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SUMMARY
Today it is known that persistant organic pollutants like
HCH, PCB and PAHs can be found in the Arctic. Evidence for contamination from radioactive
compounds, and long-range transport of heavy metals has also been documented. The organic
compounds break down slowly, are fat-soluble and accumulate in the stored fat of Arctic
animals. Thus, bio-magnification and hazardous effects on the living organisms,
populations and ecosystems may occur. The Norwegian University of Science and Technology
(Trondheim) want to contribute to clarify some of the insidious potency of these
persistent organic pollutants represent to the free-living Arctic animals. This proposal
therefore suggests to study the possible interactions between PCB-congeners and Svalbard
charr body differentiation, growth and endocrine control mechanisms. We also want to test
if the Cytochrome lA1-P450 and metallothionein can be used as biomarkers. Finally, we
propose to isolate and describe the Svabard charr (liver-) estrogen receptor in order to
prepare an in vitro system for future tests of estrogen-like agonists and antagonists,
which are documented to pollute the Svalbard environment.
INTRODUCTION
General background.
Most areas of the European Arctic have
traditionally been conceived as undisturbed ecosystems. The apparently simple structure of
these systems can provide us with new information about ecological relationships and
mechanisms in environmental adaptation. At the same time, biological resources in the
Arctic, particularly fish, are of great economic significance. The preservation of Arctic
wilderness is a common objective for all the Arctic border states. Increased insight is a
prerequisite for both the conservation and the sustainable exploitation of biological
resources in the Arctic, not least with regard to ecological processes which influence the
dynamics and diversity of ecosystems.
The Arctic Monitoring and Assessment
Programme (AMAP), established in 1991 under the Arctic Environmental Protection Strategy
(AEPS), was given the responsibility to monitor the levels and assess the effects of
selected anthropogenic pollutants in all compartments of the Arctic. In comparison with
most other areas of the world, the Arctic was found to be a clean environment. The
AMAP-results did, however, also show that, for some pollutants, combinations of different
factors give rise to concern in certain ecosystems and for some human populations (AMAP
1997).
Today it is known that persistant organic
pollutants like HCH, PCB and PAHs can be found in the Arctic. Evidence for contamination
from radioactive compounds, and long-range transport of heavy metals has also been
documented.
The organic contaminants in the Arctic
environment share many characteristics that make them especially insidious to people and
wildlife. A common characteristic of most synthetic organic chemicals found in Arctic
animals is that they break down very slowly. This persistence in the environment allows
them to accumulate in animals, and to pass through the food web. Most of the persistent
organic pollutants are also fat-soluble. They thus accumulate in the fatty tissues of
animals. Storing energy as fat is crucial for survival in cold environments, and fat is
therefore important in diets of both people and animals, which also increases the intake
of these pollutants. The combined characteristic of being fat-soluble and persistent also
make biomagnification a major concern.
In order to monitor and clearify some of the
serious biological aspects of pollutants in the Arctic, The Norwegian Polar Institute has
invited some institutions to submit proposals for scientific studies of how organisms,
populations end ecosystems are affected by exposure to radio nucleids and persistant
organic pollutants. Accordingly, the following represents a research proposal from NTNU
for such studies conducted on the Svalbard charr.
The proposal represents a "joint
venture" between Prof. K.J. Nilssen (NTNU), Prof. R. A. Andersen (NTNU) and Ph.D.
Research Fellow K-E. Tollefsen (UiO).
Research strategy
A well balanced assessment of the adverse
effects of chemical pollutants in the environment should be based on studies integrating
analytical, toxicological and ecological information. Ecological and analytical
information on pollutions are usually obtained from "field" studies, while
toxicological information is mostly obtained from laboratory studies with a limited number
of experimental animals.
The gap between field and laboratory studies
can be lessened by a better understanding of the mode of fate and action of chemicals in
the organism on one hand, and an improved knowledge of relevant physiological processes on
the other. The integration of knowledge on physiology and mode of action of chemicals may
provide relevant and sensitive parameters to monitor the toxic action of chemicals under
field conditions. Studying these parameters in the same species under laboratory and under
field conditions may also significantly improve the validity of extrapolation of toxicity
data to effects in the field.
When studying the toxicity mechanism of
chemicals suspected of causing adverse ecological effects, particular attention should be
given to potential interference in biochemical/-endocrinological control mechanisms of
physiological processes that are important for the survival of populations, such as
growth, reproduction, energy utilization, osmoregulation and disease resistance. Such
studies should be performed on laboratory animals and on wildlife species under laboratory
and semi-field conditions. Sensitive toxicity parameters may thus be obtained in a
retrospective way, and tested in field experiments. These parameters subsequently can be
used in a prospective way as biological effect monitors in an early warning system under
field conditions. This approach would also provide a better basis for extrapolation of
data obtained under laboratory conditions to field situations, and of data from one
species to another.
Svalbard charr life-cycle(s).
The charr is the only salmonid living within
the freshwater systems of the high Arctic Svalbard archipelago (74-81° NL). Exploitation
of these rigorous environments is indicative of an animal capable of yearly withstanding
long lasting periods of darkness, low water temperatures and foodshortage. One of the main
prerequisites for this "life-style" is accordingly the ability to anticipate
seasonal changes in order to begin behavioral and physiological adjustments in advance of
the time for which they are actually needed. Recent studies have verified the photoperiod
to be the zeitgeber (Nilssen 199xa,b,c), and also that this input are converted into a
biochemical messenger by the pineal gland production of melatonin. It is also possible
that this brain-hormone production can be regulated through changes in light intensity
and/or spectral composition, making the Svalbard charr a rather "sophisticated"
vertebrate (Nilssen, 199x,b).
The Svalbard charr is polymorphic and may
therefore be found in three different forms. Two of these are freshwater residents (small
and large form), while the third is anadromic. Within the high Arctic archipelago the
resident forms may be landlocked or found together with the anadrom charr in watercourses.
The bimodality and size segregation of the
resident charr are likely to be the result of a difference in food availability, or in
food selection strategy. Thus, the very low growth rate of the small resident charr could
reflect the extreme environment with poor primary production and low insect availability,
while the more typical growth of large resident charr may reflect the occurence of
cannibalism. These morphotypes, which coexists in the Svalbard freshwater systems, are
also found to differ in body proportions, spawning habits, as well as in size and colour
at sexual maturity (Gulseth and Nilssen, 1998b).
The age distribution pattern has revealed
that up to 96% of the small resident charr may be less than 10 years, whereas more than
80% of large resident charr within the same area may be older than 11 years (Gulseth and
Nilssen, 1998b). Thus, the dwarfs seem to have a reduced life expectance, while the large
resident charr have a markedly longer life span. Recent results also demonstrate that the
small resident charr population mature from an age of 4-5 years, which is 3 to 5 years
earlier than the corresponding change in the large form (Gulseth and Nilssen, 1998b).
The decision of an organism to migrate or not
is likely to be dependent on a trade-off between the benefits and costs of migration
compared to residency (Jonsson & Jonsson, 1993). The advantage conferred by migrating
is usually access to an improved food supply thereby increasing the fitness of the
migratory individuals through subsequent reproduction.
A rapid emmigration from the lake into the
river during the ice break-up has been demonstrated for fish with body lengths down to 70
mm (Gulseth & Nilssen, 1998c). The habitat change is almost completed within 14 days
(mid-July). The migrating parr stay within the river for 3 to 8 weeks, resulting in a body
growth and a body composition superior to that of the parr remaining in the lake. Thus, it
is possible that the selection for anadromy starts at a very early age at Svalbard. The
young migrating charr can, however, not benefit from the ample food resources in the sea
due to the lack of seawater tolerance (Nilssen and Gulseth, 1998).
It has been demonstrated that the Svalbard
charr may take on the anadromous summerlife at a size of 17 to 20 cm being on average 7
years of age (Gulseth and Nilssen, 1998a,b). Recent investigations have shown that
freeliving charr migrating towards the sea have an increased hypoosmoregulatory capacity
prior to their seawater entry (Nilssen et al., 1997). These results indicate both
the existence of an anticipatory development of seawater tolerance, and that environmental
factors might influence the timing of the changes taking place in the Arctic charr. The
process of smoltification has furthermore been verified for Svalbard charr reared indoor
under artificial controlled photoperiod (Iversen et al., 1998b). In contrast to
other salmonids, the repeated seawater stay of the diadromous charr is terminated 4 to 5
weeks after its marine entry (Gulseth and Nilssen, 1998a). It is also known that the
reversed migratory activity (return to freshwater) is due to changes other than
desmoltification (Nilssen et al., 1997).
During its short seawater stay, the Svalbard
charr may more than double the body weight. On average, the anadromous population studied
displayed an increase in body length superior to that of the sympatric resident charr
(Gulseth and Nilssen, 1998b). An observed decline in body growth at an age of 10 years was
related to an increase in the frequency of maturity. Results have also document a
pronounced size variability within each age class of the anadromous stock (Gulseth and
Nilssen, 1998b). This phenomenon (also to be considered as the existence of several age
classes within a single length group) could be interpreted as an indication of stability,
preventing oscillation within a simple (Arctic) system (Johnson, 1983).
The charr forms differ with respect to the
recruitment of spawners. Thus, first-time spawners may represent 35, 10 and 45 % of the
reproductive fish (spawners and immature previous spawners) within the anadromous, large
resident and small resident morphs, respectively. The studies also indicate that less than
25% of the fish have 3 or more spawning cycles during the life-time (all three morphs).
Furthermore, only one out of four reproductive fish spawn the year after the first-time
spawning. Thus, using the frequencies for maturation and sex distribution, it can be
calculated that less than 15 % of the females would reproduce in a given year. This
clearly demonstrates a low reproductive capacity, making any Svalbard charr population
vulnerabel to mis-management and occuring pollutants.
Objectives
In order to study the: "Effects of
persistant organic pollutants on body differentiation, growth, and endocrine control
mechanisms in freeliving or captive Svalbard charr (Salvelinus alpinus L.)" ,
it is proposed to carry out the following projects on Svalbard charr:
For a population living within a PCB contaminated area: to
describe growth,
fecundity and the seasonal fat cycle, and to
measure levels of 1A1-P450, and metallothionein. Also to compare the results with that of
a population from a non-contaminated (control) area.
To study the effect of å 7PCB exposure on Svalbard charr fecundity.
To study the effect of å 7PCB exposure on hormonal control mechanism for temporal
organization (melatonin-secretion) and smoltification (thyroxine- and cortisol-secretion)
in Svalbard charr.
To evaluate 1A1-P450 and metallothionein, as possible
biomarkers for accumulation of persistent organic pollutants in the Svalbard charr.
To isolate and characterise estrogen receptor (ER) and steroid
hormone binding protein (SHBP) for future (in vitro) studies using estrogen-like agonists
and antagonists polluting the Svalbard environment.
The Effect Programme granted this research
application with N.kr 200.000 in support of project 2 and 3. The original project title
"Effects of persistent organic
pollutants on body differentiation, growth, and endocrine control mechanisms in freeliving
or captive Svalbard charr (Salvelinus alpinus L.)" was therefore changed to
reflect this:
The effect of PCB’s on fecundity and
endocrine control mechanisms in captive svalbard charr.
Experimental design
To study the effect of å 7PCB exposure on Svalbard charr
fecundity.
Since 1990, Arctic charr (anadromic
strain) has regularly been collected from Svalbard and kept at Brattøra Research Center.
Rearing, handling and physiological studies of both wild fish and its offspring has
therefore become a routine operation. We will also this autumn obtain eggs from our
maturing stock.
The maturing female fish will be divided into
two (control- and exposure-) groups.
Dissolved å 7PCB (50:50 acetone and Alkamuls EL-620) will be administered to
the experimental fish by subcutanous injection, while the control-fish only receive the
vehicles. All animals will regularly be examined for maturation. Eggs from one individual
will be kept separated from those of other fish. The number of stripped eggs obtained will
be determined volumetrically for each individual. All eggs will be fertilized by use of a
mixture from 5 healthy Svalbard charr males. Egg mortality will be calculated for 3
different developmental phases: white (dead or unfertilized) eggs, the "eyed"
eggs (after the eyes are pigmented), and after hatching. Mortality after hatching will
also be registered during the yolk-sac period. Hatchability and fecundity will be
calculated for all egg-groups and mother fish.
To study the effect of å 7PCB exposure on hormonal control
mechanism for temporal organization (melatonin-secretion) and smoltification (thyroxine-
and cortisol-secretion) in Svalbard charr.
To address quantitatively the fundamental
questions about how the endocrine function of these organisms are affected by exposure to
environmental pollutants, it is imperative to perform experiments where all environmental
parameters (photoperiod, temperature and food availability) can be controlled. Such
experiments have repeatedly been performed at Brattøra Research Center, and the results
from hormonal control of temporal organisation (melatonin-secretion) and smoltification
(thyroxine- and cortisol-secretion) are known (Nilssen, 199xa,b,c).
Animals within the Arctic area are
subject to extreme seasonal changes in temperature, photoperiod and food availability.
Physiological and behavioral adjustments to these fluctuating conditions allow their
survival in this high latitude environment. However, toxicity induced by chemicals in many
instances proceeds through interference of the compound or one of its metabolites in the
sequence of hormonal controlled processes such as growth, reproduction, osmoregulation and
immunology.
While timing is required for seasonal
acclimatization, it is important that animals anticipate the forthcoming change to begin
behavioral and physiological adjustments in advance of the time they are actually needed.
Thus, several investigators have demonstrated that a temporal organization in animal
physiology and behavior involve neuroendocrine mechanisms which are affected by changes in
photoperiod. The secretion of melatonin from the pineal gland appears to be of great
importance with respect to bodily coordination in time. The gland produces melatonin in
the dark, but not in the light. The pineal gland can therefore convert neural information
about photoperiod into a biochemical output and may therefore serve both as clock and
calendar (Reiter 1993). Accordingly, circadian melatonin production has been demonstrated
to entrain cycles of growth and reproduction in several vertebrate species (Reiter 1993)
including fish (de Vlaming and Olcese 1981; Joy and Agha 1991; Nayak and Singh 1987a,b) ,
as well as the Svalbard charr (Nilssen, 199xa,b,c). If persistant organic pollutants were
to interfere with the brain (pineal-) melatonin production, or the binding between
melatonin and its receptors (through-out the body), the induction and coordination of
several bodyfunctions would most certainly be at risk.
The relation between photoperiod and pineal
activity appears to be of primary importance also for the induction and correct timing of
smoltification in salmonid species (Gern et al. 1984). Thus, it is possible that
PCB exposure, through impact on melatonin, could interfere with the development of
seawater tolerance in the anadromic Svalbard charr. It is, however, also possible that
such interference from PCB congeners can be achieved by a direct action on the charr blood
plasma levels of thyroxine and cortisol. Thus toxic effects induced by PCB’s could be
mediated through interactions with non-Ah receptor involved in the transport of thyroid
hormones, the so-called transthyretin (TTR) (Brouwer and Berg 1988). Thus, studies have
revealed that a monohydroxy-metabolite of TCB could bind with high affinity to
transthyretin. Due to the high level of structural similarity, the OH-TCB metabolite
appeared to be competitive with the thyroid hormones for the thyroxine-binding site on TTR
(Brouwer 1987). Inhibition of binding of thyroxine to TTR by the TCB metabolite would
result in more free thyroxine, which is rapidly removed from the circulation into the
tissues and may therefore cause a rapid and almost complete elimination of thyroxine from
the plasma.
A cortisol increase also represents a
necessary prerequisite for the charr smoltification (Nilssen et al., 1997). From
studies of other animals, it has been shown that PCB’s actually can interfere with
the function of adrenal cortex (Bergmann and Olsson, 1986). Since the cortisol producing
interrenal fish cells compares with cells from the mammalian adrenal cortex, one should
test whether PCB congeners could interfere with the cortisol production in the Svalbard
charr.
The Svalbard charr will be divided into two
(control- and exposure-) groups. Dissolved å 7PCB will be administered to the
experimental fish by subcutanous injection, while the control-fish only receive the
vehicles. During a short-day photoperiod (simulated winter) fish from both groups will be
sampled every hour for 24 hours. Melatonin levels will thereafter be measured by use of a
modified RIA-procedure (Nilssen et al., 1998).
At end of the simulated (3 month long) winter
period, all charrs will experience an increase in photoperiod to fullfill the stimuli for
the onset of smoltification. The fish will thereafter be sampled for blood at the time
when maximum plasma levels of thyroxine and cortisol are expected to occur, whereafter the
hormonal levels will be analysed by use of RIA-techniques (Iversen et al). Finally, both
control- and PCB-exposed groups will be tested in full strength (35 ppt.) seawater, and
the resulting levels of bloodplasma osmolality, and sodium, chloride and magnesium levels
will be used for the determination of their seawater tolerance.
BUDGET
Table 1.
The cost for the different projects are given
in (1000) Nkr. Field studies include travel (for 2 persons), logistics and chemicals.
Laboratory experiments include experimental tanks, animal rearing and chemicals.
Research activity |
NP |
NTNU |
Total |
| |
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Project 2: PCB
exposure & fecundity |
75 |
100 |
175 |
Project 3: PCB
exposure & hormones |
125 |
100 |
225 |
| |
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|
|
| |
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Grand total |
200 |
200 |
400 |
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