AN ABSTRACT OF THE THESIS OF
Tracie M. Caslin for the degree of Master of Science in Wildlife Science
presented on June 16, 1998. Title: Individual and Demographic
Responses of the Gray-taile Vole (Microtus canicaudus) to an Endocrine
Disruptor.
Redacted for Privacy
Abstract approved:
Jerry 0. Wolff
In an experimental field study, populations of gray-tailed voles
(Microtus canicaudus) were exposed to a commercial formulation of
vinclozolin, a fungicide effective for disease control on ornamental plants,
turf grasses, and fruits and vegetables. Vinclozolin has been shown in
laboratory experiments to behave as an androgen antagonist, impairing
the reproductive development in males of several species of mammals
when exposed in utero. However, when applied to grassland habitat
containing populations of gray-tailed voles, no biologically significant
impairment was observed in reproductive development of male voles
whose mothers were exposed to the treatment while the young were in
utero. Reproductive organs were sufficiently developed to result in high
reproductive rates and juvenile recruitment in the field with no effects on
population growth or demography. Under the conditions of this study,
one standard application of Curalan5'' fungicide had no measurable
reproductive or demographic consequences on gray-tailed voles.
However, the results suggest that higher application rates or several
successive applications may have negative impacts on reproductive
development and demography of wild vole populations.
©Copyright by Tracie M. Caslin
June 16, 1998
All rights reserved
Individual and Demographic Responses of the Gray-tailed Vole (Microtus
canicaudus) to an Endocrine Disruptor
by
Tracie M. Caslin
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed June 16, 1998
Commencement June 1999
Master of Science thesis of Tracie M. Caslin presented on June 16, 1998
APPROVED:
Redacted for Privacy
M
r Pro ssor, rep
enting Wildlife Science
Redacted for Privacy
Head of Department of Fisheries and Wildlife
Redacted for Privacy
Dean of Gradate School
I understand that my thesis will become part of the permanent collection
of Oregon State University libraries. My signature below authorizes
release of my thesis to any reader upon request.
Redacted for Privacy
Tracie M. Caslin, Author
ACKNOWLEDGMENTS
I am indebted to many individuals and institutions whose help
made this thesis possible.
First and foremost, I would like to thank my parents, John and
Karen Sanchez for their lasting support and encouragement throughout
my graduate career and for all that they have sacrificed so that my
brothers and I can pursue our dreams. To my brothers, Brent and
Brian, I thank them both for their unconditional support with all of my
wild dreams, for instilling with me the value of life, and for teaching me
that there are no limits to what we can do. We make a heck of a team!
I
also want to thank my partner and my best friend, Tim O'Brien, for his
remarkable patience and moral support during this last year at Oregon
State University. I am grateful for his many lessons in time-management
and for pushing me when I needed it. I thank him for his love and
encouragement and for being a part of my life.
Naturally, I would like to extend a huge thank you to my
committee members Drs. Jerry Wolff, Dan Roby, and Dan Schafer. I am
quite fortunate and thankful to have worked under the direction of such
talented individuals. A sincere appreciation is extended to my major
professor, Dr. Jerry Wolff, for his guidance, friendship, understanding,
and mentorship throughout my training. Thank you for this opportunity!
I would also like to extend a very special thank you to Dr. Robert Jarvis
for providing me with the opportunity to study at Oregon State University
and for financially supporting me throughout my last year. Thank you
also to Dr. Carl Schreck for supplying much of the laboratory and field
equipment for this project, to Dr. Dan Roby for the use of his laboratory,
and to the entire Fisheries and Wildlife Department for an incredible and
memorable two years at OSU.
Without a doubt, I am deeply indebted to those I worked with in
the field: Monica Bond, Christine Dalton, Dr. Dan Edge, Lisa Hewlett,
Bert Skil len, Kelli Walker, Guiming Wang, and Dr. Jerry Wolff. I thank
them for their hard work, support, and friendship. A very special thanks
to Bert Skil len, who should be commended for his hard work and
dedication to this project. Bert helped make this project the success that
it was. I thank Monica Bond and Christine Dalton for their sincere and
lasting friendship. I would like to send an important thank you to the
laboratories of Drs. L. Earl Gray and William R. Kelce of the EPA,
Research Triangle Park, for their interest in this project, helpful advice,
and especially for their time analyzing samples for me. A sincere thank
you goes to the statistics department, especially Shaun Wulff, for his
time and statistical talent. This work was supported by cooperative
agreement R825347-01 between the US EPA and Oregon State
University.
TABLE OF CONTENTS
Page
INTRODUCTION
1
OBJECTIVE
8
STUDY SITE
9
EXPERIMENT I
MATERIALS AND METHODS
Chemical
Study Species
Experimental Design
Application of Chemical
Field Measurements
Laboratory Measurements
Data Analysis
RESULTS
10
10
10
10
11
12
13
14
16
17
Laboratory
17
Field
20
SUMMARY OF RESULTS
EXPERIMENT II
MATERIALS AND METHODS
Experimental Design
Application of Chemical
Field Measurements
RESULTS
Laboratory
Field
20
24
24
24
24
25
25
25
25
TABLE OF CONTENTS (Continued)
SUMMARY OF RESULTS
28
DISCUSSION
32
CONCLUSION
36
BIBLIOGRAPHY
37
LIST OF FIGURES
Page
Figure
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Mean length of testes and seminal vesicles from male
necropsies for treatment and control enclosures in
Experiment I
18
Mean levels of plasma testosterone in males from treatment
and control enclosures in Experiment I
18
Mean area of the hip gland from male necropsies for
treatment and control enclosures in Experiment I
19
Proportions of females pregnant by male recruits for
treatment and control enclosures in Experiment I
21
Sex ratio of recruits for each treatment enclosure and mean
sex ratio of recruits for the four control enclosures in
Experiment I
21
Mean proportion of recruits per adult female for treatment
and control enclosures for Experiment I
22
Mean population size using MNA in treatment and control
enclosures in Experiment I
22
Mean length of testes and seminal vesicles from male
necropsies for treatment and control enclosures for
Experiment II
26
Mean area of the hip gland from male necropsies for
treatment and control enclosures in Experiment II
26
Mean levels of plasma testosterone in males from treatment
and control enclosures in Experiment II
27
Proportions of females pregnant by male recruits for
treatment and control enclosures in Experiment II
29
Sex ratio of recruits for each treatment enclosure and mean
sex ratio of recruits for the four control enclosures in
Experiment II
29
LIST OF FIGURES (Continued)
Figure
Page
13.
Mean proportion of juvenile recruits per adult female for
treatment and control enclosures for Experiment II
30
14.
Mean population size using MNA in treatment and control
enclosures in Experiment II
30
Individual and Demographic Responses of the Gray-tailed Vole
(Microtus canicaudus) to an Endocrine Disruptor
INTRODUCTION
Evidence suggests that humans and wildlife have suffered adverse
consequences from exposure to environmental chemicals that interact
with natural biological systems (Colborn et al., 1993). In 1962, Rachel
Carson's
Silent Spring
alerted the world to the dangers of pesticides.
Many pesticides were recognized to be fatally toxic to some species and to
cause widespread reproductive failure among others (Carson, 1962).
Now scientists are finding that these pesticides among many other
chemicals can have an array of unexpected effects. The chemicals are
known to interfere with the endocrine system by mimicking natural
chemical regulators. During the earliest stages of an animal's
development, contact with these chemicals can often result in abnormal
development of reproductive organs (Guillette
1995; Sharpe
Gray
et al.,
et al.,
1995; Crain
et al.,
et al.,
1994; Colborn,
1997; Baker Matta
et al.,
1998;
1998), early mortality (Fox, 1992; Schmidt, 1994; Fry, 1995),
and even altered behavior (Fox, 1992; Fry, 1995). Endocrine disrupting
chemicals that are anthropogenic in origin are often referred to as
environmental hormones.
Many environmental hormones have been linked to a decline in
wildlife populations in many areas of the world (Colborn et
al.,
1993;
2
Schmidt, 1994), primarily within agricultural areas (Hoffman et al., 1990;
Kendall and Akerman, 1992; Pimentel et al., 1992; Fry, 1995). Although
the concern with environmental hormones had been focused primarily on
the impacts on humans, most of the detectable and measurable effects of
environmental hormones has been with reproductive failures in wildlife
in nature and in laboratory experiments using animals as models (Mason
et al., 1986; Dietz et al., 1989; Peterson et al., 1993).
Most of what is known about response of mammals to endocrine
disruptors comes from using mammalian models in the laboratory.
Neonatal treatment of female mice with estrogenic compounds results in
proliferation and cornification of vaginal epithelium (Tagasugi, 1976).
Exposure to dioxin results in decreased fetal growth and neonatal
mortality in rats, guinea pigs, hamsters, rabbits, and monkeys (Couture
et al., 1990; Birnbaum, 1995; and references cited therein), thymic and
splenic atrophy, subcutaneous edema, and gastrointestinal hemorrhage
in mice, rats, and hamsters (Olson et al., 1990), cleft phallus in mice
(Olson et al., 1990), feminization of rats (Peterson et al., 1993),
mineralization of the skeleton (Migliaccio et al., 1995), decreased
testicular and sperm development (Mabley et al., 1992a; Gray et al.,
1995), delay in sexual maturation (Lundkvist, 1990), and many other
effects (reviewed in Birnbaum, 1995). The period of mammalian
embryonic development is a life stage highly susceptible to environmental
hormones (Guillette et al., 1995). Embryos are small and exhibit high
3
rates of mitosis (Guillette et al., 1995), thus numerous developmental
and physiological anomalies can result in mammals following exposure of
pregnant females to endocrine disruptors. However, it is simplistic to
assume that an individual is completely organized at birth. Current
research has indicated that postpubertal exposure to estrogen in mice
changes the shape of the pelvis to resemble a female's bone structure
and postpubertal androgens change the shape to resemble a male's bone
structure (Iguchi et al., 1995). Most of these effects occur after the
animals receive an intravenous injection or are fed large doses of a
chemical under laboratory conditions. The extent to which scientists are
able to extrapolate these laboratory results to organisms in their natural
environment is equivocal.
The effects that environmentally relevant doses of endocrinedisrupting chemicals have on wild mammal species are not well known.
Most of the field studies have been observational, retrospective, and
associative (Bennetts et al., 1946; Dietz et al., 1989; Safe, 1995),
although considerable correlational evidence is available (reviewed in
Kav lock, et al., 1996). River otters (Lutra canadensis) exposed to
organocholorine pesticides and PCB's in the Columbia Basin have
significantly smaller bacula than do otters from uncontaminated rivers
(Henney et al., 1997). Chemicals affecting reproduction and development
also have been implicated in declines of otter populations (Lutra lutra) in
England (Mason et al., 1986); in reproductive anomalies in mink (Mustela
4
vison) from the Great Lakes (Aulerich et al., 1973); in massive deaths of
harbor seals (Phoca vitulina) (Dietz et al., 1989); in reproductive failure in
sheep in Australia (Bennetts et al., 1946), and polar bears (Ursus
maritimus) in Svalbard (Norheim et al., 1992); and in disease outbreaks
of dolphins in the Mediterranean (Aguilar and Raga, 1993; Aguilar and
Borrell, 1994). In most of these wildlife cases the investigators have
simply noted gross reproductive abnormalities associated with high levels
of chemicals present in the environment.
Clearly, considerable information is available on reproductive and
developmental anomalies of species exposed to chemicals that disrupt
the normal functioning of the endocrine system. Although this
information is available, important uncertainties still remain. While the
impact of endocrine disruption on the individual is becoming better
understood (Guillette et al., 1994, 1995a,b; Tagasugi, 1976), the
sensitivity of populations to environmental hormones is an area of study
that has been virtually unexplored (reviewed in Kav lock et al., 1996).
Post hoc observations from the field may show associations between
environmental hormones and reproductive development, but do not
demonstrate cause and effect relationships. The prediction of population
level effects is also difficult to determine due to limited understanding of
the full spectrum of effects that may result from exposure to endocrine
disruptors (McTavish, 1998).
5
Examples of agents that have been shown to alter reproductive
development in various species via an endocrine mechanism include:
Hormones and drugs, including progestrogens, androgens, ecdysteroids
and farnesyl hormones (reviewed in Kav lock et al., 1996); phytoestrogens
and mycotoxins (reviewed in Kav lock et al., 1996); pollutants such as
dioxins (Couture et al., 1990; Birnbaum, 1995) and some PCBs (Sager et
al., 1987); and pesticides, including DDT (Fry and Toone, 1981), dieldrin
(Haake et al., 1987), and vinclozolin (Wong et al., 1995).
Vinclozolin is a fungicide registered in the United States and
Europe for effective disease control of fruits and vegetables, herbaceous
and woody ornamental plants, and all species of turf grass (Wong et al.,
1995; BASF, 1997). The fungicidal treatment helps to eliminate
destructive diseases such as white rot disease (Sclerotium cepivorum) of
onions (Walker, 1987), collar rot (Martin et al., 1991) and lettuce drop
(Hubbard et al., 1997) caused by Sclerotinia minor, gray mold in table
grapes induced by Botryotinia fuckeliana (Scrano et al., 1990) and in
kiwifruit by Botrytis cinerea (Michailides and Morgan, 1996), dollar spot
(Sclerotinia homoeocarpa; Lanzia sp.; Moellerodiscus sp.) on all turf grass
species and Botrytis spp. on all ornamentals. Vinclozolin and it's
primary metabolites behave as anti-androgenic compounds and their
effects on mammals have been studied extensively in the laboratory
(Gray et al., 1994; Kelce et al., 1994; Wong et al., 1995; Kevekordes et al.,
1996; Laws et al., 1996; Lodovici et al., 1997).
6
Androgens are steroid hormones that stimulate masculinization of
the fetus and induce male imprinting of the developing brain (Wong et
al., 1995). Thus, an anti-androgenic compound, also know as an
androgen antagonist, competes with and ultimately prevents the binding
of steroid hormones, or androgens, to the receptor molecule. This action
suppresses the growth and function of the male reproductive system
(Wong et al., 1995; Gray et al., 1998). The metabolite byproducts of
vinclozolin, which form as vinclozolin degrades, appear to have 10-100
times greater anti-androgenic properties than vinclozolin itself (Kelce et
al., 1994). Vinclozolin has been found to inhibit sexual differentiation in
male rats in an anti-androgenic manner. Administration of vinclozolin to
pregnant rats during the period of sexual differentiation causes a
reduction in size of seminal vesicles and in testis weights in male pups
(Gray et al., 1994). Differentiation of the Wolffian structures (e.g. the
epididymis and seminal vesicles) is testosterone mediated; a decrease in
these structures suggests a decrease in testosterone production. A
slightly reduced serum testosterone level at necropsy confirmed this
suggestion. Growth rates of male rats prior to puberty were not affected
by perinatal exposure to vinclozolin, but growth rates were reduced after
puberty (Gray et al., 1994). Male rats exposed to vinclozolin during
sexual differentiation also displayed malformations such as
areola/nipples, hypospadias (abnormal positioning of the urethra
opening), and cleft phallus resulting in the inability to inseminate
7
females (Gray et al., 1994). The laboratory experiments suggest that
vinclozolin treatment does not affect the latency for males to mount
females (Gray et al., 1994). Administration of vinclozolin to adult male
rats causes Leydig cell hyperplasia and atrophy of the prostate and
seminal vesicles (Wong et al., 1995). Lifelong fertility of females is
unaffected by vinclozolin treatments (Gray et al., 1994). Although daily
oral dosing of animal models results in metabolite levels sufficient for
antagonist activity (Wong et al., 1995), the extent to which humans and
wildlife are exposed to the widely used fungicide, or whether active
metabolites reach concentrations sufficient for anti-androgenic or
androgenic activity is unknown.
All of the studies examining the effects of the anti-androgenic
compound vinclozolin have been conducted in the laboratory with
domestic mice and rats. The developmental, behavioral, and
demographic responses of a wild population of mammals exposed to
vinclozolin in its natural environment have not been determined.
Whether laboratory results with laboratory species are representative of
wild populations in natural environments is equivocal (Cholakis et al.,
1978; Meyers and Wolff, 1995; Edge et al., 1996). A field study
measuring developmental and demographic responses of a wild species
exposed to a standard field application of vinclozolin, or any endocrine-
disrupting chemical, would further our understanding of how wild
populations respond to purported endocrine disruptors.
8
OBJECTIVE
The objective of this study was to determine if the androgen
antagonist vinclozolin, when applied once in the field at a standard
application rate for agricultural use, had any developmental effects on
the reproductive system of males within a field population of gray-tailed
voles (Microtus canicaudus) and to determine if these effects had
consequences at a population level. To meet these objectives I tested the
hypothesis that exposure to Curalan(R) fungicide, applied once at the
maximum curative application rate, will result in abnormal reproductive
development of male voles exposed in utero, thus affecting their ability to
reproduce. Specifically, the length of testes, the length of seminal
vesicles, the area of the hip gland, and levels of serum testosterone were
measured and compared between treatment and control to determine
direct effects to the male reproductive system. The exterior of male voles
was examined for evidence of hypospadias, cleft phallus, or presence of
nipples. Demographic responses were also monitored in the field to
determine if vinclozolin had any effect at the population level. These
responses included population growth, sex ratio of recruits, proportion of
recruits per adult female, and proportion of females that were
impregnated by male recruits.
9
STUDY SITE
This research was conducted at the Hyslop farm small mammal
research facility located approximately 10 km north of Corvallis, Oregon
(Wolff et al., 1994; Edge et al., 1995, 1996). Twenty-four 0.2-ha (0.5
acre) enclosures have been constructed at the site. Each enclosure is 45
x 45 m and is constructed of galvanized sheet metal approximately 90 cm
high and buried 90 cm deep to prevent escape of or entry by burrowing
animals. Each enclosure is planted with a mixture of pasture grasses.
10
EXPERIMENT I
MATERIALS AND METHODS
Chemical
Vinclozolin is a dicarboximide chemical sold under the trade
names Curalan® fungicide and Ronilan® DF fungicide which are
marketed by BASF AG. The half-life of a commercial formulation of
vinclozolin ranges from 1.2 days (Gennari
et al.,
1980) to 33.1 days (Szeto
et al.,
et al.,
1985) to 9 days (Del Re
1989). Vinclozolin degrades to
several metabolites, primarily M1, M2, and M3, one of the metabolites
being capable of converting back to vinclozolin, and all of which can be
detected in soil and plants (Szeto
et al.,
1989). Decay rates are highly
variable among plant types and parts and in response to sunlight,
temperature, and rainfall (Gennari
et al.,
1985; Szeto
et al.,
1989).
Study Species
Gray-tailed voles are a common, grassland, small mammal species
of the Willamette Valley of Oregon. Gray-tailed voles are similar in
appearance and behavior to other
related
M. montanus
Microtus
species, especially to closely
(Wolff, 1985; Verts and Carraway, 1987). Breeding
typically occurs from March through November, the gestation period is
11
21 days, modal litter size is six, and juveniles are weaned at 15 to 18
days (Peterson, 1996). Most females are able to start breeding when
they are 18 g (about 18 to 20 days) (Wolff et al., 1994). In the field
enclosures, each founding female will have about four litters and
populations will go through at least five to six generations per year.
Population size is typically 50 to 100 individuals (Schauber et al., 1997;
Edge et al., 1996). Microtus species exhibit a pheromonally induced
estrus and coitus-induced ovulation (Seabloom, 1985) and post-partum
estrus (Hasler, 1975).
Experimental Design
The experimental units consisted of eight of the 24 0.2-ha
enclosures. Four replicate enclosures were randomly assigned to the
treatment and four to the control groups. Eighty-one trap stations were
arranged in a 9 x 9 array with 5 m between stations within each
enclosure. One large-sized Sherman live trap was placed at each station.
In late April, at the beginning of the experiment, five male and 7 to
10 nulliparous female voles were introduced into the enclosures. Males
remained in the enclosures for 3 weeks, long enough to impregnate the
females. After 3 weeks, all males were removed to prevent them from
mating with the females a second time after they had given birth to their
first litter. By removing adult males, the only males left in the
12
enclosures would be those that were exposed to the treatment while they
were in utero.
Application of Chemical
The recommended application rate of 12.9 liters of Curalan®/ha
was added to the four treatment enclosures on 23 May 1997
approximately 3 weeks after the voles were introduced to the enclosures.
The fungicide was applied by a licensed applicator using a tractor and
trailer tank with 11-m spray booms. The apparatus and procedure for
the application of chemicals has been developed by the Crop Science
Department at Oregon State University and has been used successfully
in previous experiments since 1991 (Edge et al., 1996; Schauber et al.,
1997). Control enclosures received an equivalent application of water.
I attempted to time the application of the chemical to when all (or
most) females would be pregnant. Females were expected to become
pregnant 5 to 7 days after introduction into the enclosures and exposure
to males. The chemical was sprayed when the embryos were in early
development, less than 14 days into gestation. The time of sexual
differentiation is not known for voles, but is likely similar to that of rats
which is from gestation day 14 to postnatal day 3 (Gray et al., 1994).
The length of gestation in rats and voles is similar (21 days; Norris, 1997;
Keller, 1985) so I assumed that sexual differentiation would be occurring
at the time of the chemical application.
13
Field Measurements
Trapping was conducted for four consecutive days at 2-week
intervals from 12 May to 25 July. Traps were baited with oats and
sunflower seeds, and were set in the evening and checked once a day at
sunrise. All captured animals were ear-tagged for identification. Data on
the animal's weight, sex, trap location, and reproductive condition of
females were recorded for each capture. All data were number-coded for
computer entry and recorded on field data sheets. Females were
recorded as pregnant if they had swollen abdomens. Nipples were
recorded as small (not visible), medium (visible but not lactating), and
large (lactating; white mammary tissue visible around the nipple
indicating the animal is currently nursing young). Parting of the pubic
bones was recorded as closed, partly open, or wide open indicating
parturition. Females were considered in reproductive condition if they
were lactating or pregnant or had a widely parted pubic symphysis.
Values for population size, reproductive activity, and recruitment
were determined for each enclosure. Minimum Number Alive (MNA) was
used to estimate population size for each trap period. Reproductive
activity was measured by the proportion of females in reproductive
condition after male recruits reached sexual maturity. Recruitment was
measured by the number of newly tagged voles in an enclosure divided
by the total number of adult females introduced to the same enclosure at
the beginning of the experiment.
14
Laboratory Measurements
Five male recruits that were exposed to the treatment while they
were in utero were removed from each of the eight enclosures and
brought to the laboratory for necropsy. Males were removed when they
first attained a body weight of 40 ± 2 g; about 9 to 11 weeks into the
study. Male reproductive organs should be fully developed at this weight
(Nadeau, 1985). Animals were killed by cervical dislocation. Immediately
after cervical dislocation, blood samples were collected in heparinized
capillary tubes and drained into eppendorf tubes. Samples were
immediately spun at 13,700 revolutions per minute for 5 minutes in a
centrifuge to separate plasma from red blood cells. Plasma was removed
using a pipet and stored in eppendorf tubes in a freezer at -70°C.
Plasma samples were shipped on dry ice to the National Health and
Environmental Effects Research Laboratory of the U.S. Environmental
Protection Agency located at Research Triangle Park to be analyzed for
levels of testosterone by William R. Kelce, Ph.D. Plasma testosterone
levels were assayed by radioimmunoassay as described and validated by
Ewing et al. (1984). Data are presented as ng/ml and represent
testosterone levels from a single serum sample aliquot (10 ptL) from each
of the samples. Each sample was assayed in duplicate.
After blood samples were collected and prior to necropsy, each
animal was examined for evidence of sexual maturity by level of
pigmentation of the scrotum (Harman, 1978). Scrotal skin was classified
15
as dark, light, or no pigmentation. The exterior of the body of each
animal was examined for any possible malformations that are known to
occur in treated males, such as, hypospadias, cleft phallus, and presence
of nipples (Gray et al., 1994; Gray et al., 1998). During necropsy, the
epididymis was squashed and examined under a 400X microscope for
the presence of live sperm. The functional activity of the epididymides,
whether the maturation of spermatozoa or the resorption and secretion of
fluid, is highly dependent on androgen stimulation (Zaneveld, 1982). The
presence of live sperm was used as an indication that testes had
developed sufficiently for the individual to attain sexual maturation.
Organs measured that were associated with reproduction included the
length of testes, the length of seminal vesicles, and the area of the hip
glands. Spermatogenesis occurs within the testis and secretions from
seminal vesicles protect spermatozoa and enhance their lifespan and
fertilizing capacity (Bey ler and Zaneveld, 1982). Testosterone promotes
growth and development of these male sexual organs (Harman, 1978).
Hip glands in gray-tailed voles are scent glands located on the hip of
males and females and function in communication primarily during the
breeding season (Quay, 1968; Wolff and Lidicker, 1980; Wolff, 1985;
Wolff et al., 1994). The length and width of the hip glands were
measured from the inside of the skin and the gland size was
approximated using the area of an ellipse (Wolff and Lidicker, 1980).
16
Measurements were taken on both left and right organs and averaged to
obtain a mean measurement for that individual.
Data Analysis
Data for male reproductive organs (testes, seminal vesicles, and
hip glands) were first tested for significant enclosure effects and then
analyzed with a multivariate analysis of variance (MANOVA) model using
a general linear model procedure (PROC GLM) and MANOVA statement
in SAS®. Scatterplots of original data were visually reviewed for each
enclosure. Testosterone levels from the male necropsies were analyzed
by univariate statistics using the GLM procedure in SAS® after testing for
possible enclosure effects. Data for the proportions of females pregnant
by male recruits were first tested for overdispersion using an
approximate test given by p = P(x-df> deviance). The proportions of
females pregnant were analyzed by a logistic analysis (PROC GENMOD)
in SAS®, accounting for overdispersion.
Sex ratio of recruits was analyzed using a chi-square test. The sex
ratio of the treatment enclosures was compared to a mean sex ratio of
the control enclosures. A chi-square analysis was performed using
PROC FREQ in SAS®. For juvenile recruitment, the proportions of
recruits per adult female were analyzed with a one-way analysis of
variance (ANOVA) model using PROC GLM in SAS®. Population size was
17
monitored throughout the course of the experiment but was not
statistically analyzed due to the manipulations to the populations.
Probability values are based on two-sided tests.
RESULTS
Laboratory
All male recruits brought to the laboratory showed signs of sexual
maturity as indicated by the presence of dark to light pigmentation of the
scrotal skin. No male showed signs of hypospadias, cleft phallus, or
presence of nipples. The multivariate model used for the reproductive
organ data was adjusted due to a significant enclosure effect (all p 0.08)
by using the enclosure x treatment variable as the error term. The
adjusted model for male reproductive organs revealed significant
treatment effects for the length of seminal vesicles (p = 0.02; Fig. 1); the
mean length of seminal vesicles in the control animals was larger than in
the treatment animals. Control animals also had significantly higher
plasma testosterone levels (p = 0.02; Fig. 2). Mean length of testes for
control animals was slightly larger than treatment animals but was only
suggestive of a difference (p = 0.08; Fig. 1). The differences in area of the
hip gland, however, were not significant between treatments (p = 0.20;
Fig. 3).
18
F1,6 = 10.33
16-
I
E 14
E
p = 0.02
F1,6 =4.39
12 -
p = 0.08
10-
8-
6-
F-
Trtmt
Cntrl
Testes Length
Trtmt
Cntrl
Seminal Vesicle Length
Figure 1. Mean length of testes and seminal vesicles
from male necropsies for treatment and control
enclosures in Experiment I.
0.5
0.4
0.3
0.2
0.1
0
Treatment
Control
Plasma Testosterone Levels
Figure 2. Mean levels of plasma testosterone in males
from treatment and control enclosures in Experiment I.
19
Treatment
Control
Hip Gland Area
Figure 3. Mean area of the hip gland from male
necropsies for treatment and control enclosures in
Experiment I.
20
Field
The approximate test on the data for the proportion of females
pregnant by male recruits suggested that overdispersion should not be
an issue, but the data may be influenced by an underdispersion (p =
0.66). Assuming a binomial response (unscaled), the test revealed a
suggestive but inconclusive treatment effect (p = 0.12); however, after
scaling for underdispersion, a test of the difference between treatment
and control indicated a treatment effect (p = 0.05; Fig. 4).
Sex ratios did not deviate significantly from expected in any of the
treatment enclosures (all p 0.55; Fig. 5). The number of recruits per
..
adult female did not differ significantly between control and treatment
enclosures (p = 0.73; Fig. 6). The trend in population size over the
course of the experiment was similar for treatment and control
enclosures (Fig. 7); however, the data were not statistically analyzed due
to manipulations to the populations.
SUMMARY OF RESULTS
Results from Experiment I suggested that with one application of
Curalan® fungicide, Vinclozolin had only a slight effect on the
reproductive development of male gray-tailed voles that were exposed to
the chemical while in utero. However, males from treatment enclosures
21
1
0.9
p = 0.051
X21 = 3.820
(12)
8
(13)
0
(13)
0
(4)
0.8
(11)
0.7
(7) S
6 (3)
0.6
6 (5)
0.5
0.4
Control
Treatment
Female Reproductive Condition
Figure 4. Proportions of females pregnant by male
recruits for treatment and control enclosures in
Experiment I. Sample sizes are given in parentheses.
All X21
0.364
Allp 0.55
Treatment
Control
Sex Ratio of Recruits
Figure 5. Sex ratio of recruits for each treatment
enclosure and mean sex ratio of recruits for the four
control enclosures in Experiment I.
22
Treatment
Control
Juvenile Recruitment
Figure 6. Mean proportion of recruits per adult
female for treatment and control enclosures in
Experiment I.
Remove
Males for
Necropsy
26 May
9 Jun
23 Jun
7 Jul
21 Jul
Figure 7. Mean population size using Minimum Number
Alive (MNA) in treatment and control enclosures in
Experiment I.
23
were functionally capable of reproducing and the slight physiological
anomalies did not appear to have any consequences at a population
level. No differences existed between control and treatment enclosures
for sex ratio of recruits or for juvenile recruitment. Population size over
the course of the experiment was similar for treatment and control
enclosures; however, a marginally significant difference occurred for the
proportions of females pregnant between treatment and control. Based
on these results, a second experiment was conducted in an attempt to
increase the chemical exposure to the pregnant females.
A few factors during Experiment I may have decreased the
exposure of the chemical to the pregnant females, thus preventing more
severe effects from occurring to the reproductive development of males.
Within 24 hours after the chemical was applied during Experiment I,
5.40 mm of rainfall fell. Rain has the potential to wash a large percent of
the chemical off the grass and decrease the life expectancy of vinclozolin
(Szeto et al., 1989) ultimately decreasing its toxicity to the voles. Also, at
the time of chemical application the grass within each enclosure was very
tall, approximately 50 to 60 cm. Experiment II followed the same design
as Experiment I with a few modifications intended to increase the
chemical exposure to the voles.
24
EXPERIMENT II
MATERIALS AND METHODS
Experimental Design
After Experiment I was completed, all animals were removed from
the eight enclosures. Prior to introducing animals for Experiment II, the
grass in each enclosure was mowed to a height of approximately 30 cm
(20 to 30 cm shorter than in Experiment I) to increase the exposure of
the animals to the chemical. On 6 September, 13 to 15 visibly pregnant
females (probably within the second week of gestation) were removed
from enclosures being used for another study and were randomly
distributed into the eight enclosures. No males were used in this
experiment because females were already pregnant.
Application of Chemical
Because females were already pregnant, the chemical treatment
was applied on 9 September, 3 days after females were placed into the
enclosures. Most females should have been in mid pregnancy at this
time. The same application procedures for the chemical were followed as
in Experiment I. No precipitation occurred within the first 24 hours after
25
chemical application and then only 1.28 mm of precipitation occurred
within the next 4 days.
Field Measurements
Due to the colder weather, the traps were set before sunrise and
checked once a day after noon. Because of high trapping success (> 80%
of animals caught each week) trapping was conducted for 3 instead of 4
days.
RESULTS
Laboratory
All male recruits brought to the laboratory showed signs of sexual
maturity by presence of dark to light pigmentation of the scrotal skin.
No male showed any sign of hypospadias, cleft phallus, or presence of
nipples. No significant treatment effects occurred for length of testes (p =
0.36), length of seminal vesicles (p = 0.34; Fig. 8), area of the hip gland (p
= 0.88; Fig. 9), or level of testosterone (p = 0.40; Fig. 10).
Field
The approximate test on the data for the proportion of females
pregnant by male recruits suggested that overdispersion may not be an
26
Trtmt
Trtmt
Cntrl
Testes Length
Cntrl
Seminal Vesicle Length
Figure 8. Mean length of testes and seminal vesicles
from male necropsies for treatment and control
enclosures in Experiment II.
Treatment
Control
Hip Gland Area
Figure 9. Mean area of the hip gland from male
necropsies for treatment and control enclosures in
Experiment II.
27
Treatment
Control
Plasma Testosterone Levels
Figure 10. Mean levels of plasma testosterone in males
from treatment and control enclosures in Experiment
II.
28
issue (p = 0.19). The analysis of the data suggested a significant
treatment effect (p = 0.01; Fig. 11). Figure 11 is a scatterplot of the
proportions of females pregnant from each treatment and control
enclosure. No significant treatment effects were found for sex ratio (all p
0.34; Fig. 12) or juvenile recruitment (p = 0.6441; Fig. 13). The trend in
population size over the course of the experiment was similar for
treatment and control enclosures (Fig. 14); however, the data were not
statistically analyzed due to manipulations to the populations.
SUMMARY OF RESULTS
Results from Experiment II revealed no significant differences
between animals in control and treatment enclosures for variables
associated with reproductive development. These laboratory and field
results suggested that male recruits exposed to vinclozolin while in utero
were functionally capable of reproducing. Similar to Experiment I, effects
of vinclozolin were not severe enough to result in any malformations to
the external body.
Results from the field revealed that no statistical differences
existed between control and treatment enclosures for sex ratio of recruits
or for juvenile recruitment, although statistically significant differences
did occur for the proportions of females pregnant between treatment and
control populations. Similar to Experiment I, population size over the
29
o (27)
-
(16)
0.95
(36)
0.9
(23)
8
8
o (30)
(20)
0.85
0.8
X21 = 6.482
0.75
p = 0.01
(24)
0.7
Treatment
Control
Female Reproductive Condition
Figure 11. Proportions of females pregnant by male
recruits for treatment and control enclosures in
Experiment II. Sample sizes are given in
parentheses.
All X 2
0.900
All p
0.34
Sex Ratio of Recruits
Figure 12. Sex ratio of recruits for each treatment
enclosure and mean sex ratio of recruits for the four
control enclosures in Experiment II.
30
Treatment
Control
Juvenile Recruitment
Figure 13. Mean proportion of juvenile recruits
per adult female for treatment and control
enclosures in Experiment II.
80
70 60 50 -
Spray
40 -
30 20 10 0
1 Sept
7 Oct
21 Oct
3 Nov
17 Nov
Figure 14. Mean population size using Minimum Number
Alive (MNA) in treatment and control enclosures in
Experiment II.
31
course of the experiment was similar for treatment and control
enclosures.
32
DISCUSSION
Although statistically significant differences did occur between
treatment and control populations for some reproductive variables,
results from Experiment I and Experiment II provided no biologically
meaningful evidence to suggest that vinclozolin had an effect on the
reproductive development of gray-tailed voles or that any reproductive
anomalies had demographic consequences. The seminal vesicles were
the only reproductive organs with a strong difference between treatment
and control animals. Even then, the mean length of seminal vesicles for
voles in treatment enclosures in Experiment I was >12 mm, which is
greater than the 7-8 mm long seminal vesicles that are adequate for
reproduction (Wolff et al., 1994). The fact that greater than 60% of
females in both experiments became pregnant by males exposed to
vinclozolin while they were in utero also shows that the males were
capable of reproducing. Given more time, most likely males would have
impregnated all or most females in all enclosures.
Although the results of both experiments provided no biologically
meaningful evidence to support the hypothesis that in utero exposure to
vinclozolin will result in abnormal reproductive development of male
voles, trends in the data suggest that perhaps a higher or repeated
exposure of vinclozolin to wildlife during sensitive stages of reproduction
could create more severe effects on male reproductive development.
Differences in sizes of reproductive organs and the trend that occurred
33
with many of the variables where measurements from treatment animals
were lower than control animals provided evidence of slight physiological
impairment to male reproductive development. These results suggest
that with a repeated application, as often occurs on many agricultural
crops (BASF, 1997), more severe effects on reproductive development of
male voles could occur. If severe malformations were to occur in
individuals in any species of wildlife, it could be detrimental to sensitive
populations.
A lower exposure to the chemical was expected to occur in the first
experiment than in the second experiment due to the rainy weather and
the tall grass. However, results from the second experiment conducted
under dryer conditions with shorter grass suggested that this might not
have been the case. Only Experiment I revealed any statistically
significant signs of endocrine disruption. Past field experiments using
gray-tailed voles also revealed more significant effects in enclosures
treated with the insecticide azinphos-methyl when a considerable
amount of rainfall occurred within 48 hours of chemical application
(Schauber et al., 1997). One explanation could be that precipitation
washed the chemical down to the level of the animals, therefore resulting
in a higher dietary exposure by physical contact and ingestion from
grooming. A second possible explanation is that precipitation increased
absorption of metabolites into the grass by exposing the root system to
the chemical (Szeto et al., 1989) and therefore increasing the dietary
34
exposure to the animals. The reason for less significant differences
between control and treatment populations in the second experiment
may be attributable to dryer weather conditions after the chemical
application. Unfortunately, fungal pathogens thrive in moist, humid
environments and consequently vinclozolin is most commonly applied
under these conditions and throughout the growing season.
Based on observations from Experiment I and Experiment II, in
which field populations of voles were exposed to a low concentration of
vinclozolin and experienced marginally adverse effects on sexual
differentiation, it may be possible that similar effects would also occur in
other mammalian wildlife species. The sensitivity of gray-tailed voles to
some agricultural chemicals has been documented in the laboratory and,
based on LD50 values, gray-tailed voles appear to be less susceptible to 6
of 10 tested chemicals, equally to three, and more susceptible to one
than laboratory rodents (Cholakis et al., 1978). For 7 out of 10
chemicals, gray-tailed voles were at least twice as sensitive as four other
Microtus species (Cholakis et al., 1978). Previous field experiments
conducted in the Willamette Valley of western Oregon demonstrated that
the gray-tailed vole was less sensitive to chemicals than other native
small mammal species such as wild house mice (Mus musculus) and deer
mice (Peromyscus maniculatus; Meyers and Wolff, 1994). Therefore, if
vinclozolin has slight physiological effects on the reproductive
development of male gray-tailed voles, the effects to other Microtus
35
species could be less severe, but more severe for house mice and deer
mice. However, species react differently to various chemicals and
extrapolating from one species or from one chemical to other species and
chemicals is not always warranted.
36
CONCLUSION
I conducted two experiments in which one standard application of
the fungicide vinclozolin was applied to grassland habitat containing
populations of gray-tailed voles. No biologically significant impairment
was observed in reproductive development of male voles whose mothers
were exposed to the treatment while the young were in utero. In the first
experiment in which the chemical was applied to relatively tall grass and
in which precipitation occurred within 24 hours after application, a
consistent trend occurred in the data in which treatment animals had
slightly, but often not statistically, smaller reproductive organs than
control animals. This difference was not noted in the second experiment,
which was conducted in shorter grass and under drier conditions.
Therefore, the rainy conditions may have increased the exposure of the
voles to the chemical, either due to washing the chemical down to ground
level or to greater uptake by vegetation. However, male reproductive
organs were sufficiently developed in both experiments to result in high
reproductive rates and juvenile recruitment in the field with no effects on
population growth or demography. Under the conditions of this study,
one standard application of Curalan® fungicide had no measurable
reproductive or demographic consequences on gray-tailed voles.
However, the results suggest that higher application rates or several
successive applications may have negative impacts on reproductive
development and demography of wild vole populations.
37
BIBLIOGRAPHY
Aguilar A, Borrell A. 1994. Abnormally high polychlorinated biphenyl
levels in striped dolphins (Stenella coeruleoalba) affected by the
1990-92 Mediterranean epizootic. The Science of the Total Environ.
154:237-247.
Aguilar A, Raga J. 1993. The striped dolphin epizootic in the
Mediterranean Sea. Ambio. 22:524-528.
Aulerich R, Ringer R, Iwamoto S. 1973. Reproductive failure and
mortality of mink fed on Great Lakes fish. J Reprod Fert Suppl.
19:365-376.
BASF Corporation. 1997. Product Information Guide. Research Triangle
Park, NC, USA.
Baker Matta M, Cairncross C, Kocan RM. 1998. Possible effects of
polychlorinated biphenyls on sex determination in rainbow trout.
Environ Toxicol Chem. 17:26-29.
Bennetts H, Underwood E, Shier F. 1946. A specific breeding problem of
sheep in subterrainean clover pastures in western Australia. Aust
Veter J. 22:2-12.
Beyler SA, Zaneveld LJD. 1982. The male accessory sex glands. In
Zaneveld LJD, Chatterton RT, eds, Biochemistry of Mammalian
Reproduction. John Wiley & Sons, Inc., New York, NY, USA, pp 6588.
Birnbaum LS. 1995. Developmental effects of dioxins. Environ Health
Perspect. 103:89-94.
Carson R. 1962. Silent Spring. Houghton-Mifflin. Boston, MA, USA.
Cholakis JM, Wong LCK, Lee CC. 1978. Study of the chemical and
behavioral toxicology of substitute chemicals in microtine rodents.
EPA/600/02 EPA-ORD. Final Report. United States Environmental
Protection Agency, Corvallis, OR, USA.
Colborn T, vom Saal FS, Soto AM. 1993. Developmental effects of
endocrine-disrupting chemicals in wildlife and humans. Environ
Health Perspect. 101:378 -384.
Colborn T. 1995. Environmental estrogens: health implications for
humans and wildlife. Environ Health Perspect. 103:135-136.
Couture LA, Harris MW, Birnbaum LS. 1990. Characterization of the
peak period of sensitivity for the induction of hydronephrosis in
C57BL/6N mice following exposure to 2,3,7,8-TetrachlorodibenzoP-Dioxin. Fundamental Appl Toxicol. 15:142-150.
38
Crain DA, Guillette LJ, Rooney Jr AA. 1997. Alterations in
steroidogenesis in alligators (Alligator mississippiensis) exposed
naturally and experimentally to environmental contaminants.
Environ Health Perspect. 105:528-533.
Del Re A, Fontana P, Meloni GP, Natali P. 1980. Residues of vinclozolin
in grapevine musts after grey mold control. Annu Fac Agar Univ
Cattol Sacro Cuore. 20:171-181.
Dietz R, Heide-Jorensen MP, Harkonen T. 1989. Mass death of harbor
seals (Phoca vitulina) in Europe. Ambio. 18:258-264.
Edge WD, Wolff JO, Carey RL. 1995. Density-dependent responses of
gray-tailed voles to mowing. J Wildl Manage. 59:245-251.
Edge WD, Carey RL, Wolff JO, Ganio LM, Manning T. 1996. Effects of
Guthion 2S on Microtus canicaudus: a risk assessment validation.
J Appl Ecol. 33:269-278.
Ewing LL, Thompson DL Jr, Cochran RC, Lasley BL, Thompson MA,
Zirkin BR. 1984. Testicular androgen and estrogen secretion and
benign prostatic hyperplasia in the beagle. Endocrinology.
114:1308-1314.
Fox G. 1992. Epidemiological and pathological evidence of contaminantinduced alterations in sexual development in free-living wildlife. In
Colborn T, Clement C, eds, Chemically Induced Alterations in
Sexual and Functional Development: The Wildlife/ Human
Connection. Princeton Scientific Publishing, Princeton, NJ, USA, pp
147-158.
Fry DM, Toone CK. 1981. DDT-induced feminization of gull embryos.
Science. 231:919-924.
Fry DM. 1995. Reproductive effects in birds exposed to pesticides and
industrial chemicals. Environ Health Perspect. 103:165-171.
Gennari N, Zanini E, Cignetti A, Bicchi C, Amato AD, Taccheo MB,
Spessotto C, De Paoli M, Flori P, Imbroglini C, Leandri A, Conte E.
1985. Vinclozolin decay on different grapevines in four differing
italian areas. J Agric Food Chem. 33:1232-1237.
Gray LE Jr, Kelce WR, Monosson E, Ostby JS, Birnbaum LS. 1995.
Exposure to TCDD during development permanently alters
reproductive function in male LE rats and hamsters: Reduced
ejaculated and epididymal sperm numbers and sex accessory
gland weights in offspring with normal androgenic status. Toxicol
Appl Pharmacol 131:108-118.
Gray LE, Ostby JS, Kelce WR. 1994. Developmental effects of an
environmental antiandrogen: The fungicide vinclozolin alters sex
differentiation of the male rat. Toxicol Appl Pharmacol. 129:46-52.
39
Gray LE Jr, Ostby J, Wolf C, Lambright C, Kelce W. 1998. The value of
mechanistic studies in laboratory animals for the prediction of
reproductive effects in wildlife: Endocrine effects on mammalian
sexual differentiation. Environ Toxicol Chem. 17:109-118.
Guillette LJ Jr, Gross TS, Rasson GR, Matter JM, Percival HF, Woodward
AR. 1994. Developmental abnormalities of the reproductive system
of alligators (Alligator mississippiensis) from contaminated and
control lakes in Florida. Environ Health Perspect. 102:680-688.
Guillette LJ Jr. Crain DA, Rooney AA, Pickford DB. 1995a. Organization
versus activation: The role of endocrine-disrupting contaminants
(EDCs) during embryonic development in wildlife. Environ Health
Perspect. 103:157-164.
Guillette LJ Jr, Gross TS, Gross DA, Rooney AA, Percival HF. 1995b.
Gonadal steroidogenesis in vitro from juvenile alligators obtained
from contaminated and control lakes. Environ Health Perspect.
103:31-36.
Haake J, Kelley M, Keys B, Safe ST. 1987. The effects of organochlorine
pesticides as inducers of testosterone and benzo(a)pyrene
hydroxytases. Gen Pharmacol. 18:165-169.
Harman SM. 1978. Clinical aspects of aging of the male reproductive
system. In Schneider EL, ed, The Aging Reproductive System
(Aging, Volume 4). Raven Press, New York, NY, USA.
Hasler JF. 1975. A review of reproduction and sexual maturation in the
microtine rodents. Biologist. 57:52-86.
Henney C, Grove R, Hadsrom 0. 1997. Hypoplastic reproductive organs
related to xenobiotic compounds in young male river otter from
Columbia River. Proceedings, Oregon Chapter of The Wildlife
Society, Sun River, OR, USA, February 11-14, 1997, p. 17.
Hoffman DJ, Rattner BA, Hall RJ. 1990. Wildlife toxicology 3. Environ Sci
and Technol. 24:276-283.
Hubbard JC, Subbarao KV, Koike ST. 1997. Development and
significance of dicarboximide resistance in Sclerotinia minor
isolates from commercial lettuce fields in California. Plant Disease.
81:148 -153.
Iguchi T, Fukazawa Y, Bern HA. 1995. Effects of sex hormones on
oncogene expression in the vagina and on development of sexual
dimorphism of the pelvis and anococcygeus muscle in the mouse.
Environ Health Perspect. 103:79-82.
Kavlock RJ, Daston GP, DeRosa C,Fenner-Crisp P, Gray LE, Kaattari S,
Lucier G, Luster M, Mac MJ, Maczka C, Miller R, Moore J, Rolland
R, Scott G, Sheehan DM, Sinks T, Tilson HA. 1996. Research
needs for the risk assessment of health and environmental effects
40
of endocrine disruptors: A report of the U.S. EPA-sponsored
workshop. Environ Health Perspect. 104:715-740.
Kelce WR, Monosson E, Gamcsik MP, Laws SC, Gray LE Jr. 1994.
Environmental hormone disruptors: evidence that vinclozolin
developmental toxicity is mediated by antiandrogenic metabolites.
Toxicol Appl Pharmacol. 127:276-285.
Keller BL. 1985. Reproductive patterns. In Tamarin RH, ed, Biology of
New World Microtus. Amer Soc Mammal, Boston, MA, USA, pp.
725-778.
Kendall RJ, Akerman J. 1992. Terrestrial wildlife exposed to
agrochemicals: An ecological risk assessment perspective. Environ
Toxicol Chem. 11:1727-1749.
Kevekordes S, Gebel T, Pay K, Edenharder R, Dunkelberg H. 1996.
Genotoxicity of selected pesticides in the mouse bone-marrow
micronucleus test and in the sister-chromatid exchange test with
human lymphocytes in vitro. Toxicology Letters. 89:35-42.
Laws SC, Carey SA, Kelce WR, Cooper RL, Gray LE Jr. 1996. Vinclozolin
does not alter progesterone receptor (PR) function in vivo despite
inhibition of PR binding by its metabolites in vitro. Toxicology.
112:173-182.
Lodovici M, Casalini C, Briani C, Dolara P. 1997. Oxidative liver DNA
damage in rats treated with pesticide mixtures. Toxicology. 117:5560.
Lundkvist U. 1990. Clinical and reproductive effects of Clophen A50
(PCB) administered during gestation of pregnant guinea pigs and
their offspring. Toxicology. 61:249-257.
Mabley TA, Bjerke DL, Moore RW, Gendron-Fitzpatrick A, Peterson RE.
1992a. In utero and lactational exposure of male rats to 2,3,7,8tetrachlorodibenzo-p-dioxin. 3: Effects on spermatogenesis and
reproductive capability. Toxicol Appl Pharmacol. 114:118:126.
Mabley TA, Moore RW, Peterson RE. 1992b. In utero and lactational
exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin: 1.
Effects on androgenic status. Toxicol Appl Pharmacol. 114:97-107.
Martin C, Davet P, Vega D, Coste C. 1991. Field effectiveness and
biodegradation of cyclicimides in lettuce field soils. Pestic Sci.
32:427-438.
Mason C, Ford T, Last N. 1986. Organochlorine residues in British
Otters. Bull Environ Contam Tox. 36:656-661.
McTavish K, Stech H, Stay F. 1998. A modeling framework for exploring
the population-level effects of endocrine disruptors. Environ Toxicol
Chem. 17:58-67.
41
Meyers SM, Wolff JO. 1994. Comparative toxicity of azinphos-methyl to
house mice, laboratory mice, deer mice, and gray-tailed voles. Arch
Environ Contam Toxicol. 26:478-482.
Michailides TJ, Morgan DP. 1996. Using incidence of Botrytis cinerea in
kiwifruit sepals and receptacles to predict gray mold decay in
storage. Plant Disease. 80:248-254.
Migliaccio S, Newbold RR, McLachlan JA, Korach KS. 1995. Alterations
in estrogen levels during development affects the skeleton: use of
an animal model. Environ Health Perspect. 103:95-97.
Nadeau JH. 1985. Ontogeny. In Tamarin RH, ed, Biology of New World
Microtus. Amer Soc Mammal, Boston, MA, USA, pp. 254-285.
Norheim G, Skaare J, Wiig 0. 1992. Some heavy metals, essential
elements, and chlorinated hydrocarbons in polar bears (Ursus
maritimus) in Svalbard, Norway. Environ Poll. 77:51-57.
Norris DO. 1997. Vertebrate Endocrinology 37d Edition. Academic Press,
San Diego, CA, USA.
Olson JR, McGarrigle BP, Tonucci DA, Schecter A, Eichelberger H. 1990.
Developmental toxicity of 2,3,7,8-TCDD in the rat and hamster.
Chemosphere 20:1117.
Peterson JA. 1996. The effects of inbreeding and environmental stressors
on the demography of the gray-tailed vole. PhD thesis. University of
California, Berkeley, CA, USA.
Peterson RE, Theobald HM, Kimmel GL. 1993. Developmental and
reproductive toxicity of dioxins and related compounds: crossspecies comparisons. Crit Rev Toxicol. 23:283-335.
Pimentel DS, Acquay H, Biltonen M, Rice P, Silva M, Nelson J, Lipner V,
Giordano S, Horowitz A, Amore MD. 1992. Environmental and
economic costs of pesticide use. BioScience. 42:750-760.
Quay, WB. 1968. The specialized postero-lateral sebaceous glandular
regions in microtine rodents. J Mammal. 44:537-542.
Ramsey FL, Schafer DW. 1997. The Statistical Sleuth: A Course in
Methods of Data Analysis. Wadsworth Publishing Company,
Belmont, CA, USA.
Safe SH. 1995. Environmental and dietary estrogens and human health:
Is there a problem? Environ Health Perspect. 103:346-351.
Sager DB, Shih-Scaroeder W, Girard D. 1987. Effect of early postnatal
exposure to poly-chlorinated biphenyls (PCBs) on fertility in male
rats. Bull Environ Contam Tox. 38:946-953.
42
Schauber EM, Edge WD, Wolff JO. 1997. Insecticide effects on small
mammals: Influence of vegetation structure and diet. Ecological
Applications. 7:143-157.
Schmidt WA. 1994. Hormone Copycats. Final Report. National Wildlife
Federation, Great Lakes Natural Resource Center, Ann Arbor, MI,
USA.
Scrano L, Faretra F, Cariddi C, Antonacci E, Bufo SA. 1990. Evaluation
of dicarboximide residues in cold-stored grapes exposed to field
and post-harvest treatments. Pestic Sci. 31:37-44.
Seabloom RW. 1985. Endocrinology. In Tamarin RH, ed, Biology of New
World Microtus. Amer Soc Mammal, Boston, MA, USA, pp. 685724.
Sharpe RM, Fisher JS, Millar MM, Jobling S, Sumpter JP. 1995.
Gestational and lactational exposure of rats to xenoestrogens
results in reduced testicular size and sperm production. Environ
Health Perspect. 103:1136-1143.
Szeto SY, Burlinson NE, Rahe JE, Oloffs PC. 1989. Persistence of the
fungicide vinclozolin on pea leaves under laboratory conditions. J
Agric Food Chem. 37:529-534.
Tagasugi N. 1976. Cytological basis for permanent vaginal changes in
mice treated neonatally with steroid hormones. Int Rev Cytol.
44:194-224.
Verts BJ, Carraway LN. 1987. Microtus canicaudus. Mammalian Species.
267:1-4.
Walker A. 1987. Enhanced degradation of iprodinone and vinclozolin in
soil: A simple colormetric test for identification of rapid-degrading
soils. Pestic Sci. 21:233-240.
Wolff JO. 1985. Behavior. In Tamarin RH, ed, Biology of New World
Microtus. Amer Soc Mammal, Boston, MA, USA, pp. 340-372.
Wolff JO, Lidicker WZ Jr. 1980. Population ecology of the taiga vole,
Microtus xanthognathus, in interior Alaska. Can J Zool. 58:18001812.
Wolff JO, Edge WD, Bentley R. 1994. Reproductive and behavioral
biology of gray-tailed voles. J Mammal. 75:873-879.
Wong C, Kelce WR, Sar M. 1995. Androgen receptor antagonist versus
agonist activities of the fungicide vinclozolin relative to
hydroxyflutamide. J Biol Chem. 270:19998-20003.
Zaneveld LJD. 1982. The epididymis. In Zaneveld LJD, Chatterton RT,
eds, Biochemistry of Mammalian Reproduction. John Wiley 85 Sons,
Inc., New York, NY, USA, pp 37-63.