2008, Hoeger, Birgit, Dietrich, Daniel R., Schmid, Daniela, Hartmann, Andreas, Hitzfeld, Bettina C.

The detection of low levels of pharmaceuticals in aquatic environments has lately raised concerns regarding possible adverse effects of these highly active substances on aquatic organisms. The non steroidal anti inflammatory drug diclofenac (DCF) is one of the pharmaceutical substances regularly detected in surface waters and has lately been demonstrated to elicit adverse effects in salmonid species at environmentally relevant concentrations. The aim of the present study was to investigate the distribution of DCF in indigenous brown trout (Salmo trutta f. fario) following intraperitoneal (i.p.) injection of a single dose of 14C labelled DCF. A distribution kinetic over 36 h provides information on possible accumulation of DCF in different organs as well as on DCF detoxification in trout, possibly enabling identification of sites of preferential toxicity. Approximately 57% of the total single DCF dose appeared in the bile 6 h after i.p. application. Subsequently, DCF was observed to undergo enterohepatic cycling with an amount of 14C activity comparable to the 6 h bile values reappearing in bile 36 h after application. Results for 14C activity in intestine and pylori support the observation of enterohepatic cycling with a small peak in intestine at 3 h post i.p. injection and a low peak in intestine and pylori at 6 h post i.p. injection, reflecting presence of the drug substance in bile. The highest activity in intestine was found 24 h post injection coinciding with low levels in bile, followed by a gradual decrease of activity in intestine mirroring the re uptake of DCF into bile. The finding of enterohepatic cycling of DCF in brown trout is suggestive of a prolonged retention of DCF in brown trout

2006, Dietrich, Daniel R., Hitzfeld, Bettina C., O'Brien, Evelyn

2005, Höger, Birgit, Köllner, Bernd, Dietrich, Daniel R., Hitzfeld, Bettina C.

The detection of residues of various pharmaceuticals in surface waters during the last two decades has prompted concerns about possible adverse effects of this kind of pollution on aquatic organisms. The objective of the present study was to investigate effects of the non-steroidal anti-inflammatory drug diclofenac, one of the pharmaceuticals most prevalent in surface waters, on brown trout (Salmo trutta f. fario), a salmonid species native to German rivers. Brown trout were exposed to 0.5, 5 and 50 μg/L diclofenac for 7, 14 and 21 days, whereby the lowest exposure concentration is comparable with concentrations commonly found in the aquatic environment. Fish exposed to diclofenac displayed significantly reduced haematocrit levels after 7 and 14 days of exposure. After 21 days, trout were examined for histopathological alterations, whereby diclofenac exposure resulted in increased monocyte infiltration in the liver, telangiectasis in gills, and the occurrence of interstitial hyaline droplets, interstitial proteinaceous fluid and mild tubular necrosis in trunk kidney. Concurrent immunohistological analysis revealed an increase of granulocyte numbers in primary gill filaments, as well as granulocyte accumulation and increased major histocompatibility complex (MHC) II expression in kidney, suggestive of an inflammatory process in these organs. Moreover, the ability of diclofenac to hinder the stimulation of prostaglandin E2 synthesis was shown in head kidney macrophages of brown trout in vitro. These findings support the hypothesis that environmental exposure of fish to diclofenac provokes the same mechanism of action in these non-target organisms as previously described for mammalian species and can thus lead to similar (possibly adverse) effects. In general, the present study suggests that exposure of brown trout to diclofenac in concentration ranges commonly found in the environment can result in adverse effects in various organs and possibly compromise the health of affected fish populations.

2004, Höger, Birgit, Köllner, Günter, Heuvel, Michael R. van den, Hitzfeld, Bettina C., Dietrich, Daniel R.

Impairment of immune function in aquatic animals has been proposed as a possible consequence of low-level contamination of surface waters with anthropogenic substances such as through the discharge of wastewater into rivers, lakes, and oceans. The study at hand investigated the effects of chronic (32 weeks) exposure to sewage treatment plant (STP) effluent on the prevalence and distribution of different leucocyte populations in spleen samples of rainbow trout (Oncorhynchus mykiss). To simulate an infection, fish were injected intraperitoneally (ip) with inactivated Aeromonas salmonicida salmonicida, 6 weeks prior to the termination of the experiment. Immunohistological analysis revealed a marked decrease in thrombocyte numbers, an increase of monocytes, altered distribution of B-cells, and higher surface immunoglobulin expression, as well as activation of MHC class II expression in the spleen after exposure to 15% (v/v) effluent. The most prominent finding of the present study, however, was the occurrence of intraplasmatic deposits or inclusions with strong autofluorescence in spleen sections from effluent-exposed trout. In addition to effluent effects, injection of trout with A. salmonicida stimulated infiltration of monocytes, increased staining intensity on thrombocytes, and enhanced MHC class I expression in larger leucocytes surrounding melanomacrophage centres. In general, the current study demonstrates a marked, potentially adverse effect of STP effluent on spleen leucocytes and on the integrity of spleen tissue. The observed response suggests a constant unspecific stimulation of different leucocyte populations and is reminiscent of chronic inflammation.

2006, Jungblut, Anne-Dorothee, Höger, Stefan J., Mountfort, Doug, Hitzfeld, Bettina C., Dietrich, Daniel R., Neilan, Brett A.

Cyanobacteria are well known for their production of non-ribosomal cyclic peptide toxins, including microcystin, in temperate and tropical regions, however, the production of these compounds in extremely cold environments is still largely unexplored. Therefore, we investigated the production of protein phosphatase inhibiting microcystins by Antarctic cyanobacteria. We have identified microcystin-LR and for the first time [d-Asp3] microcystin-LR by mass spectrometric analysis in Antarctic cyanobacteria. The microcystins were extracted from a benthic microbial community that was sampled from a meltwater pond (Fresh Pond, McMurdo Ice Shelf, Antarctica). The extracted cyanobacterial cyclic peptides were equivalent to 11.4 ng MC-LR per mg dry weight by semi-quantitative analyses using HPLC-DAD and the protein phosphatase inhibition assay. Furthermore, we were able to identify the presence of cyanobacterial non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes in total DNA extracts from the mat community.

2005, Höger, Birgit, Hitzfeld, Bettina C., Köllner, Bernd, Dietrich, Daniel R., Heuvel, Michael R. van den

The objective of the present study was to investigate the influence of chronic exposure to municipal sewage treatment effluent at environmentally relevant concentrations on immune parameters in rainbow trout (Oncorhynchus mykiss), including the assessment of potential differences in reactivity between sexually mature male and female fish. Trout were exposed to 1.5 and 15% (v/v) secondary treated municipal sewage effluent for 32 weeks. Fish were injected intra-peritoneally either with inactivated Aeromonas salmonicida to simulate an infection or with PBS as control for this immune challenge 6 weeks prior to sampling. Exposure to effluent resulted in a decrease in A. salmonicida-specific serum antibody level and blood lymphocyte numbers in mature females, but not in male fish. Injection of A. salmonicida resulted in enhanced serum lysozyme activity in mature male trout, which were not exposed to effluent. This stimulating effect of A. salmonicida could not be found in effluent-exposed trout, again potentially revealing a suppressive effect of the effluent. An influence of sampling fish on two consecutive days was observed in many immune parameters, most likely reflecting handling stress. Leucocyte and lymphocyte numbers in peripheral blood were consistently lower in male and female fish on the second sampling day. Phagocytosis in head kidney macrophages from male trout was also influenced by sampling day, whereby a stimulation of this reaction occurred on the second day of sampling. Liver mixed function oxygenase activity was found to be enhanced in mature male trout exposed to 15% effluent. In conclusion, the study showed, that exposure to sewage treatment plant effluent, in surface water relevant concentrations, can lead to potentially adverse effects on selected immune reactions in rainbow trout. However, this study also demonstrated that both handling stress and the sex of mature fish have distinct influences on the immune response detected in male and female fish and are likely to influence measured immune parameters to the extent that subtle effluent induced changes may be difficult to detect.

2005, Jungblut, Anne-Dorothee, Hawes, Ian, Mountfort, Doug, Hitzfeld, Bettina C., Dietrich, Daniel R., Burns, Brendan P., Neilan, Brett A.

This study investigated the diversity of cyanobacterial mat communities of three meltwater ponds Fresh, Orange and Salt Ponds, south of Bratina Island, McMurdo Ice Shelf, Antarctica. A combined morphological and genetic approach using clone libraries was used to investigate the influence of salinity on cyanobacterial diversity within these ecosystems without prior cultivation or isolation of cyanobacteria. We were able to identify 22 phylotypes belonging to Phormidium sp., Oscillatoria sp. and Lyngbya sp. In addition, we identified Antarctic Nostoc sp., Nodularia sp. and Anabaena sp. from the clone libraries. Fresh (17 phylotypes) and Orange (nine phylotypes) Ponds showed a similar diversity in contrast to that of the hypersaline Salt Pond (five phylotypes), where the diversity within cyanobacterial mats was reduced. Using the comparison of identified phylotypes with existing Antarctic sequence data, it was possible to gain further insight into the different levels of distribution of phylotypes identified in the investigated cyanobacterial mat communities of McMurdo Ice Shelf.

2006, Höger, Birgit, Taylor, Sean, Hitzfeld, Bettina C., Dietrich, Daniel R., Heuvel, Michael R. van den

Rainbow trout (Oncorhynchus mykiss) were exposed to 1.5 and 15% v/v secondary treated sewage effluent for 32 weeks in flow-through mesocosms. The exposure encompassed the full period of reproductive development for rainbow trout. Trout did not show any evidence of a dose-dependent change in growth. Fish exposed to 15% effluent were the only group to show mortality (5%) over the duration of the experiment. Trout at the highest effluent concentration had significantly higher liver size than reference water fish. Both male and female trout in the 15% exposure group also exhibited significantly higher gonad weight than the reference group. In female trout, this gonad size increase could be explained by higher egg numbers. Female and male trout both displayed a significant increase in plasma 17β-estradiol levels after exposure to 15% effluent, while neither sex had dose-dependent differences in plasma testosterone. Male trout displayed elevated vitellogenin levels and reduced plasma 11-ketotestosterone concentration after exposure to 15% effluent. Chemical examination of steroidal compounds, including both estrogens and androgens, in the wastewater revealed that only estrone was detectable at a mean concentration of 4.5 ng/L. It is assumed that the effects observed in trout exposed to 15% effluent were consistent with stimulation of reproductive development due to very low levels of estrogens. Overall, long-term exposure to treated sewage effluent containing low levels of estrogen did not have significant negative implications for reproductive development in rainbow trout.

2005, Höger, Stefan J., Hitzfeld, Bettina C., Dietrich, Daniel R.

Toxin-producing cyanobacteria (blue-green algae) are abundant in surface waters used as drinking water resources. The toxicity of one group of these toxins, the microcystins, and their presence in surface waters used for drinking water production has prompted the World Health Organization (WHO) to publish a provisional guideline value of 1.0 μg microcystin (MC)-LR/l drinking water. To verify the efficiency of two different water treatment systems with respect to reduction of cyanobacterial toxins, the concentrations of MC in water samples from surface waters and their associated water treatment plants in Switzerland and Germany were investigated. Toxin concentrations in samples from drinking water treatment plants ranged from below 1.0 μg MC-LR equiv./l to more than 8.0 μg/l in raw water and were distinctly below 1.0 μg/l after treatment. In addition, data to the worldwide occurrence of cyanobacteria in raw and final water of water works and the corresponding guidelines for cyanobacterial toxins in drinking water worldwide are summarized.

2005, Höger, Birgit, Köllner, Bernd, Dietrich, Daniel R., Schmid, Daniela, Linke, Annika, Metzger, Jörg, Hitzfeld, Bettina C.

Diclofenac gehört zu den am häufigsten in der aquatischen Umwelt gefundenen Pharmaka. Der konstante Eintrag von Diclofenac in Oberflächengewässer über Kläranlagen führt zu einer chronischen Exposition aquatischer Lebewesen mit Konzentrationen im niedrigen μg/L Bereich. Da Pharmaka die Eigenschaft besitzen spezifisch in bestimmte Regulationsmechanismen einzugreifen, können schon niedrige Konzentrationen zu leichten Veränderungen physiologischer Vorgänge führen. In der vorliegenden Studie konnte gezeigt werden, dass die Exposition von Bachforellen (Salmo trutta f. fario) mit Diclofenac-Konzentrationen im Bereich der in Oberflächengewässern vorliegenden Werte, zu verminderten Hämatokrit-Werten und histopathologischen Veränderungen in Kieme, Niere und Leber führen kann. Verteilungskinetiken mit einer einmaligen Dosis i.p.-injiziertem 14C-Diclofenac zeigten, dass Diclofenac in Fischen einen enterohepatischen Zyklus eingeht und somit nicht vollständig ausgeschieden wird. Entsprechend liegt die Vermutung nahe, dass Diclofenac unter chronischen Expositionsbedingungen in den Bachforellen akkumulieren kann. Versuche zur Hemmung der Prostaglandin E2-Synthese in Kopfniere-Makrophagen deuten darauf hin, dass Diclofenac in Bachforellen denselben Wirkmechanismus verfolgt, wie bereits für Säugetiere beschrieben.