Richard Burki

Characterization of the estrogenicity of Swiss midland rivers using a recombinant yeast bioassay and plasma vitellogenin concentrations in feral male brown trout

In our study, we aim to characterize the estrogenicity of 18 independent rivers that receive effluent from sewage treatment works. During the winter and summer of 2003, we collected multiple water samples and measured environmental estrogens with an in vitro yeast-based reporter gene assay; estrogenicity was expressed as ng 17beta-estradiol equivalents (EEQ) per L of water. Estradiol equivalents values in winter ranged from 0.3 to 2.0 ng/L and, in summer, from 0.4 to 7.0 ng/L. Winter and summer EEQ values were not correlated with each other or with the dilution factor of the effluent in the river. Variation in EEQ values was large and correlated from winter to summer. Part of this variation in estrogenicity is explained by water flow rates; variation is larger at reduced flow rates. We measured plasma vitellogenin concentrations in immature male brown trout. At five sites, vitellogenin concentrations exceeded 1 microg/ml; however, at the majority of the sites, plasma vitellogenin concentrations were below 0.5 microg/ml. Our data indicate that the exposure of brown trout to environmental estrogens in Swiss midland rivers is low. However, some sites show reoccurring higher EEQ values and, at some sites, plasma vitellogenin concentrations in male fish clearly are elevated.

Endocrine disrupting compounds: Can they target the immune system of fish?

Endocrine disruption, in particular disruption by estrogen-active compounds, has been identified as an important ecotoxicological hazard in the aquatic environment. Research on the impact of endocrine disrupting compounds (EDCs) on wildlife has focused on disturbances of the reproductive system. However, there is increasing evidence that EDCs affect a variety of physiological systems other than the reproductive system. Here, we discuss if EDCs may be able to affect the immune system of fish, as this would have direct implications for individual fitness and population growth. Evidence suggesting an immunomodulatory role of estrogens in fish comes from the following findings: (a) estrogen receptors are expressed in piscine immune organs, (b) immune gene expression is modulated by estrogen exposure, and (c) pathogen susceptibility of fish increases under estrogen exposure.

Assessment of estrogenic exposure in brown trout (Salmo trutta) in a Swiss midland river: Integrated analysis of passive samplers, wild and caged fish, and vitellogenin mRNA and protein

This field study examined the vitellogenin (VTG) biomarker response under conditions of low and fluctuating activities of environmental estrogenicity. The present study was performed on immature brown trout (Salmo trutta) exposed to the small river Luetzelmurg, which is located in the prealpine Swiss midland region and receives effluents from a single sewage treatment plant (STP). To understand better factors influencing the relationship between estrogenic exposure and VTG induction, we compared VTG levels in caged (stationary) and feral (free-ranging) fish, VTG levels in fish from up- and downstream of the STP, and two different methods for quantifying VTG (enzyme-linked immunosorbent assay vs real-time reverse transcription-polymerase chain reaction), and we used passive samplers (polar organic chemical integrative sampler [POCIS]) to integrate the variable, bioaccumulative estrogenic load in the river water over time. The POCIS from the downstream site contained approximately 20-fold higher levels of bioassay-derived estrogen equivalents than the POCIS from the upstream site. In feral fish, this site difference in estrogenic exposure was reflected in VTG protein levels but not in VTG mRNA. In contrast, in caged fish, the site difference was evident only for VTG mRNA but not for VTG protein. Thus, the outcome of VTG biomarker measurements varied with the analytical detection method (protein vs mRNA) and with the exposure modus (caged vs feral). Our findings suggest that for environmental situations with low and variable estrogenic contamination, a multiple-assessment approach may be necessary for the assessment of estrogenic exposure in fish.

Proliferative kidney disease (PKD) of rainbow trout: temperature- and time-related changes of Tetracapsuloides bryosalmonae DNA in the kidney.

Proliferative kidney disease (PKD) of salmonids, caused by Tetracapsuloides bryosalmonae, can lead to high mortalities at elevated water temperature. We evaluated the hypothesis that this mortality is caused by increasing parasite intensity. T. bryosalmonae-infected rainbow trout (Oncorhynchus mykiss) were reared at different water temperatures and changes in parasite concentrations in the kidney were compared to cumulative mortalities. Results of parasite quantification by a newly developed real-time PCR agreed with the number of parasites detected by immunohistochemistry, except for very low or very high parasite loads because of heterogenous distribution of the parasites in the kidney. Two experiments were performed, where fish were exposed to temperatures of 12, 14, 16, 18 or 19 degrees C after an initial exposure to an infectious environment at 12-16 degrees C resulting in 100% prevalence of infected fish after 5 to 14 days of exposure. While mortalities differed significantly between all investigated water temperatures, significant differences in final parasite loads were only found between fish kept at 12 degrees C and all other groups. Differences in parasite load between fish kept at 14 degrees C to 19 degrees C were not significant. These findings provide evidence that there is no direct link between parasite intensity and fish mortality.

Sensitivity of brown trout reproduction to long-term estrogenic exposure

A decline in brown trout (Salmo trutta fario) catches has been reported in Switzerland, but at present the causative factors have not been clearly identified. Estrogen-active endocrine disrupters (EEDs) have been suggested as one possible explanation, since they are widespread in the aquatic environment and often found at elevated concentrations. In the present study the effects of long-term estrogenic exposure on the reproductive capability of brown trout were investigated. Adult fish were continuously exposed to an environmentally relevant mixture of the natural estrogens estrone (E1), 17beta-estradiol (E2) and the xenoestrogen 4-nonylphenol (NP); the average measured concentrations over the entire exposure time (n=9) were 14.0 ng/l (Min 8.1 and Max 20.6) for E1, 2.1 ng/l (Min 1.3 and Max 4.1) for E2 and 111.0 ng/l (Min 106.7 and Max 115.9) for NP. A solvent control served as negative control, and up to 10-fold higher mixture concentration than the environmentally relevant concentration served as positive control. The fish were exposed for 150 days from the onset of gonadal recrudescence until sexual maturation. Plasma vitellogenin (Vtg) was significantly induced by both concentrations of the estrogenic mixture, whereas effects on growth and fertility were only observed in fish exposed to the high mixture treatment. Fertilization success and offspring hatchability in brown trout exposed to the high mixture treatment were significantly reduced to 9% and 6%, respectively. Developmental time from fertilization until hatching, the percentage of larvae with malformations and survival of larvae, however, were not affected. The results suggest that a combination of estrogen-active compounds at environmentally relevant concentrations would not adversely affect those parameters of brown trout reproductive capability measured in this study. Plasma Vtg in male brown trout appeared to be more sensitive to (xeno)estrogen exposure than the measured reproductive effects.

Molecular crosstalk between a chemical and a biological stressor and consequences on disease manifestation in rainbow trout

The aim of the present study was to examine the molecular and organism reaction of rainbow trout, Oncorhynchus mykiss, to the combined impact of two environmental stressors. The two stressors were the myxozoan parasite, Tetracapsuloides bryosalmonae, which is the etiological agent of proliferative kidney disease (PKD) and a natural stressor to salmonid populations, and 17β-estradiol (E2) as prototype of estrogen-active chemical stressors in the aquatic environment. Both stressors, the parasite and estrogenic contaminants, co-exist in Swiss rivers and are discussed as factors contributing to the decline of Swiss brown trout populations over the last decades. Using a microarray approach contrasting parasite-infected and non-infected rainbow trout at low or high estrogen levels, it was observed that molecular response patterns under joint exposure differed from those to the single stressors. More specifically, three major response patterns were present: (i) expression responses of gene transcripts to one stressor are weakened by the presence of the second stressor; (ii) expression responses of gene transcripts to one stressor are enhanced by the presence of the second stressor; (iii) expression responses of gene transcripts at joint treatment are dominated by one of the two stressors. Organism-level responses to concurrent E2 and parasite treatment - assessed through measuring parasite loads in the fish host and cumulative mortalities of trout - were dominated by the pathogen, with no modulating influence of E2. The findings reveal function- and level-specific responses of rainbow trout to stressor combinations, which are only partly predictable from the response to the single stressors.

Pathogenic infection confounds induction of the estrogenic biomarker vitellogenin in rainbow trout

To examine the behavior of the estrogenic biomarker vitellogenin (VTG) under the combined impact of estrogens and pathogens, parasite-infected or noninfected rainbow trout were exposed to two doses of 17β-estradiol (E2). Infected and E2-exposed fish showed significantly lower hepatic VTG mRNA levels than healthy fish. Transcriptome data suggest that this was due to energetic constraints. Reduced responsiveness of the VTG biomarker in parasitized fish might obscure detection of low-level field exposure.