INFLUENCE OF EMBRYONIC EXPOSURE TO
HEXABROMOCYCLODODECANE (HBCD) ON THE CORTICOSTERONE
RESPONSE AND “FIGHT OR FLIGHT” BEHAVIOURS OF CAPTIVE
AMERICAN KESTRELS
Demetrios Kobiliris
Department of Natural Resource Sciences
McGill University, Montreal
June 2010
A thesis submitted to McGill University in partial fulfilment of
the requirements of the degree of Masters of Science (M.Sc.)
© Demetrios Kobiliris 2010
TABLE OF CONTENTS
LIST OF FIGURES ……………………………………………………….......................iv
ACKNOWLEDGEMENTS ………………………………………………………….….vi
CONTRIBUTIONS OF CO-AUTHORS ………………………………………………. x
ABSTRACT ……………………………………………………………………………..xi
RÉSUMÉ ………………………………………………………………………………..xii
CHAPTER 1 – Literature review and research rationale and aim and objectives
Brominated Flame Retardants ………………………...………………………….1
Chemical structure and properties of BFRs ………………………………….…...2
HBCD concentrations in predatory bird species ………………………………….2
Effects of HBCD on non-avian vertebrate species ……………………………….3
Stress response and corticosterone ……………………………………………….4
The American kestrel (F. sparverius) …..………………………………………...6
Aims and objectives of thesis …………………………………………………….8
CONNECTING STATEMENT …………………………………………………………..9
CHAPTER 2 – Effects of HBCD on corticosterone and associated behaviours in juvenile
American kestrels following embryonic exposure to environmentally relevant
concentrations
Abstract ………………………………………………………………………….10
Introduction ……………………………………………………………………...11
Materials and methods …………………………………………………………..14
Study site and details ……………………………………………………14
ii
Exposure and dosing concentrations of HBCD …………………………14
Chemical analysis of HBCD in eggs..………………………………………..16
Animal husbandry ……………………………………………………….18
Corticosterone response…………………………………………………18
Corticosterone radioimmunoassay analysis……………………………..19
Hunting behaviours and predator-response behaviours ………………..20
Statistical analysis ………………………………………………………22
Results …………………………………………………………………………..23
Corticosterone response ………………………………………………...23
Hunting behaviour ………………………………………………………24
Response to predators …………………………………………………...25
Discussion ………………………………………………………………………25
FINAL SUMMARY AND CONCLUSION ……………………………………………37
LITERATURE CITED ……………………………………………………………….....39
APPENDIX A – Animal Care Committee Approval Certificates ……………………....46
iii
LIST OF FIGURES
CHAPTER 2:
Fig. 1. The plasma corticosterone concentrations and response of (A) male and
(B) female American kestrel nestlings (d 25) to a stress challenge protocol
following their in ovo exposure to environmentally relevant concentrations
of hexabromocyclododecane (HBCD). ………………………………….31
Fig. 2. Flying activity levels of male juvenile American kestrels during hunting
behaviour trials are correlated with in ovo hexabromocyclododecane
(HBCD) concentrations. .………………………………….…………….32
Fig. 3. Differences in the hunting behaviours of juvenile American kestrels
between those kestrels exposed in ovo to hexabromocyclododecane
(HBCD) and control birds: (A) head-bobbing of males, (B) head-snapping
of males and (C) females visiting the cricket arena. Statistical analysis
involved ANCOVA with in ovo HBCD concentrations used as the
covariate. .………………………………………………………………..33
Fig. 4. Differences in predator avoidance behaviours of juvenile female American
kestrels when exposed in ovo to hexabromocyclododecane (HBCD or to
control vehicle: (A) kleeing calls and (B) time delay in first response to
predator. Statistical analysis involved ANCOVA with in ovo HBCD
concentrations used as the covariate. ……………………………………35
iv
Fig. 5. The response time of female juvenile American kestrels to a mounted
predator is correlated with in ovo hexabromocyclododecane (HBCD)
concentrations. ……………..……..……………………………………..36
v
ACKNOWLEDGEMENTS
I would like to thank my supervisors, Professor David M. Bird and Adjunct
Professor Kimberly J. Fernie, who helped formulate the original design of this study,
tirelessly and patiently guided me throughout the project with their invaluable support
and advice and substantially helped me with data analysis, thesis writing and revising. I
owe both of them my gratitude for allowing me to embark on the adventurous and often
difficult voyage of the scientific study of birds. Thanks to Dr. Bird, I was fortunate
enough to gain employment at the Avian Science and Conservation Centre of Macdonald
Campus, McGill University, where I gained much hands-on experience with the
American kestrel, my study subject, as well as making good friends. In addition, I was
provided with a contract for my research from Environment Canada through Dr.
Kimberly Fernie.
To the first person I met when I arrived in Canada, Ian J. Ritchie, I would like to
extend my deepest gratitude. Ian was not only the dedicated technician for my
experimental project at the Avian Centre, making sure that everything was planned and
organized, but is also one of the best persons I have come to know, always ready to share
his vast experience and knowledge of birds and always ready to listen and offer his
intuitive ideas on any given topic. This project would not have been feasible without his
help and guidance.
My fellow graduate students, collectively known as the “Birdcagers”, were
always there to provide advice and ideas on my research topic, and I am most grateful to
them for their support, help and friendship. I especially want to thank the following
vi
Birdcagers: Lina Bardo, Shawn Craik and Kristen Keyes for sharing their knowledge and
love of birds, but also for being such good friends; Katie Sullivan for helping me with
the initial setup of my experiment; Sarah Marteinson for discussing various aspects of
my project as well as helping me with blood-sampling of kestrels and data analysis; and
Marie-Anne Hudson, Barbara Frei, Dave Fishman, Tiffany Gilchrist, Dominique Chabot,
Raphaël Goulet, Samuel Denault, Marcel Gabhauer and Mike Ross for allowing me to
learn so much from them.
Jöelle Guillet and Janina Heim, my two assistants for blood-sampling and
behavioural observations of kestrels at the Avian Centre, were enthusiastic and basically
invaluable in helping me complete the experimental part of my study and I am indebted
to them for their work with me.
Dr. Robert Letcher of the National Wildlife Research Centre, Environment
Canada in Ottawa, supervised the chemical analysis of the in ovo HBCD egg samples and
hence provided the HBCD concentrations for my research. These chemical data were
critical to completing my thesis research. France Maisonneuve oversaw the analysis of
the corticosterone blood samples in her laboratory at the National Wildlife Research
Centre, Environment Canada in Ottawa, and provided the resulting data and analytical
information, without which much of the findings would not have been possible.
I would also like to thank Luciano Germani, a McGill IMS technician, who
provided the video camera for recording the first part of my behavioural experiments, but
also for being a great person, willing to listen and help so many other times.
Financial support for this study was obtained through Dr. David Bird’s NSERC
Discovery grant, Ecotoxicology and Wildlife Health (Science and Technology Branch) as
vii
well as the Chemical Management Plan of Environment Canada through Dr. Kimberly
Fernie and work at the Avian Science and Conservation Centre.
It has been a privilege for me to have been one of the last students of Dr. Rodger
Titman, who was for me an excellent teacher, an inspirational researcher and scientific
thinker and an amazing person, full with understanding and wisdom.
Dr. Chris Buddle, Dr. Terry Wheeler and Dr. Pierre Dutilleul, as well as various
fellow graduate students of the Department of Natural Resource Sciences (NRS)
provided important input to my project at various points during my studies and I thank
them deeply for this.
Dr. Ian Strachan was extremely helpful during the final process of my thesis
submission acting as unit head of NRS.
The French translation of the thesis abstract was made possible through the help
of CH-Kay Traductions Scientifics Inc. and McGill graduate student Eric Cristensen, and
I thank them for their contribution.
I am very grateful to Marie Kubecki of NRS for providing efficient assistance and
support for my studies from start to finish.
Dr. Paschalis Giannoulis helped me enormously during my studies and stay at
McGill, providing his support, friendship and scientific knowledge.
I would like to thank ornithologist Ben Hallmann, for having inspired me to study
birds, as well as ornithologist, Thodoros Kominos, and friend, Yiouli Hasapoyianni, for
encouraging me to pursue graduate studies on this topic.
I am very grateful to my former professors from the University of Aegean,
Department of Environment, Dr. Michalis Angelidis, Dr. Ioannis Hatzopoulos and
viii
Androniki Hatzopoulou-Karasmanoglou, for their kindest words of reference and support
regarding my graduate studies at McGill.
My best friends, Nikos Pappas and Yiannis Maroulakis, have always supported
me and encouraged me both before and during my studies in Canada and, although being
so far away, I am indebted to them for their friendship.
Finally, I dedicate this thesis to the true heroes of my life, my parents, Yiannis
and Stamata Kobiliris, and my brother Vangelis, who believed in me even at times when
I could not, and who have always supported me in the pursuit of my dreams and
embraced me with their love.
ix
CONTRIBUTIONS OF CO-AUTHORS
This thesis contains a single manuscript that will be submitted to a refereed
journal for publication. I am the primary author of this manuscript and my cosupervisors, David M. Bird and Kimberly J. Fernie, will be second and last authors,
respectively. In addition, Ian J. Ritchie and Robert J. Letcher will be third and fourth
authors, respectively. Kim Fernie and J. Laird Shutt were the principal investigators of
this research under the auspices of Environment Canada. Kim Fernie and David Bird both
assisted in the development of my research ideas and provided guidance and funding for
myself and the overall research. In addition, Kim Fernie helped substantially with data
analysis, as well as thesis writing and editing, and provided critical funding through
Environment Canada. David Bird also provided financial support via his NSERC
discovery grant and the Avian Science and Conservation Centre and contributed to the
writing and editing of the thesis. Without the invaluable assistance of Ian Ritchie with his
over 30 years of experience at the Avian Science and Conservation Centre, this study
would not have been possible. Robert Letcher supervised the analysis of the in ovo
HBCD egg samples and France Maisonneuve oversaw the analysis of the corticosterone
blood samples in their respective laboratories at the National Wildlife Research Centre in
Ottawa, and provided the resulting data and analytical information, without which much
of the findings would not have been possible.
x
ABSTRACT
Hexabromocyclododecane (HBCD) is a brominated flame retardant commonly used in
industrial and household products to reduce the spread of fire and the risk of death.
Similar to polybrominated diphenyl ethers (PBDEs), HBCD is a ubiquitous and persistent
environmental contaminant, highly lipophilic and bioaccumulative, and has been detected
throughout various ecosystems and phyla, thus posing potential cause for concern for top
consumer species, including birds of prey. Captive adult American kestrels (Falco
sparverius) were exposed by diet to HBCD at an estimated daily concentration of 0.8 μg
HBCD / μL safflower oil per cockerel (or 800 ng/ g ww / day / pair); their offspring, used
in this study, were exposed in ovo only to environmentally relevant HBCD
concentrations of 164.13 ± 18.26 ng/g ww or to background concentrations in the control
eggs (0.4 ± 0.04 ng/g ww). The in ovo exposed HBCD group of male nestling kestrels
showed a reduced corticosterone response. Moreover, in ovo HBCD concentrations were
correlated with reduced flying activities of juvenile males during hunting behaviour trials
and delayed response times of juvenile female kestrels during predator avoidance
behaviour trials, suggesting an ongoing effect of HBCD on corticosterone levels. These
findings show that embryonic exposure to environmentally relevant concentrations of
technical mixture HBCD influences the corticosterone response, hunting success and
avoidance of potential predators in captive American kestrels.
xi
RÉSUMÉ
L'Hexabromocyclododécane (HBCD) est un ignifuge bromé couramment utilisé dans les
produits industriels et ménagers pour réduire les risques d'incendies et de mortalité. À
l'instar des éthers diphényliques polybromés (EDPB), le HBCD est un polluant
omniprésent et persistant de l'environnement, fortement lipophile et bioaccumulable,
décelé dans divers écosystèmes et phylums et donc potentiellement préoccupant pour les
espèces du haut de la chaîne alimentaire, dont les oiseaux de proie. L'étude consiste à
exposer par leur alimentation des crécerelles d'Amérique (Falco sparverius) adultes en
captivité à une concentration quotidienne estimée de 0,8 mg HBCD / μL d'huile de
carthame par coquelet (c.-à-d. 800 ng / g pf/ jour / paire); leur progéniture, utilisée dans
cette étude, ont été exposés in ovo à des concentration écologiquement pertinentes de
HBCD 164,13 ± 18,26 ng / g pf ou des concentrations de fond (0,4 ± 0,04 ng / g pf) dans
le cas des œufs de contrôle . Chez les oisillons mâles du groupe exposé au HBCD in ovo,
il y a réduction de la sécrétion de corticostérone. En outre, les concentrations de HBCD
in ovo sont corrélées à une réduction des activités de vol chez les mâles juvéniles durant
des essais de comportement de chasse et des temps de réaction accrus chez les femelles
juvéniles durant des essais de comportement d’évitement de prédateurs, ce qui suggère un
effet continu du HBCD sur les niveaux de corticostérone. Ces constatations démontrent
que l’exposition embryonnaire à des concentrations écologiquement pertinentes de
HBCD dans un mélange technique a une incidence sur le taux de sécrétion de
corticostérone, le taux de réussite des activités de chasse et l’évitement de prédateurs
potentiels chez la crécerelle d’Amérique.
xii
CHAPTER 1 – Literature review and research rationale and aim and objectives
Brominated Flame Retardants
Brominated flame retardants (BFRs) are used in plastics, textiles, electronic
circuitry and equipment, construction, industrial and household products to reduce the
risk of injuries and property damage related to fire (de Wit 2002). BFRs are classified
into five major classes, one of which is the polybrominated diphenyl ethers (PBDEs),
involving 209 individual congeners. Another class of BFRs are the cyclododecanes,
including hexabromocyclododecane (HBCD) which is commonly used in Europe and less
so in North America (Birnbaum and Staskal 2004).
PBDEs come in three major commercial mixtures: penta-BDE, octa-BDE, and
deca-BDE. In 2001, the global market demand of these three mixtures was 11%, 6%, and
83%, respectively (La Guardia et al. 2006). Penta-BDE and octa-BDE were banned by
the European Union in 2004 (Costa and Giordano 2007) and production was voluntarily
stopped at the same time in North America by the U.S. producer (Renner 2004); their use
was also banned in Canada in 2006 (Canada Gazette 2006).
HBCD remains widely used in Europe. The commercial technical HBCD mixture
consists of three technical stereo-isomers: alpha-, beta- and predominantly gammaHBCD (Birnbaum and Staskal 2004).
1
Chemical structure and properties of BFRs
BFRs are considered to be persistent, environmental contaminants. Many PBDEs
and non-PBDE BFRs are additive flame retardants, i.e. they are mixed with, but not
bound to, other polymers to make final products (Birnbaum and Staskal 2004).
Consequently, they separate from these products, leaching into the environment whereby
they can be transported long distances, e.g., the Arctic, via atmospheric deposition and
through the aquatic ecosystem. They are highly stable in nature with an ability to
withstand degradation by acids or bases, heat, light, and reducing or oxidizing agents
(Rahman et al. 2001). These PBDE and some non-PBDE compounds are highly
lipophilic and bioaccumulative, and some PBDEs have been classified as “endocrine
disruptors.” The aforementioned characteristics of PBDEs and HBCD are critically
important and of particular concern to species at the top of the food chain such as birds of
prey (Law et al. 2003).
HBCD concentrations in predatory bird species
HBCD concentrations have been detected in the tissues, including the eggs, of a
number of predatory bird species. In peregrine falcons (Falco peregrinus), geometric
means of HBCD concentrations measured in eggs ranged from 100-110 ng/g lipid weight
(lw) in Sweden (Johansson et al. 2009) and median HBCD concentrations were 2.4 ng/g
lw in south Greenland (1986-2003; Vorkamp et al. 2005). HBCD was detected in only
two of 40 eggs of Little Owls (Athene noctua) in Belgium, and these HBCD
concentrations were 20 and 50 ng/g lw (Jaspers et al. 2005). In South Africa, the highest
in ovo HBCD concentration detected was 71 ng/g lw in the egg of an African sacred ibis
2
(Threskiornis aethiopicus; Polder et al. 2008). In the Arctic, HBCD concentrations in
dead or dying glaucous gulls (Larus hyperboreus) ranged from 200–15000 ng/g lw in the
liver and 5-500 ng/g lw in the brain (Sagerup et al. 2009). In the Great Lakes of North
America, α-HBCD concentrations were detected in the eggs of herring gulls (L.
argentatus) and ranged from 2.1 to 20 ng/g wet weight (ww) (Gauthier et al. 2007).
Although the commercial technical mixture of HBCD is dominated by the γ-HBCD
isomer, birds appear to metabolize the mixture so that α-HBCD is the only isomer
detected in tissues, including eggs.
Effects of HBCD on non-avian vertebrate species
Although HBCD has been detected in the tissues of many avian species, the
potential effects of such exposure is currently unknown for birds, including the very
sensitive embryonic stage. However, several studies have determined effects of HBCD
exposure on laboratory rats and mice.
Administration of HBCD via the diet to male and female rats for two generations
at levels of 1500 ppm and beyond affected the thyroid system, organs, growth and
viability of first and second generation rats (Ema et al. 2008). Thyroid effects of this high
level of HBCD exposure included an increased incidence of rats having decreased thyroid
follicle size, increased serum thyroid-stimulating hormone (TSH) levels but decreased
serum thyroxine (T4) levels, and an increase in absolute and relative weights of the
thyroid glands. Similarly, absolute and relative weights of the livers were also increased,
yet body weight and body weight gain were reduced in association with the rats’ reduced
3
food consumption, including that of the pups, which also demonstrated reduced viability
(Ema et al. 2008). A one-generation reproduction study in Wistar rats exposed via diet to
levels ranging from 0.1-100 mg HBCD/kg body weight affected bone mineral density,
immune function, and testes mass in the F1 animals (van der Ven et al. 2009).
The embryonic exposure of weanling rats to HBCD exerts developmental brain
effects, as well as changes in their thyroid glands as adults, when they were exposed as
embryos to HBCD concentrations beyond 100 ppm (Saegusa et al. 2009). A
complementary study involving young Wistar pups, littermates of those in a study by van
der Ven et al. (2009), also demonstrated effects of embryonic exposure to HBCD on their
auditory function as well as adverse effects on dopamine-dependent neurobehaviours
such as their righting responses (Lilienthal et al. 2009). While studies investigating the
effects of HBCD exposure are limited to date, there is clear evidence that HBCD at levels
beyond those found in predatory bird eggs affect neurobehaviours and some aspects of
the endocrine function, e.g. thyroid system, in laboratory animals. Whether HBCD has
impacts on other hormones in any vertebrate species is not yet known.
Stress response and corticosterone
Upon perception of a stress factor, animals can exhibit stress responses
orchestrated by the catecholamine hormones and the glucocorticoid hormones. The
catecholamine hormones (i.e. epinephrine and norepinephrine), which are produced and
stored in advance, are released very rapidly from the adrenal medullary cells (i.e.
chromaffin) and nerve terminals of the sympathetic nervous system. The release of the
4
catcholamines affects an individual’s behaviour, metabolism and cardiovascular system,
in what is known as the initial “fight or flight” response or catecholamine response. Thus,
the catecholamine response allows the individual to react within seconds to a sudden
threatening event (Wingfield et al. 1998), such being attacked by a predator, and then
resume their normal activity within minutes. The glucocorticoid hormones (i.e. cortisol
and corticosterone), are synthesized in the adrenal glands located above the kidneys, as
the final product of a hormonal cascade, called the hypothalamo-pituitary-adrenal axis
(HPA). The HPA hormonal cascade is initiated in the hypothalamus by the detection of a
stressor, and ends with the release of glucocorticoids in the blood serum. Most species
rely on either cortisol (i.e. fish and most mammals) or corticosterone (i.e. birds, reptiles,
amphibians and some rodents) in response to a stressor. Since the HPA involves multiple
glands producing multiple hormones, including the glucocorticoids, upon perception of
the stressor, the glucocorticoid response is slower, more sustained, and can only be
detected after more than 3-5 minutes from its initiation (Romero and Butler 2007). The
release of the glucocorticoids including corticosterone, affects immediate and long-term
behaviour, fuel metabolism, reproduction, growth and the immune system. Thus, the
glucocorticoid response allows the individual to pursue longer-term behavioural
strategies such as hiding, waiting out or fleeing from a persistent stressor, following on
from the initial “fight or flight” response (Romero and Butler 2007).
In birds, corticosterone is the principal glucocorticoid for mediating response to a
stress factor, and the HPA represents one of the most important regulatory pathways in
allowing them to adapt physiologically and behaviourally to unpredictable changes in the
5
environment by an endocrine mechanism known as the “corticosterone response”. Upon
perception of a stress factor, including a bird’s response to a predator or potential prey
item, the hypothalamus and other organs within the HPA release appropriate hormones,
including adrenocorticotropic hormone (ACTH), that in turn stimulates the adrenal cortex
to produce and release corticosterone into the blood serum, thereby stimulating
behavioural reactions and corresponding locomotory activity.
A study by Love et al. (2003b) involving male captive American kestrels exposed
to environmentally relevant concentrations (10 mg/kg body weight) of polychlorinated
biphenyls (PCBs) in their diet, and subjected to a standardized capture/handling/restraint
protocol to simulate their response to a physical stressor (Wingfield 1994), exhibited
lower baseline and stress-induced corticosterone levels in comparison to control birds of
the same age. Thus, it was shown that environmentally relevant levels of PCBs can
impair the corticosterone response of kestrels, possibly resulting in the birds becoming
increasingly prone to environmental stressors and potentially unable to adequately
respond to stimuli such as predators and prey.
The American kestrel (F. sparverius)
The American kestrel is a small bird of prey of the Falconidae family and the
most abundant and widespread falcon species in North America as well as in the northern
parts of South America. Their preferred habitat is open country with low-ground
vegetation and they have adapted to living close to human settlements as secondary
cavity-nesters, exhibiting a high-threshold for human disturbance (Smallwood and Bird
6
2002). They are sexually dimorphic both in plumage and in size, with females being 1030% larger than males (Smallwood and Bird 2002), and they produce semi-altricial
young that are easily sexed at 12 days of age (Bardo and Bird 2009).
The American kestrel feeds upon rodents, small birds and insects and, as well as
being preyed upon by larger birds of prey such as peregrine falcons and Cooper’s hawks
(Accipiter cooperii) (Smallwood and Bird 2002, Farmer et al 2006). Kestrels often use a
“sit-and-wait” hunting technique while perching, although they occasionally revert to
“hover-hunting”. When they locate potential prey, kestrels typically bob and snap their
heads (head-bobbing, head-snapping), and/or wag their tails (tail-wagging) to signify
their intention to fly to and pounce on their prey using their talons or beak (Balgooyen
1976, Smallwood and Bird 2002). Their ability to successfully hunt and capture their
prey is critical to their survival, and equally so is their ability to successfully detect and
avoid being captured by potential predators.
Due to its great accessibility by researchers, high tolerance of humans and
ecological affinity to larger predatory birds, such as the peregrine falcon and the bald
eagle (Haliaeetus leucocephalus) (Wiemeyer and Lincer 1987, Bardo and Bird 2009), the
American kestrel has proved to be an ideal candidate species to study the effects of
HBCD on birds in general as well as birds of prey. Its biology is well documented (Bird
1982, Bird and Bardo 2009) and it has been used in many toxicological studies (Bird and
Bardo 2009), including determination of the effects of other BFRs such as PBDEs (Fernie
et al. 2005, 2006, 2008, 2009).
7
Aims and objectives of thesis
The overall aim of my M.Sc. thesis research was to identify the potential effects
of in ovo exposure to environmentally relevant concentrations of the technical mixture of
HBCD on plasma corticosterone concentrations, predatory behaviours and predatory
responses of captured American kestrels.
Pairs from a captive colony with known pedigree (Bird 1982) were exposed by
diet to HBCD or to safflower oil as a control vehicle. Their nestlings were then used to
investigate the potential effects on their corticosterone levels and associated behavioural
responses in terms of capturing prey and exposure to predators. These results will help to
provide an important understanding of how exposure to environmentally relevant
concentrations of HBCD, a widely used BFR that has become a ubiquitous, persistent
contaminant found in bird eggs throughout the world, might affect the health and wellbeing of predatory birds.
8
CONNECTING STATEMENT
In the first chapter, a literature review of brominated flame retardants (BFRs) and
hexabromocyclododecane (HBCD) was presented, which included their use, chemical
structure and properties, release and occurrence in the environment, and the findings,
trends and effects in wildlife. To my knowledge there has not been any investigation into
the effects of HBCD on the corticosterone response in any vertebrate species.
Corticosterone is important for a number of functions, which includes the stress response
to unpredictable environmental events, such as successful hunting of potential prey and
avoidance of potential predators. Chapter 2 details my study investigating the
corticosterone response, hunting behaviours and predator avoidance behaviours in captive
American kestrels exposed in ovo to an environmentally relevant level of HBCD.
9
Chapter 2: Effects of hexabromocyclododecane (HBCD) on corticosterone and
associated behaviours in juvenile American kestrels following embryonic exposure
to environmentally relevant concentrations.
Abstract
Hexabromocyclododecane (HBCD) is a brominated flame retardant commonly used in
industrial and household products to reduce the risk of property fire, injury and death.
HBCD is a ubiquitous and persistent environmental contaminant, highly lipophilic and
bioaccumulative, detected throughout various ecosystems and phyla, thus posing
potential cause for concern for top consumers, including predatory birds. Captive adult
American kestrels (Falco sparverius) were exposed by diet to HBCD at an estimated
daily concentration of 0.8 μg HBCD / μL safflower oil per cockerel (or 800 ng/ g ww /
day / pair); their offspring, used in this study, were exposed in ovo only to
environmentally relevant HBCD concentrations of 164.13 ± 18.26 ng/g ww or to
background concentrations in the control eggs (0.4 ± 0.04 ng/g ww). The in ovo exposed
HBCD group of male nestling kestrels showed a reduced corticosterone response and
modifications in some hunting behaviours. Moreover, in ovo HBCD concentrations were
correlated with reduced flying activities of juvenile males during hunting behaviour trials
using live crickets, and with delayed response times of the juvenile female kestrels during
predator avoidance behaviour trials using a mounted hawk, suggesting an ongoing effect
of HBCD on corticosterone levels. These findings show that embryonic exposure to
environmentally relevant concentrations of technical HBCD influences the corticosterone
response, hunting success and avoidance of potential predators in American kestrels.
10
Keywords: American kestrels, HBCD, corticosterone response, hunting success, predator
avoidance
Introduction
Brominated flame retardants (BFRs) are additives used in the manufacturing of
plastics, textiles, electronic circuitry and equipment, construction, industrial and
household products; they essentially extend combustibility times of materials and most
importantly, reduce the risk of death of humans. They are considered to be persistent
environmental contaminants, mixed with, but not bound to, other polymers (Birnbaum
and Staskal 2004). Consequently, BFRs separate from manufactured products, leaching
into the environment whereby they can be transported long distances via atmospheric
deposition and the aquatic ecosystem. They are highly stable in nature (Rahman et al.
2001), and some PBDE and non-PBDE compounds have high lipophilicity and so
bioaccumulate in biota. Like other ubiquitous chemicals in the environment such as
organochlorines, PBDEs have been classified as “endocrine disruptors.”
Some of the major BFR commercial mixtures include penta-brominated diphenyl
ethers (PBDEs), octa-BDEs, and deca-BDE. Of these, penta-BDE and octa-BDE were
banned by the European Union in 2004 (Costa and Giordana 2007) and production was
voluntarily stopped at the same time in North America by the U.S. producer (Renner
2004); their use was also banned in Canada in 2006 (Canada Gazette 2006). Another
BFR technical mixture, hexabromocyclododecane (HBCD), is widely used in Europe and
11
to some extent in North America, although its production is currently being reconsidered
by the United Nations Environment Program (UNEP).
HBCD concentrations have been detected in wildlife tissues, including the eggs of
a number of predatory bird species. They have been found in eggs of peregrine falcons
(Falco peregrinus) in Sweden (Johansson et al. 2009) and south Greenland (1986-2003;
Vorkamp et al. 2005), in two eggs of Little Owls (Athene noctua) in Belgium (Jaspers et
al. 2005), in the egg of an African sacred ibis (Threskiornis aethiopicus) in South Africa
(Polder et al. 2008), in the eggs of herring gulls (Larus argentatus) in the Great Lakes of
North America (Gauthier et al. 2007), as well as in the liver and brain of dead or dying
glaucous gulls (L. hyperboreus) in the Arctic (Sagerup et al. 2009).
Although HBCD has been detected in the tissues of many avian species, the
potential effects of such exposure is currently unknown for birds, especially the effects of
in ovo exposure during the very sensitive embryonic stage. However, several studies have
determined effects of HBCD exposure on laboratory rats and mice. The exposure of rats
to HBCD by diet affected thyroid follicle size, serum thyroid-stimulating hormone (TSH)
and serum thyroxine (T4) levels, weights of the thyroid glands and livers, weight gain,
and viability (Ema et al. 2008), as well as bone mineral density, immune function, and
testes mass (van der Ven et al. 2009). Embryonic exposure of weanling rats to HBCD
was found to exert developmental brain effects, as well as changes in their thyroid glands
as adults (Saegusa et al. 2009), while embryonic exposure to HBCD affected the auditory
function and dopamine-dependent neurobehaviours, i.e. righting response, in young
12
Wistar pups (Lillenthal et al. 2009). Although studies investigating the effects of HBCD
exposure are limited to date, there is clear evidence that HBCD at levels beyond those
found in predatory bird eggs affect neurobehaviours and thyroid function in laboratory
animals. Whether HBCD affects any other endocrine function in vertebrates is not yet
known.
Corticosterone is one of the principal avian hormones responsible for the
regulation of fuel metabolism, immune reactions, responses to environmental stressors, as
well as stimulating the hunting of prey and the avoidance of predators. The endocrine
mechanism known as the “corticosterone response” allows birds (and other vertebrates)
to adapt physiologically and behaviourally to the pursuit of prey and to encounters with
predators. Environmental contaminants, such as polychlorinated biphenyls (PCBs), are
known to impair the corticosterone response of birds, including American kestrels (F.
sparverius) exposed to environmentally relevant concentrations of PCBs (Love et al.
2003b).
The American kestrel is a small falcon species of the Falconidae family that preys
upon insects and small mammals and birds, as well as being preyed upon by larger birds
of prey such as peregrine falcons and Cooper’s hawks (Accipiter cooperii) (Smallwood
and Bird 2002, Farmer et al. 2006). Their survival is highly dependent upon both their
ability to successfully hunt and capture their prey and to successfully detect and avoid
being captured by potential predators.
13
Although HBCD concentrations have been well documented in eggs of many
avian species, including those of birds of prey, the effects of such HBCD exposure on
birds remains unknown. Moreover, the impact of HBCD on the corticosterone response
of birds has also not been studied to date. As part of a larger study investigating the
effects of brominated flame retardants on birds (Fernie et al. 2005, 2006, 2008, 2009),
here we examine the potential effects of in ovo exposure to an environmentally relevant
concentration of the technical HBCD mixture on plasma corticosterone concentrations,
predatory behaviours and predatory responses of captive juvenile American kestrels.
Materials and Methods
Study site and details
This study was conducted at the Avian Science and Conservation Centre of
McGill University (Montreal, Quebec) using captive American kestrels of known age and
pedigree from a colony that has been used for toxicological studies for over thirty years
(Bird 1982, Bardo and Bird 2009). Male and female adult kestrels were randomly
assigned to one of two groups: the HBCD exposure group (0.8 ppm; 19 pairs), or the
control group (safflower oil only; 11 pairs). Each group of kestrels was fed their regular
daily diet of frozen / thawed day-old cockerels ad libitum.
Exposure and dosing concentrations of HBCD
Because the HBCD levels in wild American kestrel eggs were unknown when this
study began, dosage concentrations were calculated based on the concentrations of
HBCD residues recently found in herring gull eggs collected in the Great Lakes Basin
14
(Gauthier et al. 2007). Kestrels were exposed by diet to HBCD at concentrations
exceeding the background concentrations (represented by control birds) for ~75 days in
2008. Because female kestrels gain almost one-third of their body weight in the three
weeks preceding egg laying (Balgooyen 1976), exposure to HBCD began three weeks
prior to pairing and continued through courtship, egg-laying and incubation, until the first
chick (would have) hatched. Thus, the F1 generation of nestlings and juvenile birds in this
study were exposed in ovo only to HBCD in 2008.
Each of the solutions used in this experiment was prepared using 250 mL of the
same safflower oil (Master Choice®, 100% pure). The HBCD exposure treatment
involved 0.8 μg technical HBCD mixed with safflower oil to produce a final
concentration of 0.800 μg/μL; only safflower oil, prepared identically but without the
technical HBCD, was used for the control exposure. The mixtures were then stirred
slowly for 20 h in brown bottles lightly covered with aluminum foil until the HBCD
dissolution (when appropriate) and out-gassing was complete. The dosing mixtures were
injected daily into the brains of frozen / thawed day-old cockerels diet, and during the
exposure period, each pair of kestrels received three cockerels per day. The estimated
daily dietary exposure concentrations consisted of 0.8 μg HBCD / μL safflower oil per
cockerel (or 800 ng/ g ww / day / pair) for the HBCD exposure group based on a mean
cockerel weight of 40.4 ± 0.26 g. Kestrels assigned to the control group received
safflower oil only.
15
Chemical analysis of HBCD in eggs
The first egg produced by each kestrel pair was collected and used to determine
total α-HBCD burdens (representing the sum of α-, β-, and γ-ΗΒCD isomers). The
contaminant burdens in the first egg of each kestrel clutch may not reflect concentrations
in other eggs within the same clutch, although contaminant burdens, including HBCD,
did not vary with the laying order in other species (Verrault et al. 2006, van den Steen et
al. 2006). All eggs samples (N = 26) were collected in June 2008 and were stored at
−40 °C prior to the chemical analysis, which was performed at the National Wildlife
Research Centre, Environment Canada, Ottawa, Ontario.
Approximately 3 g (ww) of each freeze-dried egg sample were ground with
anhydrous sodium sulfate and then extracted with 175 mL of 50:50 dichloromethane:nhexane (DCM:HEX). Internal standards were spiked with 13C12-labeled-α-, β-, and γHBCD isomers. Prior to cleanup by gel-permeation chromatography, a 10% portion of
the column extraction elutant was used for gravimetric lipid determination. Final sample
cleanup was performed with 12 mL of 15:85 DCM:HEX using 6 mL, 0.5 g silica (SiOH),
Bakerbond disposable solid-phase extraction cartridges (VWR International,
Mississauga, Canada). The final volume of the sample extract was accurately reduced to
250 μL in isooctane.
Egg extracts were further analyzed by LC-electrospray-tandem quadrupole MS
(LC/ESI-MS(MS)) for HBCD isomers. Quantification of α-, β-, and γ-HBCD using the
corresponding 13C12-labeled internal standard permitted for the correction of any matrix-
16
dependent, ionization suppression/enhancement that may have occurred. A solvent
transfer from isooctane to methanol was necessary for LC/ESI-MS(MS) analysis. All
samples were reduced under nitrogen to dryness, reconstituted in 500 μL HPLC-grade
methanol, and filtered using Acrodisc LC 13 mm syringe filters (PALL, Mississauga,
ON, Canada) with 0.20 μm polyvinylidene fluoride (PVDF) membrane. A Waters
Alliance 2695 HPLC Pump system (Waters, Mississauga, ON, Canada) was equipped
with a Symmetry C18 reverse phase column (part no. WAT058965) having dimensions
100 × 2.1 mm, 3.5 μm. At ambient temperature of about 23 °C, the gradient mobile phase
used was water (A), methanol (B), and Acetonitrile (C), at a flow rate of 250 μL/minute.
Initially, the solvent composition was 40/40/20 A/B/C (%) held for 3.00 min, followed by
60/40 B/C for 6.50 min and finally returning back to initial conditions 40/40/20 A/B/C,
held for 8.00 min to allow for equilibration. The total run time was 17.50 min and the
column temperature did not exceed 23 °C.
Mass spectral analysis was achieved via a Waters Micromass Ultima ESIMS(MS) operating in the ESI(−) mode. The source parameters were as follows: ionspray
voltage −4000 V; curtain gas flow 25 a.u. (arbitrary units); sheath gas flow 35 a.u.; turbogas flow and temperature 30 a.u. and 500 °C, respectively. For the determination of α-, β, and γ-HBCD, the [M−H]- → Br- transition m/z 640.8 → 79.2 was accomplished via the
multiple reaction monitoring mode.
For quality control and assurance method limits of quantification (MLOQs) were
based on the criterion that an analyte response must be 10 times the standard deviation of
17
the noise. For an analyte to be detectable but not quantifiable the analyte response must
be at least is 3 times the standard deviation of the noise. Based on samples that had the
lowest BFR and DP concentrations levels, the MLOQs were estimated by calculating the
proportional concentration adjusted to a S/N = 10.
Animal husbandry
The treatment and care of the kestrels was conducted in accordance with the
Canadian Council on Animal Care guidelines (McGill University AUP#5600: Appendix
A; Olfert et al. 1993). The F0 kestrels were paired in April 2008 (N = 30 pairs), and
assigned to the HBCD exposure group (19 pairs) or the control group (11 pairs). Each
pair had previous breeding experience and was paired with another bird that was
genetically unrelated within the past six generations. During the experiment, the kestrels
were housed as a pair in their own breeding pen (1.0 m x 2.4 m x 2.4 m; W x L x H)
containing a nest box (0.3 m x 0.3 m x 0.4m; W x L x H), rope perches and a food
platform (0.15 m x 0.15 m 0.01 m; W x L x H). The in ovo exposed HBCD kestrels
hatched between 2 and 22 June 2008 and were raised by their parents until fledging.
Mimicking conditions of wild kestrels (Balgooyen 1976), the captive kestrel fledglings
remained with their parents for approximately 4 weeks before completion of the
behavioural experiment (7 – 12 August 2008).
Corticosterone response
The corticosterone challenge was conducted using 25-d old sibling nestlings that
hatched late or last in their brood in order to control for potential effects of hatching order
18
on corticosterone levels (Love et al. 2003a). The two 25-d old nestlings (N = 38) were
removed from their nest box between 09:00 and 09:30 a.m., before the birds were fed,
using a standardized capture and handling stress response protocol (Wingfield 1994,
Love et al. 2003d). Nestlings were blood-sampled to assess corticosterone levels in blood
plasma. The blood sample was taken from the jugular vein, using a heparanized 27-gauge
needle and 1 cc syringe. Approximately 30 μL of whole blood was collected within 3
minutes of capture to determine baseline levels of corticosterone prior to capture;
corticosterone levels generally do not start to increase until 3 min after capture
(Wingfield 1994). After the initial blood sampling, the bird was placed in a dark, holding
box with sufficient ventilation; the bird was removed from the box for an additional
blood sample 10 min after its initial capture. The total amount of blood taken (60 μL)
represented less than 1% of the body weight. When the blood sampling was complete,
morphometric measurements were recorded for body mass (g), tarsus length (mm), wing
length (mm), wing chord length (mm) and tail length (mm) according to Olendorff
(1972).
Corticosterone radioimmunoassay analysis
Plasma levels of corticosterone were measured using a specific
radioimmunoassay (RIA) at the National Wildlife Research Centre, Environment Canada,
Ottawa, Ontario. Reagents were brought to room temperature and plasma samples and
controls were thawed. The control set was reconstituted with 2.0 mL water and was
allowed to sit for 30 min. Plasma samples were diluted at a 1:25 ratio of plasma to steroid
diluent for the initial blood samples (pre-CORT), and at a 1:50 ratio of plasma to steroid
19
diluent for the additional blood samples (post-CORT) respectively, using RIA kits (ICN
Biomedicals, Inc., Costa Mesa, CA, Cat. No. 07-120180). During the assay of kestrel
samples, 100 μL of each diluted plasma sample was added in duplicate to labeled assay
tubes. A total of 200 μL each of 125I-labeled corticosterone and then corticosterone antiserum were added to each tube, vortexed and allowed to incubate for 2 h. Then 500 μL of
steroid precipitant was added, vortexed and centrifuged for 15 min at 2300-2500 rpm.
The supernatant was removed and analyzed with a Gamma-counter. The standard binding
curve was modified for lower expected results by adding a 12.5 ng/mL standard. The
low/high control was within the expected range given in the kit.
Hunting behaviours and predator-response behaviours
In August 2008, 2-month old juvenile kestrels (N = 51) from the HBCD in ovo
exposed group or the control group, the same birds used in the corticosterone response
trial earlier, were assigned to one of 6 groups (1 group per day) and randomly placed as
individuals in 9 identical flight pens on 6 consecutive days (7 – 12 August 2008). The
juvenile kestrels were placed in their respective flight pens, and allowed to acclimate for
one hour prior to the hunting behavioural trial followed by the predator-avoidance trial.
All behavioural observations were conducted at approximately the same time each day
over the course of six days to control for temperature and photoperiod variations. Each
flight pen (1 × 2 × 2m: L×W×H) contained one window screen (1 × 1 m: L×H), one
wooden perch (0.10 m), and one plastic transparent arena (0.54 × 0.38 × 0.30 m:
L×W×D) in the middle of the floor of the pen. Birds were observed through a one-sided
mirrored window (0.10 × 0.20 m: L×H) so as not to be disturbed by the observer.
20
Ten live crickets were placed in the plastic arena, and observations of hunting
behaviours began immediately for a period of 10 min. Location (perch, window screen,
arena inside, arena edge, ground, other position) and activity (perching, hunting, random
flying, feeding, maintenance, other) of the kestrels were recorded at 15-second intervals.
The number of incidences of specific “focal” hunting behaviours, i.e. head snapping into
fixed position, head-bobbing, tail-pumping, visiting the arena, successful-unsuccessful
capture of crickets (Balgooyen 1976), were recorded at 15-second intervals. The time of
the first successful capture of a cricket was also recorded.
One hour after the hunting behaviours were recorded, the plastic arena was
replaced with a mounted predator of similar size, i.e. red-tailed hawk (Buteo
jamaicencis), red-shouldered hawk (B. lineatus), or rough-legged hawk (B. lagopus)
placed on the floor of the pen, opposite the perch. The predator avoidance responses and
behaviours of the kestrels (N = 51) were observed immediately for 10 minutes. Location
(perch, window screen, ground, other position) and activity (perching, flying, kleeing,
maintenance, other) of the kestrels were recorded at 15-second intervals. The number of
incidences of specific “focal” predator avoidance behaviours, i.e. kleeing, defensive
posture, stationary flapping, head snapping into fixed position, head-bobbing, tailpumping, attacking, panting (Balgooyen 1976), were recorded at 15-second intervals, as
above for the hunting behaviours, in addition to recording the time of the first reaction of
the kestrel to the mounted predator. The increased number of kestrels used in the
behavioural experiments (N = 51) compared to the previously performed corticosterone
response challenge (N = 38) was due to the addition of kestrels from the control group;
21
these control kestrels had not been available at the time of the corticosterone challenge as
they were being used in another experiment by Katrina Sullivan for her M.Sc. thesis
research.
Statistical analysis
For the statistical analysis of HBCD concentrations in the F1 exposed birds, it was
assumed that all eggs of the clutch contained the same concentrations of HBCD as the
first egg that was analyzed. Because female and male kestrels may hunt different-sized
prey (Bird and Bardo 2009), we conducted all statistical analysis separately by sex. Data
were tested for normality and then log-transformed when necessary, including the in ovo
HBCD concentrations. Body condition was calculated as body mass:tibiotarsus (leg)
bone length at the time of sampling. Repeated Measures ANOVA was used to determine
significant differences in the corticosterone response of the nestlings; there were no
significant differences among the siblings so this factor was excluded from the model.
Subsequently, we included in ovo HBCD concentrations and the body condition index as
two covariates in the Repeated Measures ANOVA model. Behaviours relating to hunting
of prey or avoidance of predators were analyzed by Wilcoxon non-parametric statistical
tests or by analyses of covariance (ANCOVA) with in ovo HBCD concentrations as the
covariate; Spearman’s correlation analysis was also used to identify correlations among
behaviours and HBCD concentrations. All statistical analyses were performed with SAS
9.2 software (SAS Institute Inc. 2000-2008). The statistical level of significance was
P ≤ 0.05 and means ± standard errors are presented.
22
Results
Corticosterone response
The in ovo total HBCD concentrations measured 164.13 ± 18.26 ng/g ww in the
exposed treatment eggs and 0.4 ± 0.04 ng/g ww in the control eggs. The body condition
indices and body mass of the male and female nestling kestrels 25 d after they hatched
were similar between the treatment groups within each sex, respectively (P > 0.11).
However, the body condition of the nestlings was negatively correlated with the postchallenge plasma corticosterone concentrations (N = 33; R = -0.47 P = 0.0056), but not
the pre-challenge corticosterone concentrations. There was no correlation among the
plasma corticosterone concentrations pre- or post-challenge and the in ovo HBCD
concentrations (P > 0.1909).
The pre-challenge corticosterone concentrations of male nestlings was similar
between the HBCD in ovo exposed and control chicks, but the post-challenge
corticosterone concentrations of the HBCD exposed male nestlings (13.42 ± 1.05 ng/mL)
was significantly lower than those of the control males (17.75 ± 1.81 ng/mL; F1,15 = 4.29
P = 0.0572) (Figure 1). The corticosterone response of both the male and female nestlings
was appropriate, significantly changing over time (P < 0.0001), but the corticosterone
response of the male nestlings to the challenge was significantly affected by their in ovo
exposure to HBCD (F1,14 = 5.37 P = 0.0361). The body condition of the female nestlings,
but not the in ovo HBCD concentrations, had a significant effect on their post-challenge
corticosterone levels (F1,13 = 4.33 P = 0.0577) and a modest effect overall (F2,12 = 3.53 P
= 0.0621), whereas for the male nestlings, the in ovo HBCD concentrations but not body
23
condition had a significant overall effect on the corticosterone levels of the male nestlings
(F2,11 = 5.55 P = 0.0215).
Hunting Behaviour
In the male juvenile kestrels, their flying activity levels were negatively correlated
with their in ovo HBCD concentrations (N = 26 R = -0.4946 P = 0.0102) (Figure 2). As
there were increasing HBCD concentrations to which they were exposed to as embryos,
there was a correlative decrease in the amount of time they spent flying during the
hunting behaviour trials. Head-bobbing was performed significantly more by the males in
the HBCD-exposed group than the control males (F1,23 = 5.35 P = 0.0301) (Figure 3A),
and this behaviour was also significantly influenced by the in ovo HBCD concentrations
(covariate analysis: F1,23 = 4.88 P = 0.0374). There was also a very modest decrease in
head-snapping by the HBCD males compared to the control males (F1,23 = 3.20 P =
0.0866) (Figure 3B), which was also somewhat influenced by the in ovo HBCD
concentrations (F1,23 = 3.44 P = 0.0766). The HBCD female kestrels visited the crickets
less often than the control females (F1,22 = 4.60 P = 0.0434) (Figure 3C), and this too was
significantly influenced by the in ovo HBCD concentrations (F1,22 = 4.2 P = 0.0520). Of
note, there were no significant differences between the two groups, nor influence of the in
ovo HBCD concentrations on the number of attempts made to capture the crickets, the
hunting success rates, or the length of time to visit the crickets.
24
Response to Predators
The juvenile female kestrels showed a modest difference between the HBCD and
control groups in the length of time of their first response to the mounted predator (Chisquare = 3.28 df = 1 P = 0.0700), and the HBCD-exposed females made significantly
more “klee” calls at the predator (F1,22 = 6.59 P = 0.0176) (Figure 4A) and the frequency
of these calls was also significantly influenced by the in ovo HBCD concentrations (F1,22
= 6.34 P = 0.0195). There was a positive correlation between the in ovo HBCD
concentrations and the response time of the females to the mounted predator (N = 25 R =
0.5705 P = 0.0029) (Figure 5); with increasing exposure to HBCD levels, there was a
corresponding, increasing delay in the response of the female bird to the predator.
The male kestrels were significantly different among the controls and HBCD in
their performance of two activities. The HBCD-exposed males perched more often (F1,23
= 5.75 P = 0.0254) and flew less frequently (F1,23 = 5.57 P = 0.0271), and both perching
(F1,23 = 5.78 P = 0.0247) and flying (F1,23 = 5.55 P = 0.0274) were significantly
influenced by the in ovo HBCD concentrations.
Discussion
The results of this study indicate that the modification in the corticosterone
response of the male nestling kestrels is a function of their exposure to HBCD as
embryos, and not related to any differences in their body mass. Although not statistically
significant, a similar modified corticosterone response was observed with the female
nestlings exposed to HBCD compared to female control nestlings. The behavioural
25
responses of these nestlings one month later as juveniles to predators and prey are
consistent with the plasma corticosterone changes in the nestlings, which suggest that the
HBCD exposure has an ongoing effect on corticosterone levels. Further evidence
supporting this hypothesis is derived from the correlations of in ovo HBCD
concentrations with reduced flying activity levels of the juvenile males in the hunting
trials and delayed response times of the juvenile female kestrels to the mounted hawks.
The plasma corticosterone concentrations of the American kestrel nestlings preand post-challenge are comparable to those corticosterone concentrations reported for
nestling American kestrels of similar age (Heath and Dufty 1998, Love et al. 2003a), and
showed an appropriate increase over time following the challenge (Wingfield 1994, Love
et al. 2003d). Similar to other studies (Smith et al. 1994), body condition of the nestlings
was negatively correlated with their maximal corticosterone concentrations following the
challenge. However, there were no significant differences in body condition or body mass
of the male nestlings at the time of the challenge, and the kestrels were fed ad libitum
throughout the study. The results of our study suggest that there may be a sex-specific
corticosterone response following embryonic exposure to HBCD since only the male and
not the female nestlings demonstrated a modified corticosterone response and
significantly lower plasma corticosterone concentrations following the challenge.
However, the body condition and not the in ovo HBCD concentrations influenced the
corticosterone response and post-challenge corticosterone levels of the larger female
nestlings.
26
Several toxicology studies have identified similar mixed effects on corticosterone
following the exposure of birds to one or multiple contaminants. Mercury was correlated
with elevated corticosterone levels in common loons (Gavia immer) in Atlantic Canada
(Burgess et al. 2005). The exposure of adult male mallard ducks (Anas platyrynchos) to
Aroclor 1254 at doses of up to 500 mg/kg had no effect on corticosterone levels (Fowles
et al. 1997). In free-ranging tree swallows (Tachycineta bicolor), the corticosterone
response of the nestlings was modified at the study site that was more highly
contaminated with PCBs, while baseline corticosterone levels were lower at the site more
heavily contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (Franceschini et al. 2008).
Love et al. (2003b) reported that adult PCB-exposed kestrels exhibited significantly
lower corticosterone levels and a modified response over time compared to control
kestrels, independent of their body condition. We were unable to find any studies
reporting on possible corticosterone effects of PBDEs or other brominated flame
retardants, which are structurally similar to PCBs. However, similar to the PCB study by
Love et al. (2003b), our nestling male kestrels exposed in ovo to HBCD demonstrated
significantly lower corticosterone levels post-challenge and a modified corticosterone
response. Since rapid increases of corticosterone levels trigger locomotor behaviours
(Astheimer et al. 1992) and stimulate movement away from stressful situations (Smith et
al. 1994), we would anticipate behavioural modifications in the kestrels as a function of
the changes in their corticosterone patterns. A study involving estuarine minnows
(Fundulus heteroclitus) that were embryonically exposed to the PBDE technical mixture,
DE-71 (Timme-Laragy et al. 2006), identified a significant decrease in activity levels and
27
fright response, and significantly increased predation rates in subsequent life stages.
These decreased levels of activity and fright response are consistent with our findings.
HBCD in ovo exposed male juvenile kestrels performed more head-bobs and
fewer head-snaps in comparison to control males. When hunting, kestrels snap their
heads to a fixed position to estimate prey size and distance, and repeatedly bob their
heads vertically and more frequently with increasing interest in their potential prey
(Balgooyen 1976). In addition, the overall flying activity levels of these juvenile male
kestrels were negatively correlated with their in ovo HBCD concentrations during the
hunting behaviour trials. The female kestrels exposed in ovo to HBCD also showed
decreased activity levels, visiting the cricket arena significantly fewer times than their
control female counterparts.
The reduced activity levels of the juvenile kestrels exposed to environmentally
relevant concentrations of HBCD were also observed in their responses to potential
predators. When shown a mounted predator to assess their predator response, juvenile
female HBCD-treated kestrels performed significantly more vocal “klee” calls; klee calls
are performed by a kestrel under stressful conditions, and klee calls increase in numbers
as the bird becomes increasingly stressed (Balgooyen 1976). In addition, these HBCDexposed female kestrels demonstrated a significantly longer time delay in their first
response to the potential predator, and their overall response time was positively
correlated with their in ovo HBCD concentrations.
28
Our findings suggest that the embryonic exposure to environmentally relevant
concentrations of HBCD can influence avian hunting success in males especially, and the
avoidance of potential predators with a potentially more pronounced effect on female
birds. We cannot explain the sex-related differences in these two behaviours at this time.
The reduced levels of hunting activities and response times to predators by the HBCD in
ovo exposed kestrels are consistent with their lower post-challenge corticosterone levels
and their muted corticosterone response as nestlings. Furthermore, these results suggest
that HBCD behavioural effects are not restricted to dopamine-dependent behaviour
(Lilienthal et al. 2009).
Corticosterone is one of the primary avian hormones responsible for the stress
response and “fight or flight” behaviours and hence, governs behaviours critical to the
survival of birds. Love et al. (2003c) demonstrated that adult and 22-d old nestling
American kestrels had statistically similar corticosterone concentrations and responses,
suggesting that the results observed in our study with 25-d old nestling and juvenile
kestrels might also occur with adult kestrels exposed in ovo and by diet to HBCD.
Furthermore, we might expect concentrations of HBCD currently measured in bird eggs
to have similar effects on the avian corticosterone system with subsequent and potential
modifications in hunting behaviours and avoidance of potential predators by free-ranging
birds.
These results are particularly interesting in view of the fact that American kestrel
populations in some areas of North America are in a state of decline (Bird 2009). It is
29
well known that a number of chemical pollutants released into the environment, e.g.
organochlorines, organophosphates, heavy metals (Bardo and Bird 2009), have had
impacts upon aspects of the ecology of the kestrel. Also, Farmer et al. (2006) suggested
that this species may be under significant predation pressure, at least in some areas of its
range, from larger raptors such as Cooper’s hawks. It is not inconceivable that relatively
new chemical pollutants like brominated flame retardants, e.g. PBDEs, HBCD, may be
playing a role in this demise by subtly reducing their ability to avoid such predation, as
well as to procure prey to survive and reproduce.
30
Fig. 1. The plasma corticosterone concentrations and response of (A) male and (B)
female American kestrel nestlings (d 25) to a stress challenge protocol following their in
ovo exposure to environmentally relevant concentrations of hexabromocyclododecane
(HBCD).
(A) Corticosterone stress response of male American kestrel nestlings
Corticosterone stress response - male nestlings (d 25)
22
F1,15 = 4.29
P = 0.0572
Corticosterone ( ng / mL ) .
20
18
16
14
12
Control (N = 4)
10
HBCD (N = 15)
8
6
P = 0.3283
4
2
0
pre-CORT (t ≤ 3 min)
post-CORT (t = 10 min)
Time after capture (min)
(B) Corticosterone stress response of female American kestrel nestlings
Corticosterone stress response - female nestlings (d 25)
22
Corticosterone ( ng / mL ) .
20
P = 0.4285
18
16
14
12
Control (N = 4)
10
8
6
HBCD (N = 15)
P = 0.6229
4
2
0
pre-CORT (t ≤ 3 min)
post-CORT (t = 10 min)
Time after capture (min)
31
Fig. 2. Flying activity levels of male juvenile American kestrels during hunting behaviour
trials are correlated with in ovo hexabromocyclododecane (HBCD) concentrations.
10
N = 26, R = -0.4946, P = 0.0102
8
flight frequency
(times / 15sec)
6
4
2
0
-2
0
100
200
300
400
in ovo HBCD concentrations
(ng/g ww)
32
Fig. 3. Differences in the hunting behaviours of juvenile American kestrels between those
kestrels exposed in ovo to hexabromocyclododecane (HBCD) and control birds: (A)
head-bobbing of males, (B) head-snapping of males and (C) females visiting the cricket
arena. Statistical analysis involved ANCOVA with in ovo HBCD concentrations used as
the covariate.
(A) Hunting of prey behaviour trials: males head-bobbing Mean # head-bobs
Hunting behaviour: males - head-bobbing
120
100
80
60
40
20
0
-20
-40
-60
F1,23 = 5.35
P = 0.0309
Control (N=11)
HBCD (N=15)
Treatment
(B) Hunting of prey behaviour trials: males head-snapping
Hunting behaviour: males - head-snapping
Mean # head-snaps
100
F1,23 = 3.20
P = 0.0866
80
60
Control (N=11)
HBCD (N=15)
40
20
0
-20
Treatment
33
(C) Hunting of prey behaviour trials: females visiting cricket arena
Hunting behaviour: females - cricket arena visits
Mean # arena visits
6
F1,22 = 4.60
P = 0.0434
5
4
3
2
1
0
-1
-2
-3
Control (N=10)
HBCD (N=15)
Treatment
34
Fig. 4. Differences in predator avoidance behaviours of juvenile female American
kestrels when exposed in ovo to hexabromocyclododecane (HBCD or to control vehicle:
(A) kleeing calls and (B) time delay in first response to predator. Statistical analysis
involved ANCOVA with in ovo HBCD concentrations used as the covariate.
(A) Predator avoidance behaviour trials: females’ kleeing calls
Predator avoidance: females - kleeing
0.7
F1,22 = 6.59
P = 0.0176
Mean # klee calls
0.6
0.5
0.4
Control (N = 10)
0.3
HBCD (N = 15)
0.2
0.1
0
Treatment
(B) Predator avoidance behaviour trials: females time delay in first response
Mean time delay to first
response (min)
Predator avoidance: females - time delay first response
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Control (N = 10)
HBCD (N = 15)
Treatment
35
Fig. 5. The response time of female juvenile American kestrels to a mounted predator is
correlated with in ovo hexabromocyclododecane (HBCD) concentrations.
1.4
N = 26, R = 0.5705, P = 0.0029
response time to predator
(min)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
50
100
150
200
250
300
in ovo HBCD concentrations
(ng/g ww)
36
FINAL SUMMARY AND CONCLUSION
This study investigated the potential effects of in ovo exposure to environmentally
relevant concentrations of the technical mixture of HBCD on plasma corticosterone
concentrations, predatory behaviours and predatory responses of captive American
kestrels. Corticosterone is one of the principal hormones that govern the stress response
and the “fight or flight” response of birds and other vertebrates. The endocrine
mechanism known as the “corticosterone response” allows birds to adapt physiologically
and behaviourally to environmental stimuli and stressors such as the pursuit of prey and
encounters with predators.
The corticosterone response of the male nestling kestrels in the HBCD
embryonically exposed group was suppressed compared to the control group and a
similar non-significant modified corticosterone response was observed with the female
HBCD-exposed nestlings. HBCD exposure likely had an ongoing effect on corticosterone
levels of these birds one month later, as juveniles, based on modifications of their
behavioural responses to predators and prey. The flying activity of male juveniles in the
hunting trials was negatively correlated with their in ovo HBCD concentrations.
Moreover, during hunting trials, HBCD-treated males performed more head-bobs and
fewer head-snaps, and HBCD-treated females visited the cricket arena less frequently
than their control counterparts. The response time of the juvenile female kestrels to the
mounted hawks was positively correlated with their in ovo HBCD concentrations.
Moreover, during predator avoidance trials, HBCD-treated females performed more
vocal “klee” calls and demonstrated a longer time delay in their first response to the
37
potential predator than the control females. These results suggest a sex-specific effect of
embryonic exposure to environmental levels of HBCD, influencing corticosterone
response and hunting success especially in males, and avoidance of potential predators
especially in female American kestrels. The sex-related differences in these physiological
and behavioural responses cannot be explained at this time.
HBCD is a ubiquitous and persistent environmental contaminant, highly lipophilic
and bioaccumulative, detected in various ecosystems and phyla, thus posing potential
cause for concern for top consumers, including predatory birds. Although HBCD
concentrations have been documented in eggs of many avian species, including those of
birds of prey, the effects of such HBCD exposure on birds remained unknown. Moreover,
the impact of HBCD on the corticosterone response of birds had also not been studied to
date. The results of this study provide an important understanding of some of the
physiological changes of the corticosterone response and the subsequent changes in
behavioural responses to prey and potential predators, induced by in ovo exposure of
American kestrels to environmentally relevant levels of HBCD. Through this research,
we now have a better understanding of the potential effects of HBCD concentrations
currently measured in bird eggs of free-ranging birds. Whether our findings may serve to
help explain the decline of some populations of American kestrels in North America
remains to be investigated.
38
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APPENDIX A – Animal Care Committee Approval Certificates
46