René Schönenberger

Light‐induced redox cycling of iron in circumneutral lakes

The light-induced redox cycling of Fe II /Fe III was studied both in laboratory experiments and in the field in two circumneutral Swiss lakes: Greifensee, a eutrophic, natural water body, and Melchsee, an oligotrophic, artificial mountain lake. To determine Fe II

The endocrine disrupting potential of sediments from the Upper Danube River (Germany) as revealed by in vitro bioassays and chemical analysis

IntroductionThe present study was part of a comprehensive weight-of-evidence approach with the goal of identifying potential causes for the declines in fish populations, which have been observed during the past decades in the Upper Danube River.MethodsThe specific goal was the investigation of the endocrine disrupting potential of sediment extracts from different sites along the Danube River. Parallel to the identification and quantification of target estrogens, two in vitro bioassays were employed to assess the estrogenic potential (yeast estrogen screen, YES) of the sediment samples and to evaluate their effects on the production of testosterone (T) and E2 (H295R Steroidogenesis Assay). Using a potency balance approach, the contribution of the measured compounds (Chem-EEQs) to the total endocrine activity measured by the YES (YES-EEQs) was calculated.Results and discussionOf the nine sediment extracts tested five extracts exhibited significant estrogenic activities in the YES, which suggested the presence of ER agonists in these samples. The xenoestrogens nonylphenol (NP) and bisphenol A (BPA) and the natural estrogen estrone (E1) were detected while concentrations of 17β-estradiol (E2) and ethinylestradiol (EE2) were less than their respective limits of quantification in all sediment extracts. A comparison of the measured YES-EEQs and the calculated Chem-EEQs revealed that as much as 6% of estrogenic activity in extracts of most sediments could be explained by two xeno- and one natural estrogen. Exposure of H295R cells to sediment extracts from four different locations in the Danube River resulted in significantly increased concentrations of E2, but only slight inhibition of T synthesis. Furthermore, application of the H295R Steroidogenesis Assay provided evidence for endocrine disrupting potencies in sediment samples from the Upper Danube River, some of which were not detectable with the YES. In conclusion, differential endocrine activities were associated with several sediments from the Upper Danube River. Further investigations will have to show whether the observed activities are of biological relevance with regard to declines in fish populations in the Upper Danube River.

Multiple-endpoint assay provides a detailed mechanistic view of responses to herbicide exposure in Chlamydomonas reinhardtii

The release of herbicides into the aquatic environment raises concerns about potential detrimental effects on ecologically important non-target species, such as unicellular algae, necessitating ecotoxicological risk assessment. Algal toxicity tests based on growth, a commonly assessed endpoint, are integrative, and hence do not provide information about underlying toxic mechanisms and effects. This limitation may be overcome by measuring more specific biochemical and physiological endpoints. In the present work, we developed and applied a novel multiple-endpoint assay, and analyzed the effects of the herbicides paraquat, diuron and norflurazon, each representing a specific mechanism of toxic action, on the single celled green alga Chlamydomonas reinhardtii. The endpoints added to assessment of growth were pigment content, maximum and effective photosystem II quantum yield, ATP content, esterase and oxidative activity. All parameters were measured at 2, 6 and 24h of exposure, except for growth and pigment content, which were determined after 6 and 24h only. Effective concentrations causing 50% of response (EC50s) and lowest observable effect concentrations (LOECs) were determined for all endpoints and exposure durations where possible. The assay provided a detailed picture of the concentration- and time-dependent development of effects elicited by the analyzed herbicides, thus improving the understanding of the underlying toxic mechanisms. Furthermore, the response patterns were unique to the respective herbicide and reflected the different mechanisms of toxicity. The comparison of the endpoint responses and sensitivities revealed that several physiological and biochemical parameters reacted earlier or stronger to disturbances than growth. Overall, the presented multiple-endpoint assay constitutes a promising basis for investigating stressor and toxicant effects in green algae.

Water temperature and concomitant waterborne ethinylestradiol exposure affects the vitellogenin expression in juvenile brown trout (Salmo trutta)

Environmental estrogens have the potential to considerably affect the reproduction and development of aquatic vertebrates by interfering with the endocrine system. In addition to the potential risk of environmental estrogens, increasing water temperatures as a result of global warming have become a serious problem in many rivers and streams. To assess the degree of estrogenic exposure, the analysis of the estrogen-dependent protein vitellogenin (Vtg) is a frequently used biomarker in field studies. 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 an environmentally relevant concentration of ethinylestradiol (EE(2)) and held them either at low or high temperatures (12 and 19 degrees C, respectively), but also at temperature cycles of 12-19 degrees C in order to simulate the field situation. The EE(2) exposure caused a 7-74-fold increase of hepatic Vtg mRNA. The synthesis of Vtg mRNA was clearly stimulated in fish held at higher water temperatures (12-19 degrees C and 19 degrees C, respectively). 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 (ERalpha) after exposure to waterborne EE(2). The gene expression of estrogen receptor beta-1 (ERbeta-1) and the glucocorticoid receptor (GR) in the liver of EE(2) exposed fish, however, showed no treatment-related alterations. In line with observed constant bile cortisol concentrations, our data do not indicate corresponding stress related effects on hepatic Vtg production. The present survey, however, clearly demonstrates that increased temperature significantly elevates the estrogen-induced expression of Vtg and therefore has to be considered when interpreting environmental monitoring studies.

Linking proteome responses with physiological and biochemical effects in herbicide-exposed Chlamydomonas reinhardtii.

Exposure to a toxicant causes proteome alterations in an organism. In ecotoxicology, analysis of these changes may allow linking them to physiological and biochemical endpoints, providing insights into subcellular exposure effects and responses and, ultimately mechanisms of action. Based on this, useful protein markers of exposure can be identified. We investigated the proteome changes induced by the herbicides paraquat, diuron, and norflurazon in the green alga Chlamydomonas reinhardtii. Shotgun proteome profiling and spectral counting quantification in combination with G-test statistics revealed significant changes in protein abundance. Functional enrichment analysis identified protein groups that responded to the exposures. Significant changes were observed for 149-254 proteins involved in a variety of metabolic pathways. While some proteins and functional protein groups responded to several tested exposure conditions, others were affected only in specific cases. Expected as well as novel candidate markers of herbicide exposure were identified, the latter including the photosystem II subunit PsbR or the VIPP1 protein. We demonstrate that the proteome response to toxicants is generally more sensitive than the physiological and biochemical endpoints, and that it can be linked to effects on these levels. Thus, proteome profiling may serve as a useful tool for ecotoxicological investigations in green algae.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

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.

Experimental study and steady-state simulation of biogeochemical processes in laboratory columns with aquifer material.

Packed bed laboratory column experiments were performed to simulate the biogeochemical processes resulting from microbially catalyzed oxidation of organic matter. These included aerobic respiration, denitrification, and Mn(IV), Fe(III) and SO(4) reduction processes. The effects of these reactions on the aqueous- and solid-phase geochemistry of the aquifer material were closely examined. The data were used to model the development of alkalinity and pH along the column. To study the independent development of Fe(III)- and SO(4)-reducing environments, two columns were used. One of the columns (column 1) contained small enough concentrations of SO(4) in the influent to render the reduction of this species unimportant to the geochemical processes in the column. The rate of microbially catalyzed reduction of Mn(IV) changed with time as evidenced by the variations in the initial rate of Mn(II) production at the head of the column. The concentration of Mn in both columns was controlled by the solubility of rhodochrosite (MnCO(3(S))). In the column where significant SO(4) reduction took place (column 2), the concentration of dissolved Fe(II) was controlled by the solubility of FeS. In column 1, where SO(4) reduction was not important, maximum dissolved Fe(II) concentrations were controlled by the solubility of siderite (FeCO(3(S))). Comparison of solid-phase and aqueous-phase data suggests that nearly 20% of the produced Fe(II) precipitates as siderite in column 1. The solid-phase analysis also indicates that during the course of experiment, approximately 20% of the total Fe(III) hydroxides and more than 70% of the amorphous Fe(III) hydroxides were reduced by dissimilatory iron reduction. The most important sink for dissolved S(-II) produced by the enzymatic reduction of SO(4) was its direct reaction with solid-phase Fe(III) hydroxides leading initially to the formation of FeS. Compared to this pathway, precipitation as FeS did not constitute an important sink for S(-II) in column 2. In this column, the total reacted S(-II) estimated from the concentration of dissolved sulfur species was in good agreement with the produced Cr(II)-reducible sulfur in the solid phase. Solid-phase analysis of the sulfur species indicated that up to half of the originally produced FeS may have possibly transformed to FeS(2).

Internal exposure of whitefish (Coregonus lavaretus) to estrogens.

Gonad malformations have been found in fish all over the world. Particularly in Lake Thun (Switzerland) a high prevalence of gonad deformations in whitefish has been observed. Very often, a link between exposure to endocrine disrupting compounds and altered gonad morphology exists. Hence, we analyzed the estrogenic burden in bile and muscle from whitefish (coregonids) from Lake Thun and linked it to observed gonad malformations. Estrogenicity in bile, measured with the yeast estrogen screen (YES) was exclusively caused by the natural steroids estrone and 17beta-estradiol. Estrogenicity determined in muscle tissue using YES was similar in cases and controls, and between the sexes. Furthermore, endocrine active compounds in the lake water were investigated using passive sampling devices to monitor tributaries and the main outflow of Lake Thun. Here, we found accumulated estrogenicity. With target chemical analysis small amounts of estrone and bisphenol A were determined. We conclude, that the whitefish from Lake Thun are not suffering from (xeno)estrogens. The present study contributed substantially to the search for the cause for gonad malformations in Lake Thun whitefish, even though the cause of the malformations remains yet to be discovered.

TfdDII, one of the two chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4), cannot efficiently convert 2-chloro-cis, cis-muconate to trans-dienelactone to allow growth on 3-chlorobenzoate

Abstract.Ralstonia eutropha JMP134 (pJP4) harbors two functional gene clusters for the degradation of chlorocatechols, i.e. tfdCDEF (in short: tfdI) and tfdDIICIIEIIFII (in short: tfdII), which are both present on the catabolic plasmid pJP4. In this study, we compared the function of both gene clusters for degradation of chlorocatechols by constructing isolated and hybrid tfdI-tfdII clusters on plasmids in R. eutropha, by activity assays of Tfd enzymes, and by HPLC/MS of individual enzymatic catalytic steps in chlorocatechol conversion. R. eutropha containing the tfdII cluster alone or hybrid tfd-clusters with tfdDII as sole gene for chloromuconate cycloisomerase were impaired in growth on 3-chlorobenzoate, in contrast to R. eutropha harboring the complete tfdI cluster. Enzyme activities for TfdDII and for TfdEII were very low in R. eutropha when induced with 3-chlorobenzoate. By contrast, a relatively high enzyme activity was found for TfdFII. Spectral conversion assays with extracts from R. eutropha strains expressing tfdDII all showed accumulation of a compound with a similar UV spectrum as 2-chloro-cis,cis-muconate from 3-chlorocatechol. HPLC analysis of in vitro assays in which each individual step in 3-chlorocatechol conversion was reproduced by sequentially adding cell extracts of an Escherichia coli expressing one Tfd enzyme only demonstrated that TfdDII was unable to cause conversion of 2-chloro-cis,cis-muconate. No accumulation of intermediates was observed with 4-chlorocatechol. From these results, we conclude that at least TfdDII is a bottleneck in conversion of 3-chlorocatechol and, therefore, in efficient metabolism of 3-chlorobenzoate. This study showed the subtle functional and expression differences between similar enzymes of the tfd-encoded pathway and demonstrated that extreme care has to be taken when inferring functionality from sequence data alone.