PeCDF/TCDF concentrations
Concentrations of TCDD, PeCDF and TCDF in liver and adipose increased with
dose. Bioaccumulation factors suggested that TCDD and PeCDF bioaccumulated in both
liver and adipose tissue, although to different degrees, and that TCDF was rapidly eliminated
from the animal. Results from a toxicokinetic study of PeCDF and TCDF in mink (Zwiernik
et al., 2008b, Moore et al., 2009) are similar to those reported here, in that PeCDF
accumulated in the liver of the mink to a much greater extent than did TCDF. This suggested
75

hepatic sequestration of PeCDF, perhaps by binding of the congener to hepatic CYP1A2
protein, which has been shown to occur in rodents (Brewster et al., 1987, Brewster et al.,
1988, DeVito et al., 1997, Diliberto et al., 1999). The lesser concentrations of TCDF in
livers of mink suggested an efficient elimination and/or metabolism of the congener. The
half-life of PeCDF was estimated to be approximately 8 d, whereas the half-life of TCDF
was less than 0.5 d in mink (Fur Commision U.S.A., 2003). These values are less than those
reported for rodents. The half-life of TCDF is approximately 2 d in mice (Brewster et al.,
1987), and the half-life of PeCDF in the rat is more than 60 d (Brewster et al., 1987).

Conclusions
Results of the present study indicate that TEQ concentrations provided by TCDD,
PeCDF or TCDF, which were expected to result in complete reproductive failure in mink
based on studies using environmentally derived mixtures of TCDD-like chemicals with
calculated sum of TEQ, had no significant effects on reproductive performance of adult
female mink or growth and viability of their offspring through 27 weeks of age.
Additionally, minimal and, in some cases, inconsistent effects were seen on more subtle
individual health endpoints, including organ masses and morphology. Hepatic and adipose
concentrations of the three congeners suggested that PeCDF is preferentially sequestered in
the liver more so than in adipose tissue relative to TCDD and that TCDF is rapidly
eliminated from the animal. Although the results of the present study are insufficient to
calculate the relative potency of PeCDF and TCDF to TCDD for reproductive endpoints,
histological data presented in Bursian et al. (2012) as well as comparisons with parallel
studies suggest that the current TEF values may not accurately predict the toxic potency of

76

TCDD, PeCDF and TCDF as compared with PCB 126 or environmental contaminant
mixtures composed largely of PCB 126.

ACKNOWLEDGEMENT
This research received financial support from an unrestricted grant from The Dow Chemical
Company.

77

APPENDIX

78

SUPPLEMENTAL DATA
a,b

Table S1. Effects of TCDD, PeCDF, and TCDF on adult female mink absolute organ mass (g) .
Dose
(ng/kg
Dietary
Thyroid
body
c
Liver
treatment
wt/d)
n
gland
Thymus
Heart
Spleen
Control
0
9
41.17
0.063
0.52
6.72
1.95
3.12
0.011
0.17
0.56
0.54
TCDD
2.1
8
51.86 A
0.088
0.95
8.33
2.27
3.31
0.011
0.18
0.60
0.58
4.6
9
50.08 A
0.094
0.78
6.56
3.25
3.12
0.011
0.17
0.56
0.54
6.0
8
47.96
0.070
0.69
7.91
2.19
3.31
0.011
0.18
0.60
0.58
8.4
9
48.23
0.074
0.50
7.67
3.04
3.31
0.011
0.18
0.60
0.58
PeCDF
13
9
42.95
0.069
0.58
8.36 A
3.59 A
3.12
0.011
0.17
0.56
0.54
25
9
45.59
0.121 A
0.68
8.17
2.63
3.12
0.011
0.17
0.56
0.54
30
9
51.89 A
0.084
0.96
7.57
3.31
3.12
0.011
0.17
0.56
0.54
49
8
53.07 A
0.084
0.60
7.71
3.26
3.31
0.012
0.18
0.60
0.58

79

Table S1. Cont’d.

TCDF

52

9

116

9

214

9

246

9

Control vs Congener
p-value TCDD
p-value PeCDF
p-value TCDF

50.93 A
3.12
45.69
3.12
55.01 A
3.12
53.11 A
2.96

0.093
0.011
0.060
0.011
0.089
0.011
0.079
0.011

1.14
0.17
0.44
0.17
0.80
0.17
0.56
0.16

8.02
0.56
7.17
0.56
8.97
0.56
7.03
0.53

3.06
0.54
2.81
0.54
3.76 A
0.54
3.23
0.52

0.0190
0.0420
0.0050

0.1566
0.0363
0.1754

0.2731
0.3208
0.2552

0.1618
0.0542
0.0897

0.2303
0.0439
0.0401

80

Table S1. Cont’d.

2.1

8
9
8

8.4
PeCDF

n
9

6.0

TCDD

Dose
(ng/kg
bw/d)
0

4.6

Dietary
treatment
Control

9

13

9

25

9

30

9

49

8

Adrenal
glands
0.161
0.030
0.171
0.031
0.167
0.030
0.176
0.031
0.162
0.031
0.160
0.030
0.202
0.030
0.209
0.030
0.062
0.031

Kidneys
8.14
0.34
8.24
0.37
8.40
0.34
9.18
0.37
7.67
0.39
7.77
0.34
8.00
0.34
8.28
0.34
8.72
0.37

81

Lymph
Node
0.47
0.11
0.78 A
0.11
0.79 A
0.11
0.65
0.11
0.64
0.11
0.61
0.11
0.69
0.11
0.81 A
0.11
0.76
0.11

Brain
7.86
0.21
7.62
0.23
7.22
0.21
7.99
0.23
7.74
0.23
8.06
0.21
7.71
0.21
7.86
0.21
8.03
0.23

Reproductive
Tract
0.87
0.18
1.00
0.17
1.22
0.16
1.11
0.17
1.07
0.18
1.00
0.16
1.32
0.16
1.29
0.17
1.24
0.17

Table S1. Cont’d.

TCDF

52

9

116

9

214

9

246

9

Control vs Congener
p-value TCDD
p-value PeCDF
p-value TCDF

0.270
0.030
0.174
0.030
0.190
0.030
0.180
0.028

8.46
0.34
8.33
0.34
9.03
0.34
7.90
0.33

0.93 A
0.11
0.61
0.11
0.72
0.11
0.61
0.10

7.76
0.21
7.61
0.21
7.87
0.21
7.74
0.20

1.34
0.16
1.05
0.16
1.13
0.16
1.20
0.15

0.8225
0.5141
0.2069

0.5526
0.8917
0.4500

0.0461
0.0436
0.0405

0.3641
0.8252
0.6191

0.1436
1.0000
0.1171

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8-pentachlorodibenzofuran; and
TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Data are presented as least squares mean with standard error
beneath.
c
Means that are significantly different than the control mean at p < 0.05 are designated with an A.

82

a

Table S2. Effects of TCDD, TCDF, and PeCDF on adult female mink relative organ mass (% of body mass).
Dose
Body mass
(ng/kg
b
necropsy
Dietary
body
treatment wt/d) n
(g)
Control
0
9
788
70
TCDD
2.1
8
931
74
4.6
9
968
70
6.0
8
949
74
8.4
9
877
70
PeCDF
13
9
860
70
25
9
969
70
30
9
1028 A
70
49
8
1037 A
74

c,d

Liver
5.25
4.71-5.80
5.65
4.95-6.34
5.29
4.76-5.83
5.13
4.42-5.83
5.61
4.88-6.18
5.08
4.52-5.64
4.87
4.03-5.70
5.18
4.41-5.96
5.15
4.64-5.66

Thyroid
gland
0.008
0.005-0.012
0.009
0.007-0.012
0.011
0.007-0.014
0.007
0.006-0.009
0.008
0.006-0.010
0.008
0.007-0.010
0.014
0.004-0.025
0.009
0.006-0.012
0.008
0.007-0.010

83

Thymus
0.06
0.03-0.09
0.09
0.04-0.14
0.07
0.05-0.10
0.07
0.04-0.10
0.06
0.04-0.08
0.06
0.04-0.09
0.07
0.04-0.09
0.09
0.06-0.11
0.06
0.05-0.07

Heart
0.86
0.77-0.94
0.90
0.79-1.01
0.73
0.53-0.92
0.84
0.76-0.93
0.89
0.66-1.17
0.98
0.81-1.16
0.85
0.73-0.97
0.77
0.66-0.88
0.76
0.64-0.88

Spleen
0.25
0.22-0.28
0.24
0.22-0.26
0.33
0.26-0.40
0.23
0.18-0.28
0.34 A
0.30-0.42
0.42
0.04-0.80
0.27
0.23-0.31
0.32
0.24-0.40
0.30
0.22-0.39

Table S2. Cont’d.

TCDF

52

9

116

9

214

9

246

9

Control vs Congener
p-value TCDD
p-value PeCDF
p-value TCDF

1052
70
841
70
947
70
877
70

4.96
4.16-5.76
5.58
4.61-6.54
5.99
4.92-7.05
6.16
5.53-6.79

0.009
0.007-0.010
0.008
0.005-0.010
0.010
0.007-0.012
0.010
0.007-0.013

0.10
0.05-0.14
0.05
0.04-0.07
0.08
0.05-0.11
0.06
0.05-0.08

0.78
0.64-0.91
0.88
0.72-1.03
0.96
0.84-1.08
0.82
0.73-0.90

0.30
0.24-0.36
0.35
0.14-0.55
0.41
0.26-0.56
0.34
0.26-0.43

0.0703
0.0196
0.0728

0.6617
0.8683
0.1050

0.2572
0.3180
0.3669

0.5972
0.3850
0.2343

0.2701
0.0479
0.1547

0.0004
0.6408
0.2455

84

Table S2. Cont’d.

Dose
Body mass
(ng/kg
b
necropsy
Dietary
body
(g)
treatment wt/d) n
Control
0
9
788
70
TCDD
2.1
8
931
74
4.6
9
968
70
6.0
8
949
74
8.4
9
877
70
PeCDF
13
9
860
70
25
9
969
70
30
9
1028 A
70
49
8
1037 A
74

Adrenal
glands
0.021
0.016-0.027
0.018
0.014-0.023
0.019
0.013-0.025
0.019
0.014-0.024
0.019
0.013-0.024
0.020
0.014-0.025
0.022
0.015-0.030
0.021
0.018-0.025
0.017
0.012-0.022

Kidneys
1.06
0.92-1.20
0.91
0.76-1.05
0.91
0.74-1.09
0.99
0.83-1.15
0.86
0.73-1.00
0.92
0.81-1.03
0.86
0.70-1.02
0.85
0.70-1.00
0.87
0.76-0.97

85

Lymph Node
0.06
0.04-0.07
0.08
0.06-0.10
0.08
0.06-0.10
0.06
0.04-0.09
0.07
0.05-0.08
0.07
0.05-0.08
0.07
0.06-0.08
0.07
0.06-0.09
0.07
0.05-0.09

Brain
1.03
0.87-1.19
0.85
0.70-1.00
0.81
0.57-1.05
0.86
0.71-1.01
0.91
0.75-1.03
0.96
0.83-1.10
0.83
0.68-0.98
0.82
0.64-1.00
0.81
0.66-0.97

Reproductive
Tract
0.12
0.10-0.13
0.13
0.11-0.15
0.12
0.10-0.14
0.12
0.10-0.13
0.12
0.10-0.14
0.11
0.09-0.13
0.13
0.10-0.16
0.13
0.02-0.14
0.11
0.08-0.15

Table S2. Cont’d.

TCDF

52

9

116

9

214

9

246

9

Control vs Congener
p-value TCDD
p-value PeCDF
p-value TCDF

1052
70
841
70
947
70
877
70

0.026
0.005-0.047
0.021
0.017-0.026
0.020
0.017-0.023
0.022
0.016-0.028

0.84
0.71-0.97
1.02
0.83-1.21
0.98
0.82-1.15
0.93
0.81-1.06

0.09
0.07-0.11
0.07
0.06-0.08
0.08
0.05-0.10
0.07
0.06-0.08

0.77
0.65-0.89
0.93
0.82-1.04
0.85
0.75-0.95
0.96
0.76-1.15

0.13
0.09-0.16
0.12
0.10-0.14
0.12
0.10-0.13
0.13
0.10-0.16

0.0703
0.0196
0.0728

0.8862
0.5203
0.9907

0.2381
0.0766
0.1549

0.2169
0.4772
0.2012

0.2269
0.0842
0.0621

0.7519
0.5409
0.8898

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8-pentachlorodibenzofuran;
and TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Body mass data are presented as least squares mean with standard error beneath.
c

Relative organ mass data are presented as mean with 95% confidence interval beneath.

d

Means that are significantly different than the control mean at p < 0.05 are designated with an A.

86

Table S3. Effects of TCDD, PeCDF, and TCDF on female mink kit absolute organ mass (g) at six weeks of
a,b
age .
Dietary
treatment
Control
TCDD

PeCDF

Dose
(ng/kg
bw/d) n
0
5
2.1
4.6
6.0

0
0
6

8.4
13

1
4

25
30
49

0
0
1

Liver
16.70
4.90
----16.16
3.52
11.03
18.43
4.92
----2.76

Thyroid
gland Thymus
0.044
0.491
0.043
0.163
--------0.032
0.457
0.039
0.133
0.011
0.114
0.125
0.616
0.048
0.172
--------0.028
0.065

Heart
2.41
0.49
----1.94
0.35
1.35
2.49
0.50
----0.62

87

Spleen
0.97
0.55
----1.82
0.41
0.90
1.53
0.56
----0.21

Adrenal
glands Kidneys
0.108
3.33
0.039
0.66
--------0.070
3.03
0.036
0.48
0.084
2.72
0.174
3.31
0.044
0.67
--------0.088
1.13

Lymph
Node
0.230
0.072
----0.224
0.066
0.119
0.415
0.080
----0.057

Brain
8.33
0.55
----7.60
0.40
7.45
8.44
0.55
----5.13

Table S3. Cont’d.

TCDF

52
116

0
2

214 1
246 1
Control vs Congener
p-value TCDD
p-value PeCDF
p-value TCDF

--12.10
6.10
9.43
20.75

--0.026
0.067
0.031
0.047

--0.123
0.231
0.229
0.234

--1.46
0.61
1.11
1.98

--1.60
0.71
0.73
1.37

--0.049
0.062
0.069
0.058

--1.96
0.83
1.87
2.83

--0.161
0.114
0.246
0.235

--7.29
0.69
8.26
7.67

0.4914
0.3739
0.5257

0.7620
0.6653
0.8966

0.3837
0.5143
0.2528

0.2476
0.2589
0.1991

0.7604
0.8610
0.8712

0.6435
0.7427
0.4727

0.5009
0.2776
0.2299

0.6345
0.9684
0.8882

0.3189
0.1541
0.4278

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8 pentachlorodibenzofuran;
and TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Data are presented as least squares mean with standard error beneath.

88

Table S4. Effects of TCDD, PeCDF, and TCDF on female mink kit relative organ mass (% of body mass) at
a
six weeks of age .

Dietray
treatment
Control
TCDD

PeCDF

Dose
(ng/kg
body
wt/d)
0

n
5

2.1
4.6
6.0

0
0
6

8.4
13

1
4

25
30
49

0
0
1

Body mass
b
necropsy
(g)
246
63
----213
37
146
254
63
----51

Thyroid
gland
0.021
0.014-0.028
----0.017
0.004-0.030
0.008
0.07
0-0.265
----0.055

c,d

Liver
7.36
6.03-8.70
----7.55
5.87-9.22
7.55
7.98
6.74-9.21
----5.44

89

Thymus
0.20
0.06-0.34
----0.21
0.12-0.29
0.08
0.28
0.08-0.05
----0.13

Heart
1.12
0.97-1.27
----0.91 A
0.80-1.02
0.93
1.16
0.83-1.49
----1.23

Spleen
0.40
0.15-0.66
----0.80 A
0.57-1.03
0.61 A
0.63
0.32-0.94
----0.41

Table S4. Cont’d.

TCDF

52
116

0
2

214
246

1
1

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

--140
56
152
188

--8.50
0-17.33
6.20
11.03

--0.020
0-0.115
0.020
0.025

--0.09
0-0.55
0.15
0.12

--1.02
0-2.42
0.73
1.05

--1.09 A
0-4.31
0.48
0.73

0.6420
0.3398
0.5535

0.9929
0.2004
0.1845

0.6457
0.7254
0.9009

0.3952
0.4616
0.6678

0.0509
0.8035
0.2136

0.0375
0.3325
0.1745

90

Table S4. Cont’d.

Dietray
treatment
Control
TCDD

PeCDF

Dose
(ng/kg
body
wt/d)
0

n
5

2.1
4.6
6.0

0
0
6

8.4
13

1
4

25
30
49

0
0
1

Body mass
b
necropsy
(g)
246
63
----213
37
146
254
63
----51

Adrenal
glands
0.055
0.013-0.096
----0.033
0.027-0.039
0.058
0.100
0-0.275
----0.173

Kidneys
1.53
1.37-1.69
----1.45
1.25-1.64
1.86
1.54
1.08-2.01
----2.22

91

Lymph
Node
0.10
0.05-0.15
----0.10
0.08-0.13
0.08
0.20
0-0.44
----0.11

Brain
4.07
2.73-5.41
----4.06
2.47-5.66
5.10
4.19
2.11-6.27
----10.10 A

Table S4. Cont’d.

TCDF

52
116

0
2

214
246

1
1

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

--140
56
152
188

--0.035
0-0.035
0.045
0.031

--1.39
1.16-1.62
1.23
1.50

--0.11
0-0.32
0.16
0.12

--5.31
0-12.80
5.43
4.08

0.6420
0.3398
0.5535

0.2747
0.4403
0.8143

0.1677
0.1132
0.3871

0.8517
0.4047
0.6064

0.7158
0.0558
0.4976

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8-pentachlorodibenzofuran,
and TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Body mass data are presented as least squares mean with standard error beneath.
c

Relative organ mass data are presented as mean with 95% confidence interval beneath.

d

Means that are significantly different than the control mean at p < 0.05 are designated with an A.

92

a,b

Table S5. Effects of TCDD, PeCDF, and TCDF on male mink kit absolute organ mass (g) at six weeks of age

PeCDF

2.1
4.6

1
2
2

8.4

TCDD

n
8

6.0

Dietary
treatment
Control

Dose
(ng/kg
body
wt/d)
0

8

13

5

25
30

0
3

49

0

c

Liver
15.58
2.93
14.87
23.45
5.85
15.42
5.85
13.78
2.93
18.68
3.70
--22.18
4.78
---

Thyroid
gland
0.041
0.007
0.051
0.038
0.012
0.053
0.017
0.039
0.007
0.032
0.008
--0.033
0.008
---

Thymus
0.282
0.120
0.367
0.603
0.196
0.485
0.196
0.283
0.120
0.430
0.136
--0.561
0.160
---

Heart
2.22
0.35
1.76
2.53
0.71
1.84
0.71
1.70
0.35
2.50
0.45
--2.78
0.58
---

93

Spleen
0.88
0.30
0.95
2.22
0.52
1.11
0.52
1.26
0.30
1.34
0.35
--2.66
0.42
---

Adrenal
Lymph
glands Kidneys Node
0.081
2.93
0.227
0.012
0.62
0.053
0.061
2.73
0.177
0.107
4.01
0.361
0.024
1.23
0.107
0.095
3.57
0.247
0.024
1.23
0.107
0.067
2.80
0.204
0.012
0.62
0.053
0.084
3.39
0.293
0.015
0.78
0.067
------0.076
3.69
0.340
0.019
1.01
0.087
-------

.

Brain
9.08
0.44
8.18
8.57
0.75
8.84
1.05
8.21
0.44
9.21
0.51
--9.63
0.61
---

Table S5. Cont’d.

TCDF

52
116
214
246

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

7 26.95 A
3.13
5 27.31 A
3.70
4 20.20
4.14
4 20.28
4.14

0.067
0.007
0.043
0.008
0.054
0.008
0.050
0.008

0.792
0.121
0.513
0.136
0.279
0.138
0.411
0.138

3.12
0.38
3.13
0.45
2.47
0.50
2.41
0.50

1.88
0.31
1.78
0.35
1.58
0.37
1.30
0.37

0.095
0.013
0.116
0.015
0.086
0.017
0.065
0.017

5.54
0.66
4.47
0.78
3.67
0.87
3.32
0.87

0.387
0.057
0.461
0.067
0.289
0.075
0.256
0.075

10.25
0.46
9.53
0.51
8.23
0.53
9.61
0.53

0.7631
0.2756
0.0425

0.7311
0.3879
0.1156

0.4439
0.3034
0.1485

0.6087
0.4305
0.2140

0.2742
0.1005
0.0734

0.9120
0.9909
0.5403

0.7188
0.5099
0.1056

0.7961
0.2683
0.0846

0.3519
0.6262
0.7175

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8-pentachlorodibenzofuran;
and TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Data are presented as least squares means with standard error beneath.
c

Means that are significantly different than the control mean at p < 0.05 are designated with an A.

94

Table S6. Effects of TCDD, PeCDF, and TCDF on male mink kit relative organ mass (% of body mass) at
a
six weeks of age .
Body
Dose
mass
(ng/kg
b
necropsy
Dietary
body
Thyroid
c
Liver
treatment
(g)
wt/d)
n
gland
Thymus
Heart
Spleen
Control
0
8
208
7.72
0.021
0.14
1.13
0.43
25
6.73-8.71
0.017-0.025
0.10-0.18
0.99-1.28
0.29-0.56
TCDD
2.1
1
186
8.00
0.027
0.20
0.95
0.51
4.6
2
253
9.46
0.015
0.21
1.02
0.88
42
1.17-17.75
0-0.036
0-1.40
0.11-1.93
0.84-0.92
6.0
2
174
9.23
0.022
0.26
1.16
0.63
42
0-21.01
--0-0.97
0-4.44
0.21-1.04
8.4
8
191
7.26
0.017
0.15
0.96
0.65
25
6.37-8.16
0.013-0.021
0.09-0.21
0.71-1.21
0.45-0.84
241
7.42
0.013
0.17
0.98
0.52
PeCDF
13
5
27
6.24-8.59
0.006-0.020
0.13-0.22
0.75-1.21
0.27-0.76
25
0
------------30
3
279
7.88
0.012
0.20
1.01
0.77
31
5.73-10.04
0.007-0.017
0.13-0.27
0.74-1.28
0.30-1.24
49
0
-------------

95

Table S6. Cont’d.

TCDF

52

7

116

5

214

4

246

4

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

312
33
309
39
217
43
225
43

8.46
7.39-9.54
8.66
6.93-10.39
8.95
5.80-12.10
9.09
8.03-10.14

0.022
0.019-0.025
0.014
0.011-0.016
0.030
0-0.070
0.022
0.012-0.032

0.27
0.09-0.46
0.16
0.08-0.23
0.14
0.05-0.23
0.17
0.07-0.27

0.97
0.76-1.18
1.00
0.83-1.18
1.10
0.44-1.76
1.07
0.96-1.18

0.61
0.45-0.79
0.58
0.45-0.71
0.66
0-1.38
0.58
0.55-0.62

0.6869
0.2807
0.0983

0.1359
0.8102
0.3892

0.2330
0.0868
0.2418

0.4967
0.1524
0.0938

0.5789
0.5704
0.6835

0.1538
0.1348
0.5371

96

Table S6. Cont’d.

PeCDF

2.1
4.6

1
2
2

8.4

TCDD

n
8

6.0

Dietary
treatment
Control

Dose
(ng/kg
body
wt/d)
0

8

13

5

25
30

0
3

49

0

Body
mass
necropsy
(g)
208
25
186
253
42
174
42
191
25
241
27
--279
31
---

Adrenal
glands
0.042
0.031-0.053
0.032
0.040
0-0.155
0.052
0-0.130
0.038
0.032-0.044
0.034
0.022-0.047
--0.027
0-0.060
---

Kidneys
1.48
1.31-1.65
1.47
1.66
0-5.12
2.51
0-17.16
1.51
1.38-1.64
1.34
1.14-1.54
--1.33
1.15-1.50
---

97

Lymph
Node
0.11
0.09-0.14
0.10
0.14
0.00-0.28
0.15
0.03-0.26
0.10
0.07-0.14
0.12
0.10-0.14
--0.12
0.05-0.18
---

Brain
4.66
3.99-5.34
4.40
3.63
0-14.68
3.63
--4.80
3.61-5.99
3.80
2.72-4.87
--3.58
1.74-5.43
---

Table S6. Cont’d.

TCDF

52

7

116

5

214

4

246

4

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

312
33
309
39
217
43
225
43

0.031
0.026-0.036
0.038
0.021-0.055
0.046
0-0.096
0.029
0.024-0.034

1.72
1.19-2.26
1.48
1.20-1.75
1.67
1.34-2.00
1.46
1.17-1.76

0.12
0.08-0.15
0.14
0.10-0.18
0.14
0.12-0.16
0.11
0.07-0.15

3.63
2.59-4.67
3.24
2.46-4.02
4.06
2.52-5.59
4.51
2.42-6.59

0.6869
0.2807
0.0983

0.5331
0.3063
0.4373

0.2728
0.4595
0.6485

0.4901
0.9486
0.3460

0.7259
0.4007
0.1939

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8-pentachlorodibenzofuran;
and TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Body mass data are presented as least squares mean with standard error beneath.
c

Relative organ mass data are presented as mean with 95% confidence interval beneath.

98

Table S7. Effects of TCDD, PeCDF, and TCDF on juvenile female mink absolute organ mass (g) at 27
a,b
weeks of age .

7
8
16

8.4
PeCDF

2.1

6.0

TCDD

n
17

4.6

Dietary
treatment
Control

Dose
(ng/kg
body
wt/d)
0

7

13

13

25

7

30

8

49

1

Liver
58.66
2.45
54.63
3.80
58.74
3.53
58.69
2.56
56.31
3.67
58.04
2.74
56.00
3.84
61.42
3.46
56.37

Thyroid
glands
0.080
0.021
0.084
0.033
0.081
0.031
0.091
0.022
0.080
0.033
0.086
0.024
0.083
0.033
0.206
0.031
0.076

99

c

Thymus
1.210
0.084
0.807 A
0.134
0.903 A
0.123
0.702 A
0.092
0.753 A
0.126
1.060
0.096
0.581 A
0.136
0.758 A
0.120
0.582 A

Heart
8.27
0.49
8.35
0.78
8.75
0.71
9.35
0.54
8.05
0.72
8.64
0.56
8.84
0.79
7.93
0.69
7.39

Spleen
3.21
0.19
2.38
0.31
3.23
0.29
3.04
0.21
3.32
0.30
2.53
0.22
2.64
0.31
3.38
0.28
3.40

Table S7. Cont’d.

TCDF

52

4

116

12

214

8

246

9

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

51.93
4.68
59.72
2.97
55.88
3.40
58.62
3.32

0.088
0.044
0.077
0.025
0.094
0.031
0.070
0.029

0.584 A
0.155
0.871 A
0.106
0.755 A
0.115
0.745 A
0.117

9.09
0.88
7.92
0.62
8.59
0.66
8.29
0.68

2.26
0.38
3.58
0.24
3.12
0.28
3.62
0.27

0.4490
0.7644
0.4346

0.8811
0.3320
0.9445

< 0.0001
0.0002
< 0.0001

0.9126
0.6483
0.9519

0.2911
0.3936
0.7851

100

Table S7. Cont’d.

7
8
16

8.4
PeCDF

2.1

6.0

TCDD

n
17

4.6

Dietary
treatment
Control

Dose
(ng/kg
body
wt/d)
0

7

13

13

25

7

30

8

49

1

Adrenal
glands
0.293
0.025
0.353
0.039
0.287
0.036
0.292
0.026
0.366
0.039
0.306
0.028
0.358
0.039
0.325
0.036
0.270

Kidneys
8.20
0.31
7.99
0.49
7.89
0.44
7.95
0.33
7.85
0.45
8.04
0.35
7.90
0.49
7.92
0.43
8.30

101

Lymph Node
2.10
0.18
1.13 A
0.30
1.44 A
0.27
1.19 A
0.20
1.50 A
0.28
1.57 A
0.21
1.26 A
0.30
1.79
0.26
1.76

Brain
8.65
0.17
8.54
0.26
8.75
0.24
8.40
0.18
8.21
0.25
8.36
0.19
8.57
0.27
8.11
0.24
8.24

Reproductive
Tract
1.82
0.11
1.41
0.18
1.67
0.17
1.39 A
0.12
1.54
0.18
1.70
0.13
1.52
0.18
1.63
0.17
1.57

Table S7. Cont’d.

TCDF

52

4

116

12

214

8

246

9

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

0.281
0.051
0.355
0.030
0.306
0.036
0.271
0.034

7.88
0.55
8.72
0.38
8.17
0.41
7.28
0.42

1.18 A
0.34
1.45 A
0.23
1.37 A
0.26
1.47 A
0.26

9.14
0.31
8.21
0.21
8.28
0.23
8.21
0.23

1.33
0.24
1.63
0.14
1.32 A
0.17
1.61
0.16

0.3089
0.5775
0.7611

0.6358
0.9506
0.9911

0.0003
0.0252
0.0008

0.3103
0.1970
0.3421

0.0269
0.2229
0.0198

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8-pentachlorodibenzofuran;
and TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Data are presented as least squares mean with standard error beneath.
c

Means that are significantly different than the control mean at p < 0.05 are designated with an A.

102

Table S8. Effects of TCDD, PeCDF, and TCDF on juvenile female mink relative organ mass (% of body mass) at 27
a
weeks of age .

Dietary
treatment
Control
TCDD

Dose
(ng/kg
body
wt/d)
0
2.1
4.6
6.0
8.4

PeCDF

13
25
30
49

Body
mass
b
necropsy
(g)
n
17
1164
54
7
999
87
8
990
78
16
1013
60
7
952
79
13
1123
63
7
938
90
8
1043
79
1
967

c,d

Liver
5.13
4.84-5.42
5.44
4.84-6.04
5.66
5.35-5.98
5.76
5.26-6.26
5.95
5.16-6.73
5.21
4.82-5.60
6.20
4.26-8.15
5.87
5.32-6.42
5.83

Thyroid
gland
0.007
0.006-0.009
0.008
0.006-0.011
0.008
0.006-0.010
0.009
0.006-0.011
0.008
0.007-0.010
0.008
0.007-0.009
0.010
0.004-0.015
0.019
0-0.042
0.008

103

Thymus
0.11
0.09-0.12
0.08
0.06-0.11
0.09
0.06-0.11
0.07 A
0.06-0.08
0.08
0.06-0.10
0.09
0.08-0.11
0.07
0.04-0.09
0.07
0.05-0.09
0.06

Heart
0.71
0.65-0.77
0.83
0.70-0.97
0.85
0.77-0.93
0.92 A
0.87-0.96
0.86
0.72-0.99
0.78
0.72-0.84
0.96 A
0.74-1.18
0.76
0.68-0.84
0.76

Spleen
0.28
0.24-0.31
0.24
0.20-0.28
0.31
0.24-0.39
0.30
0.26-0.33
0.35
0.32-0.38
0.23
0.02-0.26
0.30
0.17-0.42
0.32
0.27-0.36
0.35

Table S8. Cont’d.

TCDF

52

4

116

12

214

8

246

9

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

1025
80
1144
58
1019
61
1035
64

5.05
4.58-5.51
5.26
4.95-5.57
5.49
5.09-5.89
5.67
5.18-6.16

0.008
0.005-0.012
0.007
0.005-0.008
0.009
0.007-0.012
0.007
0.005-0.009

0.06 A
0.01-0.11
0.08
0.06-0.09
0.07
0.06-0.09
0.07
0.06-0.09

0.89
0.63-1.15
0.70
0.62-0.78
0.84
0.72-0.96
0.80
0.70-0.89

0.22
0.12-0.32
0.32
0.27-0.36
0.31
0.27-0.34
0.35
0.30-0.40

0.1609
0.2672
0.2256

0.0833
0.1689
0.1712

0.7000
0.2096
0.2983

0.0287
0.0784
0.0167

0.0203
0.0370
0.0778

0.0602
0.0754
0.0248

104

Table S8. Cont’d.

Dietary
treatment
Control
TCDD

Dose
(ng/kg
body
wt/d)
0
2.1
4.6
6.0
8.4

PeCDF

13
25
30
49

Body
mass
b
necropsy
(g)
n
17
1164
54
7
999
87
8
990
78
16
1013
60
7
952
79
1123
13
63
7
938
90
8
1043
79
1
967

Adrenal
glands
0.026
0.021-0.030
0.035
0.024-0.047
0.028
0.020-0.036
0.029
0.025-0.032
0.038 A
0.033-0.043
0.027
0.020-0.034
0.039
0.025-0.053
0.032
0.022-0.042
0.028

Kidneys
0.69
0.66-0.73
0.80
0.72-0.89
0.76
0.71-0.82
0.79
0.72-0.87
0.83
0.71-0.95
0.73
0.68-0.77
0.87
0.61-1.14
0.76
0.71-0.80
0.86

105

Lymph Node
0.18
1.14-0.22
0.11
0.08-0.14
0.14
0.11-0.17
0.11 A
0.10-0.13
0.16
0.08-0.24
0.14
0.11-0.17
0.14
0.08-0.20
0.17
0.13-0.20
0.18

Brain
0.76
0.70-0.83
0.86
0.76-0.96
0.85
0.79-0.91
0.85
0.74-0.96
0.88
0.74-1.01
0.76
0.69-0.83
0.95
0.66-1.24
0.79
0.64-0.94
0.85

Reproductive
Tract
0.16
0.14-0.18
0.14
0.11-0.17
0.16
0.14-0.19
0.13
0.12-0.15
0.16
0.13-0.19
0.15
0.14-0.16
0.16
0.11-0.21
0.15
0.10-0.20
0.16

Table S8. Cont’d.

TCDF

52

4

116

12

214

8

246

9

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

1025
80
1144
58
1019
61
1035
64

0.027
0.018-0.037
0.031
0.026-0.037
0.030
0.021-0.039
0.027
0.021-0.032

0.78
0.52-1.04
0.77
0.72-0.81
0.81
0.74-0.88
0.71
0.65-0.77

0.11
0.07-0.16
0.13
0.11-0.15
0.13
0.09-0.17
0.14
0.11-0.18

0.90
1.67-1.13
0.73
0.68-0.78
0.82
0.74-0.89
0.80
0.74-0.85

0.13
0.06-0.20
0.14
0.12-0.16
0.13
0.11-0.15
0.16
0.11-0.21

0.1609
0.2672
0.2256

0.0528
0.1341
0.5388

0.0685
0.1457
0.1443

0.0549
0.4172
0.1754

0.3792
0.3862
0.1443

0.1205
0.9578
0.5977

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8-pentachlorodibenzofuran;
and TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Body mass data are presented as least squares mean with standard error beneath.
c

Relative organ mass data are presented as mean with 95% confidence interval beneath.

d

Means that are significantly different than the control mean at p < 0.05 are designated with an A.

106

Table S9. Effects of TCDD, TCDF, and PeCDF on juvenile male mink absolute organ mass (g) at 27
a,b
weeks of age .

7
6
6

8.4
PeCDF

2.1

6.0

TCDD

n
12

4.6

Dietary
treatment
Control

Dose
(ng/kg
body
wt/d)
0

9

13

10

25

3

30

7

49

8

c

Liver
76.85
3.35
69.54
4.27
71.30
4.60
62.43 A
4.60
70.04
3.95
68.91
3.52
69.16
6.29
85.77
4.18
70.94
4.03

Thyroid
gland
0.094
0.011
0.089
0.015
0.086
0.016
0.074
0.016
0.065
0.013
0.063
0.012
0.098
0.022
0.091
0.015
0.087
0.014

107

Thymus
0.768
0.097
0.718
0.125
0.668
0.135
0.476
0.135
0.788
0.110
0.661
0.104
0.605
0.188
0.885
0.124
0.635
0.117

Heart
12.61
0.71
10.95
0.90
11.47
0.96
11.41
0.96
10.52
0.77
12.00
0.73
11.92
1.29
12.61
0.86
10.00
0.85

Spleen
2.86
0.68
2.43
0.87
2.92
0.88
1.90
0.88
3.19
0.68
2.55
0.63
6.64
0.92
4.07
0.70
3.22
0.86

Table S9. Cont’d.

TCDF

52

9

116

12

214

10

246

9

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

68.34
3.75
78.44
3.25
69.25
3.60
75.67
3.69

0.114
0.013
0.104
0.011
0.095
0.012
0.107
0.013

0.569
0.110
1.080
0.095
0.752
0.105
0.757
0.109

12.11
0.78
12.85
0.68
11.45
0.76
10.55
0.76

2.95
0.64
3.22
0.62
3.01
0.75
3.42
0.63

0.0405
0.4424
0.3144

0.2357
0.4881
0.4179

0.3424
0.5204
0.8888

0.0652
0.2114
0.2258

0.7210
0.1363
0.7500

108

Table S9. Cont’d.

7
6
6

8.4
PeCDF

2.1

6.0

TCDD

n
12

4.6

Dietary
treatment
Control

Dose
(ng/kg
body
wt/d)
0

9

13

10

25

3

30

7

49

8

Adrenal
glands
0.32
0.09
0.30
0.12
0.27
0.13
0.26
0.13
0.29
0.10
0.64
0.10
0.34
0.18
0.35
0.12
0.37
0.11

Kidneys
9.81
0.44
10.09
0.55
10.66
0.59
8.69
0.59
9.30
0.47
9.28
0.44
9.25
0.76
10.14
0.52
9.55
0.52

109

Lymph Node
1.56
0.16
1.46
0.20
1.35
0.22
0.73 A
0.22
1.15
0.17
1.33
0.16
0.92
0.29
1.60
0.19
1.20
0.19

Brain
11.02
0.25
10.11 A
0.31
10.67
0.32
10.22
0.32
9.73 A
0.26
10.50
0.24
9.52 A
0.40
10.32
0.28
9.85 A
0.29

Reproductive
Tract
0.89
0.14
0.89
0.17
0.98
0.18
0.60
0.18
0.94
0.14
0.78
0.13
0.63
0.21
0.98
0.15
0.77
0.17

Table S9. Cont’d.

TCDF

52

9

116

12

214

10

246

9

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

0.34
0.10
0.39
0.09
0.30
0.10
0.38
0.10

9.89
0.47
10.64
0.41
9.62
0.47
9.48
0.46

1.34
0.18
1.78
0.15
1.33
0.17
1.74
0.17

10.87
0.26
9.86 A
0.23
9.65 A
0.26
9.71 A
0.25

0.90
0.14
1.42
0.13
0.93
0.15
0.95
0.14

0.7016
0.3517
0.7810

0.7909
0.5690
0.9063

0.0358
0.1028
0.8248

0.0083
0.0022
0.0018

0.8211
0.5412
0.3611

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8-pentachlorodibenzofuran;
and TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Data are presented as least squares mean with standard error beneath.
c

Means that are significantly different than the control mean at p < 0.05 are designated with an A.

110

Table S10. Effects of TCDD, PeCDF, and TCDF on juvenile male mink relative organ mass (% of body mass) at 27
a
weeks of age .

7
6
6

8.4
PeCDF

2.1

6.0

TCDD

n
12

4.6

Dietary
treatment
Control

Dose
(ng/kg
body
wt/d)
0

9

13

10

25

3

30

7

49

8

Body mass
b
necropsy
(g)
1536
79
1355
96
1295
103
1072 A
103
1225
81
1306
71
1093 A
128
1497
85
1058 A
82

c,d

Liver
5.10
4.79-5.42
5.19
4.28-6.11
5.54
4.73-6.36
5.99
5.23-6.75
5.97
5.50-6.45
5.38
4.62-6.15
6.67
1.78-11.56
5.70
5.35-6.06
6.83 A
6.01-7.64

Thyroid
gland
0.006
0.005-0.008
0.007
0.006-0.007
0.007
0.004-0.009
0.007
0.005-0.009
0.006
0.004-0.007
0.005
0.003-0.006
0.009
0.002-0.017
0.006
0.004-0.008
0.008
0.007-0.009

111

Thymus
0.05
0.03-0.07
0.05
0.04-0.07
0.05
0.02-0.08
0.04
0.03-0.05
0.06
0.05-0.07
0.05
0.04-0.06
0.05
0.02-0.08
0.06
0.04-0.08
0.06
0.04-0.08

Heart
0.86
0.73-0.99
0.81
0.69-0.93
0.89
0.72-1.05
1.09
0.89-1.28
0.87
0.79-0.95
0.92
0.83-1.00
1.17
0.02-2.32
0.85
0.67-1.02
0.98
0.80-1.16

Spleen
0.19
0.16-0.21
0.18
0.16-0.20
0.22
0.20-0.25
0.18
0.16-0.20
0.26 A
0.21-0.31
0.19
0.17-0.22
0.78
0-3.25
0.27
0.23-0.32
0.31
0.25-0.38

Table S10. Cont’d.

TCDF

52

9

116

12

214

10

246

9

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

1357
90
1574
79
1323
90
1302
87

4.99
4.50-5.48
4.98
1.77-5.19
5.31
4.87-5.75
5.85 A
5.48-6.22

0.008
0.004-0.012
0.007
0.005-0.008
0.008
0.005-0.011
0.008
0.006-0.010

0.04
0.03-0.05
0.07
0.05-0.08
0.06
0.04-0.07
0.06
0.05-0.07

0.89
0.78-0.99
0.82
0.71-0.92
0.88
0.79-0.98
0.82
0.71-0.94

0.21
0.14-0.28
0.20
0.17-0.24
0.24
0.19-0.28
0.27
0.24-0.29

0.0287
0.0963
0.0021

0.0757
0.0156
0.0192

0.5700
0.5088
0.0157

0.4088
0.0731
0.7603

0.1251
0.7055
0.2945

0.0068
0.0948
0.1447

112

Table S10. Cont’d.

Dietary
treatment
Control
TCDD

Dose
(ng/kg
body
wt/d)
0
2.1
4.6
6.0
8.4

PeCDF

13
25
30
49

Body mass
b
n necropsy (g)
12
1536
79
7
1355
96
6
1295
103
6
1072 A
103
9
1225
81
1306
10
71
3
1093 A
128
7
1497
85
8
1058 A
82

Adrenal
glands
0.021
0.018-0.024
0.022
0.019-0.025
0.021
0.019-0.023
0.024
0.021-0.028
0.024
0.020-0.028
0.046 A
0.001-0.091
0.034
0-0.079
0.024
0.013-0.034
0.034 A
0.027-0.041

Kidneys
0.66
0.60-0.72
0.75
0.07-0.84
0.83 A
0.070-0.95
0.83
0.74-0.92
0.76
0.70-0.82
0.72
0.65-0.80
0.86 A
0.56-1.17
0.68
0.62-0.74
0.91 A
0.82-0.10

113

Lymph Node
0.10
0.09-0.12
0.10
0.07-0.14
0.10
0.06-0.14
0.07
0.05-0.09
0.10
0.08-0.12
0.10
0.07-0.12
0.08
0.08-0.09
0.11
0.08-0.13
0.11
0.08-0.14

Brain
0.75
0.64-0.87
0.76
0.65-0.86
0.84
0.68-0.99
0.99
0.82-1.17
0.83
0.69-0.97
0.83
0.70-0.96
0.93
0.09-1.78
0.69
0.62-0.77
0.96
0.80-1.13

Reproductive
Tract
0.06
0.05-0.06
0.07
0.06-0.07
0.07
0.06-0.09
0.06
0.05-0.06
0.08
0.06-0.10
0.06
0.05-0.06
0.06
0.05-0.07
0.07
0.06-0.07
0.07 A
0.06-0.09

Table S10. Cont’d.

TCDF

52

9

116

12

214

10

246

9

Control vs
Congener
p-value TCDD
p-value PeCDF
p-value TCDF

1357
90
1574
79
1323
90
1302
87

0.024
0.015-0.032
0.025
0.020-0.030
0.023
0.018-0.028
0.030
0.023-0.036

0.73
0.65-0.80
0.68
0.65-0.70
0.75
0.68-0.82
0.74
0.67-0.80

0.09
0.07-0.12
0.11
0.09-0.14
0.10
0.08-0.12
0.14
0.11-0.16

0.82
0.65-0.98
0.64
0.56-0.71
0.77
0.64-0.89
0.76
0.70-0.82

0.06
0.05-0.07
0.09
0.06-0.12
0.07
0.05-0.10
0.07
0.06-0.09

0.0287
0.0963
0.0021

0.3834
0.3271
0.3046

0.0457
0.1837
0.0004

0.1602
0.0938
0.7383

0.1805
0.1691
0.0886

0.3963
0.2387
0.0314

a

TCDD is 2,3,7,8-tetrachlorodibenzo-p-dioxin; PeCDF is 2,3,4,7,8-pentachlorodibenzofuran;
and TCDF is 2,3,7,8-tetrachlorodibenzofuran.
b
Body mass data are presented as least squares mean with standard error beneath.
c

Relative organ mass data are presented as mean with 95% confidence interval beneath.

d

Means that are significantly different than the control mean at p < 0.05 are designated with an A.

114

CHAPTER 4

CONCLUSIONS AND RECOMMENDATIONS FOLLOWING TWO LABORATORY
FEEDING STUDIES EVALUATING THE EFFECTS OF TCDD AND TCDD-LIKE
COMPOUNDS ON MINK (MUSTELA VISON)

CONCLUSIONS
Toxic equivalency factors (TEFs) are consensus values that reflect the relative
toxicity of individual 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like congeners to TCDD.
Relative effect potency (REP) is determined for individual polychlorinated dibenzo-p-dioxin
(PCDD), polychlorinated dibenzofuran (PCDF), and polychlorinated biphenyl (PCB)
congeners for producing toxic or biological effects relative to a reference compound, usually
TCDD (Van den Berg et al., 2006). These effect concentrations then provide Toxicity
Reference Values (TRVs), which are used by risk managers to assess risk of harm for living
organisms. A compound must meet certain criteria to be included in the TEF concept. These
criteria include (1) a structural relationship to PCDDs and PCDFs; (2) ability to bind to the
AhR receptor; (3) ability to elicit AhR-mediated biochemical toxic responses; and (4)
persistent and accumulates in the food chain (Ahlborg et al., 1994; Van den Berg et al., 1998,
2006). Studies of TCDD-like chemicals are then conducted and/or evaluated to establish
TEFs. However, uncertainties remain. Many of the TEFs are based on in vitro studies and
thus do not take into account the potential differences in accumulation, disposition and
metabolism in animals (Blankenship et al., 2008). Toxic equivalency factors based on
biochemical effects such as enzyme induction in rodents may misrepresent an
environmentally relevant endpoint if applied to reproduction in an environmentally relevant

115

and sensitive wildlife receptor. As a result, it may be necessary to consider species-specific
TEFs for mink.
Results from the present study suggest that the TEFs for TCDF and PeCDF may not
accurately reflect their relative toxicity in mink. The assigned TEF of 0.1 for 2,3,7,8tetrachlorodibenzofuran (TCDF) suggests that TCDF elicits a relative toxic response that is
10% that of TCDD at a specific dose and is equivalent in toxicity to other TCDD-like
compounds assigned a TEF of 0.1, specifically, 3,3’, 4,4’, 5-pentachlorobiphenyl (PCB 126).
The TEF for 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) is 0.3, which suggests that at a
specific dose, it is 30% as toxic as TCDD and three times as toxic as both TCDF or PCB 126.
However, results from mink feeding studies described herein indicate that the effects of
TCDF, PeCDF and TCDD on tissue morphology, reproduction and offspring viability and
growth following exposure are less than what would be expected based on TCDDnormalized TEFs. Conversely, results of previous mink exposure studies completed at the
same facility with similar methods using PCB 126-dominated PCB mixtures indicate that
PCB 126 may be more toxic to mink than TCDF and PeCDF, and perhaps provides a more
suitable reference compound for mink risk assessments than TCDD.
Taking into consideration the TEFs assigned to TCDF, PCB 126, PeCDF and TCDD,
concentrations normalized to TEQs should elicit relative responses indicating that TCDD is
more toxic than PeCDF, PeCDF is more toxic than TCDF and PCB 126, and TCDF and
PCDF 126 are similar in toxicity. The present reproductive feeding study used doses of
TCDF, PeCDF and TCDD that should have resulted in reproductive impairment based on
TEFs, however, there were no adverse effects on reproduction or offspring viability at any of
the doses. In contrast, PCB 126-dominated mixtures had effects on mink reproduction and
116

offspring survival and growth at TEQ doses at or below those used in the present study,
indicating discrepancies in the TEF approach. In the case of mink, either the current TEF
approach does not adequately reflect the actual toxicity of PCB 126 relative to other TCDDlike compounds, or the toxicity of PCB 126 should be standardized outside the TCDD-centric
TEF approach.
The kinetic study described herein provides evidence that might in part explain this
apparent discrepancy between dietary TEQ concentrations and the apparent lack of effects
induced by TCDF and PeCDF. In the reported 180 d feeding study, PeCDF accumulated in
liver of mink to a much greater extent than did TCDF when administered as a single
congener or in combination with TCDF, which suggested an efficient metabolism and/or
elimination of TCDF. In addition, enzyme induction resulting from exposure to the
combination of the two furan congeners might not have been additive and perhaps was due
primarily to the action of only one of the congeners. Based on liver concentration data
indicating greater concentration of PeCDF compared to TCDF, it is possible that enzyme
induction in those animals receiving a mixture of the two congeners was due primarily to
PeCDF. Alternatively, TCDF might also have contributed to the increase in enzyme
activities, but due to metabolism, its concentration in the liver was less than that of PeCDF.
While exposure to PeCDF and TCDF in the 180 d study established EROD and MROD
activity as a biomarker of exposure to TCDD-like compounds, the doses and time of
exposure were not enough to address further questions of environmental relevance. For
example, mink in the Tittabawassee River are exposed to TCDF and PeCDF (Zwiernik et al.,
2008) in utero, during lactation and through the growth period to adulthood; therefore, it was

117

not possible to realistically extrapolate any effects from the 180 d feeding study to answer
questions related to reproduction and offspring viability.
The second feeding study attempted to answer this question by assessing dose-andtime dependent effects of TCDD, PeCDF and TCDF exposure in utero, during lactation and
throughout the growth period to 27 weeks of age. The highest dose of each congener
(TCDD, PeCDF, TCDF) was expected to cause reproductive effects based on results of
laboratory studies in which mink fed TEQ-normalized concentrations of PCBs (Heaton et al.,
1995a,b, Tillitt et al., 1996, Bursian et al., 2006a,b,c, Beckett et al., 2008) at similar levels
experienced decreased litter size and/or reduce offspring viability. However, dietary TEQ
concentrations for TCDD, PeCDF and TCDF as high as 8.4 ng TEQTCDD/kg body wt/d, 49
ng TEQPeCDF/kg body wt/d and 246 ng TEQTCDF/kg body wt/d, respectively, had no
significant effect on reproductive performance of mink or viability of their offspring, and
therefore a Lowest Observable Adverse Effect Level (LOAEL) could not be defined for these
specific environmentally relevant endpoints. The lack of an effect of TCDF on reproduction
and survival and growth is consistent with results from another reproductive feeding study,
where no significant effects on reproduction or survival endpoints were reported for dietary
concentrations as high as 242 ng TEQTCDF/kg feed (Zwiernik et al., 2009). Hochstein et al.
(2001) attempted a mink reproduction study utilizing TCDD at dietary concentrations as high
as 1,400 ng TEQTCDD/kg feed, however, an effect on reproduction could not be clearly
determined because of subnormal reproductive performance of the control group, which was
attributed to the fact that the trial was conducted indoors. While results of Zwiernik et al.
(2009) are consistent with the lack of reproductive effects from the TCDF reproductive study
118

described herein, another study completed at the same facility utilizing similar methods as
the TCDF reproductive study were not.
Similar to the methods of the two TCDF studies discussed herein, feed containing
PCB-contaminated fish collected from the Housatonic River was fed to mink at dietary
concentrations of 50.4 ng TEQPCBs, PCDDs/Fs/kg for 147 to 164 d (Bursian et al., 2006a).
This dietary TEQ concentration (90% TEQs contributed by non-ortho- and mono-ortho PCBs
with PCB 126 being the predominant contributor) resulted in decreased survival of kits at 6
weeks of age and decreased body weight at 3 weeks. The TEF for PCB 126 is equal to that
of TCDF while it is 3x less than PeCDF. However, when the Housatonic River study is
compared to the present reproductive feeding study, results suggest that TCDF is less toxic
than PCB 126 and PCB 126 is more toxic than PeCDF.
2,3,7,8-Tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran
(PeCDF) were fed to mink at concentrations expected to cause significant morphological,
reproductive, and/or survival effects (Zwiernik et al., 2009, Moore et al., 2009, 2012) relative
to its TEF of 0.1. A lack of adverse effects at dietary TEQ concentrations that exceed TRVs
leads to the conclusion that the TEF for TCDF is not reflective of its toxicity. Other studies
support this conclusion as TCDF is rapidly cleared from the mink (Zwiernik et al. 2008,
Bursian et al., 2012). Results from these studies and similar studies performed at the same
facility (Bursian et al., 2006b,c, 2012) also suggest that the current TEF values might not
accurately predict the toxic potency of TCDF and PeCDF, while PCB 126 may be the most
toxic TCDD-like compound to mink.

119

RECOMMENDATIONS

Evaluate TCDD-like compound interactions and the relative toxicity of TCDD-like
compounds in mink
Since compound interactions may increase or decrease toxicity, additional studies
evaluating environmentally relevant or site-specific mixtures may be necessary to understand
TCDD-like compound interactions. The TEQ approach assumes additive toxicity, however,
the administration of two environmentally relevant TCDD-like compounds to mink, which
exceeded TRVs under controlled conditions, did not result in the expected toxic response.
This presents challenges to risk managers, particularly where environmental exposures that
include TCDF are assumed additive. Given the disparity, it may be appropriate to further
evaluate interactions among environmentally relevant PCDDs, PCDFs, and PCBs in mink.
This conclusion challenges the additive assumption of the TEQ method in the case of mink,
while results from a mink reproductive feeding study following the sole administration of
TCDF at TEQ normalized concentrations expected to elicit effects on reproduction and
offspring viability presents another challenge to risk managers applying current TEFs for
mink.

120

In order to definitively examine the TEFs assigned to TCDF and other TCDD-like
compounds for a sensitive, environmentally relevant wildlife receptor, it is important to
evaluate the relative toxicity of TCDD-like compounds solely and directly to TCDD as well
as to PCB 126 under controlled conditions. Results from this study and other reproductive
feeding studies performed at the same facility with similar methodology suggest that the TEF
for PCB 126 is underestimated and should be reevaluated relative to TCDD or the toxicity of
PCB 126 should be standardized outside the TCDD-centric TEF approach. It may be
appropriate at this time to research the relative potency of TCDD-like compounds to TCDD
and to PCB 126 via side-by-side sole administration of these compounds to mink to evaluate
sensitive endpoints under controlled conditions including enzyme induction and jaw lesions
(Bursian et al., 2012) as well as less sensitive endpoints of reproductive performance and
offspring viability in order to establish species-specific TEFs. This information would
provide risk managers with environmentally relevant TRVs for mink and lead to
management actions, which represent ecologically relevant effects.

121

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