Thesis #762

In recent years, the annual catches of brown trout and other native fish species
have been declining in Switzerland about 60%. One hypothesis was that the
reduced catch is linked to estrogen-active chemicals entering the aquatic
environment via waste water effluents. These so–called environmental estrogens
have the potential to mimic the actions of endogenous hormones and impair the
reproductive fitness of fish. The present thesis aimed to assess the reproductive
health of brown trout in Swiss rivers and to link putative reproductive disturbances
with the exposure to waterborne estrogens. In this context, we tracked field as well
as laboratory based approaches.
In order to assess whether the reproductive health of feral brown trout is disturbed,
we applied two different sampling strategies - namely passive and active
monitoring approaches. In the first approach, we sampled feral fish at three sites
along four rivers with a well documented catch decline. These rivers are affected
by inputs of wastewater effluents. The sampling was conducted during two years;
we measured plasma vitellogenin (Vtg) concentrations and surveyed gonadal
histology. In general, our data indicate that effects of environmental estrogens in
Swiss rivers are low. In only 5% of the analyzed males, we found plasma Vtg
concentrations higher than 1 μg/mL. Also the incidence of ovarian atresia was low
and we found no male intersex fish. In contrast to males, females caught along
two rivers had spermatogenic activity in ovarian tissue. However, this intersex
condition does not appear to be linked to environmental estrogens. In our second
field trial, we developed a mini–caging method to suit the hydrological conditions
in small rivers and to improve upon the often poor survival of salmonids in caging
trials. After three weeks of exposure, we measured plasma yolk protein and linked
the Vtg concentrations with the bioaccumulation of estrogens in bile of caged
fish. Because of the estrogenicity of river water is highly variable and it is difficult to
obtain an average measure of the estrogenicity we additionally tested the use of
passive sampling by means of polar organic chemical integrative samplers
(POCIS). The POCISs were positioned upstream and downstream of wastewater
treatment works. Concurrently, water grab samples were taken at each site.
Concentrations of estrogens were determined using a yeast-based reporter gene
assay and chemical analysis. Results from grab sampling, passive sampling, and
bioaccumulation were correlated; however, plasma vitellogenin concentrations
were elevated at only 1 of 5 sites. The POCISs provided an integrated and
biologically meaningful measure of estrogenicity in that they accumulated
estrogens in a pattern similar to that of brown trout. The mini caging appears a
significant methodological advance; no fish were lost, moreover, all fish survived in
excellent health. On the basis of our field data, we conclude that impaired
reproductive health does not appear to be a major factor contributing to the
marked decline of brown trout catches in the four investigated rivers. In addition to
the potential risk of environmental estrogens, increasing water temperatures as a
result of global warming has become a serious problem in many Swiss rivers and
streams. In particular low mountain range rivers frequently reach temperatures
that are suboptimal for many salmonid species. In our field surveys, we used the
analysis of Vtg as an indicator of estrogenic exposure. Little, however, is known
regarding the potential interaction between ambient water temperature and the
Vtg production induced by waterborne environmental estrogens. In order to test
the influence of temperature on Vtg synthesis, we exposed juvenile brown trout to
ethinylestradiol (EE2) and hold them either at low or high temperatures (12°C and
19°C, respectively), but also at temperature cycles of 12°-19°C to simulate the field
situation. The EE2 exposure caused a 7 to 74-fold increase of hepatic Vtg mRNA
and the synthesis Vtg mRNA was clearly stimulated in fish hold at higher water
temperatures. On the protein level, Vtg showed a similar pattern; the higher the
temperature, the higher the concentration of Vtg in the plasma. The experiment
further revealed a temperature dependent increasing amount of hepatic
estrogen receptor alpha mRNA after exposure to waterborne EE2. The gene
expression of estrogen receptor beta-1 and the glucocorticoid receptor in the liver
of EE2 exposed fish, however, showed no treatment related alterations. In line with
observed constant bile cortisol concentrations, our data do not indicate any stress
related effects on hepatic Vtg production. The present experiment, however,
clearly demonstrated that ambient temperature significantly change the
estrogen-induced expression of Vtg and therefore may alter the interpretation of
environmental monitoring studies under field conditions.
Changing water temperature alters the permeability of the gills and result in a
disturbed mineral balance in fish. The branchial sodium pump (Na+/K+–ATPase)
enables teleosts to cope with such varying environmental conditions and
compensates for the temperature–related loss of ions by active ion uptake from
the ambient water. Estrogens have the potential to interfere with the endocrine
regulation of Na+/K+–ATPase and may affect the molecular expression of sodium
pump mRNA and related branchial steroid receptors (mineralocorticoid and
glucocorticoid receptor). In the light of a recently observed warming of Swiss rivers
as well as the occurrence of estrogen-active chemicals in river water, such
interactions may have detrimental effects on the general health of brown trout in
Switzerland. To test the influence of temperature on the regulation of Na+/K+–
ATPase we used the same juvenile brown trout as described above in the Vtg
study. Data obtained from quantitative PCR evidenced a significant down
regulation of Na+/K+-ATPase gene expression in gills from estrogen–treated brown
trout held at low and fluctuating temperatures. However, the expression of Na+/K+–
ATPase in estrogen-treated fish from the EE2–high temperature group were not
significant lower than the control groups – indicating a response to the elevated
water temperatures. No significant effects on the number of immunoreactive
chloride cells were found; though, estrogen treatment tend to reduce the protein
abundance of Na+/K+–ATPase in the gills. The synthesis of mineralocorticoid
receptor mRNA correlated significantly with the expression of Na+/K+–ATPase. In
contrast, bile cortisol levels and the glucocorticoid receptor gene expression were
not affected by estrogen treatment alone or in combination with elevated
temperatures. This suggests that the expression of Na+/K+–ATPase is probably
regulated via the mineralocorticoid receptor. In addition, the lack of cortisol
response as well as the absence of effects on higher levels of biological
organization (e.g. histology or condition factor) suggests that the temperature
regimes used in the present study were insufficient to cause stressful conditions in
brown trout.