Angela Krüger

Bisphenol A induces feminization in Xenopus laevis tadpoles

To evaluate possible estrogenic effects of bisphenol A (BPA) in an amphibian model, Xenopus laevis tadpoles were exposed to BPA and 17beta-estradiol (E2) during larval development. After metamorphosis, the gonadal phenotype was determined by gross morphology, and testes were further examined histologically to validate the results. BPA treatment altered the normal sex ratio toward females depending on the BPA concentrations added. Chemical analysis showed a time-dependent decline of BPA during semistatic exposure, indicating that BPA is taken up and metabolized to some extent by tadpoles. In addition, tadpoles were exposed to BPA and E2 for 2 weeks during sensitive stages of sexual differentiation. Afterward, the expression of an estrogenic biomarker, estrogen receptor (ER) mRNA, was assessed by semiquantitative RT-PCR. Both BPA and E2 up-regulated ER mRNA significantly. In conclusion, these results show clear evidence that BPA induces feminization in X. laevis tadpoles, revealing an estrogenic potency of BPA that influences sexual development in amphibians.

Enrichment and cultivation of pelagic bacteria from a humic lake using phenol and humic matter additions.

Individual bacterial populations are known to respond differently toward substrate availability. To test how the availability of either pure phenol or natural humic matter (HM) selects for specific pelagic bacteria phylotypes from a humic lake (Lake Grosse Fuchskuhle, northeastern Germany), we used culture-dependent and -independent approaches. Using a batch approach, the bacterial community composition (BCC) differed depending on both the quantity and the quality of added substrates. Using a dilution-to-extinction approach, distinct BCC were detected by eliminating less abundant species. Most bacteria that were common in the lake were favored by phenol, and yet different subsets of the native BCC were enriched by HM. Specific bacterial groups with different growth requirements were consistently present, negatively influenced, or positively enriched following substrate additions. This study comprises the first explicit demonstration that bacteria such as Methylobacterium, Methylophilus, and Methylosinus spp. can be enriched on phenol or HM. Our isolation approaches led to the successful cultivation of a variety of native bacteria from the lake, such as Novosphingobium (Alphaproteobacteria) and Flexibacter (Bacteroidetes), or phenol-utilizing bacteria such as members of Actinobacteria or Burkholderia (Betaproteobacteria). Enrichment and cultivation on phenol and HM as substrates revealed highly specialized bacterial communities that resemble those found in many HM-rich lakes.

The Synthetic Gestagen Levonorgestrel Disrupts Sexual Development in Xenopus laevis by Affecting Gene Expression of Pituitary Gonadotropins and Gonadal Steroidogenic Enzymes

In the present study, Xenopus laevis tadpoles were chronically exposed to four concentrations of the synthetic gestagen Levonorgestrel (LNG; 10(-11), 10(-10), 10(-9), and 10(-8)M) starting at Nieuwkoop and Faber (NF) stage 48 until completion of metamorphosis. At NF 58 and 66, brain-pituitary and gonad samples were taken for gene expression analyses of gonadotropins and gonadal steroidogenic enzymes. Exposure to 10(-9) and 10(-8)M LNG until NF 58 repressed messenger RNA (mRNA) expression of luteinizing hormone (LH) β in both genders. This decrease was persistent after further treatment until NF 66 in the 10(-8)M LNG treatment. Expression of follicle-stimulating hormone (FSH) β was affected sex-specifically. No effect was present in NF 58 females, whereas LNG at 10(-9) and 10(-8)M significantly increased FSHβ mRNA levels in males. In NF 66 females, 10(-9)M LNG treatment increased FSHβ gene expression, whereas a decrease was observed in NF 66 males exposed to 10(-8)M LNG. In gonads, expression of steroid-5-alpha-reductase was affected sex-specifically with increased mRNA levels in females but repressed levels in males. Gene expression of further gonadal steroidogenic factors was decreased by 10(-8)M LNG in both genders at NF 66. Assessment of gonad gross morphology and histology revealed poorly developed testes in the 10(-8)M LNG treatment. Our results reveal considerable effects of chronic LNG exposure on sexual development of amphibians. The persistent inhibition of LHβ expression concomitant with decreased mRNA levels of gonadal steroidogenic enzymes is suggested to result in the disruption of reproduction in adult amphibians.

Agricultural land-use affects the nutritional quality of stream microbial communities

We investigated how the lipid composition (fatty acids and sterols) of benthic microbial mats, which represent an important basal food resource for stream food webs, differs between tropical streams located in protected pristine and agricultural Cerrado savannah areas. The total microbial biomass and lipid composition differed significantly between pristine and agricultural streams in parallel with differences in water quality and hydrodynamic characteristics. Agricultural streams exhibited lower total biomass of benthic microbial mats than pristine streams. However, the higher concentrations of essential polyunsaturated fatty acids, such as linoleic acid (LIN, 18:2ω6), α-linolenic acid (ALA, 18:3ω3), and eicosapentaenoic acid (EPA, 20:5ω3), that were observed in agricultural streams suggest enhanced lipid complexity and a higher nutritional quality of the microbial community relative to pristine streams. Meanwhile, pristine stream microbial communities had higher total concentrations of saturated fatty acids and cholesterol than those of agricultural streams, reflecting their heterotrophic microbial communities. Moreover, stream morphotype and associated differences in the hydrodynamic characteristics affected the community composition and thereby also the lipid composition of microbial mats. Land-use-induced changes in the total biomass and lipid composition of microbial communities may affect the trophic transfer of energy in stream food webs, leading to changes in the composition and productivity of primary consumers and their predators, and thereby affecting stream ecosystem functioning.

Land-use impacts on fatty acid profiles of suspended particulate organic matter along a larger tropical river

Land-use change, such as agricultural expansion and urbanization, can affect riverine biological diversity and ecosystem functioning. Identifying the major stressors associated with catchment land-use change is a prerequisite for devising successful river conservation and restoration strategies. Here, we analyzed land-use effects on the fatty acid (FA) composition and concentrations in suspended particulate organic matter (SPOM) along a fourth-order tropical river, the Rio das Mortes. Thereby, we aimed at testing the potential of fatty acids in riverine suspended particulate organic matter (SPOM-FAs) as indicators of land-use change in tropical catchments, and at identifying major human impacts on the biochemical composition of SPOM, which represents an important basal energy and organic matter resource for aquatic consumers. River water SPOM and total FA concentrations ranged between 2.8 and 10.2mg dry weight(DW)L(-1) and between 130.6 and 268.2μg DW L(-1), respectively, in our study. Urbanization was the only land-use category correlating with both FA composition and concentrations, despite its low contribution to whole catchment (1.5-5.6%) and riparian buffer land cover (1.7-6.6%). Higher concentrations of saturated FAs, especially C16:0 and C18:0, which are the main components of domestic sewage, were observed at sampling stations downstream of urban centers, and were highly correlated to urbanization, especially within the 60m riparian buffer zone. Compared to water chemical characteristics (inorganic nutrients, dissolved oxygen, pH, and specific conductance) and river habitat structural integrity, FA variables exhibited a higher variability along the investigated river and were more strongly correlated to urban land use, suggesting that SPOM-FA profiles may be an efficient indicator of urban land-use impacts on larger tropical rivers. High total FA concentrations in the SPOM of urbanized tropical rivers may represent high-energy biochemical subsidies to food webs, potentially leading to changes in functional ecosystem characteristics, such as bacterial and suspension-feeder production.

Sterol Composition of Freshwater Algivorous Ciliates Does Not Resemble Dietary Composition

Algivorous ciliates represent an important link in freshwater food webs, as they transfer energy and biochemical matter from their algal prey to mesozooplankton predators. However, it is still unknown how dietary composition influences the biochemical composition of ciliates. We analyzed the sterol composition of the algivorous ciliates Balanion planctonicum and Urotricha farcta and compared it to the sterol composition of their diet—the cryptomonad Cryptomonas phaseolus. The sterol composition of the ciliates did not resemble that of their algal diet. Ergosterol [(22E)-ergosta-5,7,22-trien-3β-ol] was the sterol in highest concentration in C. phaseolus, whereas stigmasterol [(24S)-24-ethylcholesta-5,22E-dien-3β-ol] was dominant in both B. planctonicum and U. farcta. Moreover, the occasionally higher sterol concentrations in the ciliates than in their algal diet suggest sterol accumulation by the ciliates. We conclude that dietary sterol composition influences the composition of the two algivorous ciliates studied, but species-specific differences in metabolism probably determine the ultimate sterol composition of the ciliates.

Prerequisites for the Restoration of the European Atlantic Sturgeon, Acipenser sturio and the Baltic Sturgeon (A. oxyrinchus × A. sturio) in Germany

Acipenser sturio was once a prevalent fish species in all the major rivers of Northern Germany. From the end of the nineteenth century, the population sizes have decreased rapidly. The last large population was observed in the River Eider, where the last specimen was caught in 1969. Under a cooperation agreement with the French Cemagref, the Leibniz-Institute of Freshwater Ecology and Inland Fisheries in Berlin received juvenile A. sturio of Gironde origin in 1996 and initiated an ex-situ measure. The main scientific results found since then have been: (1) first gonad maturation occurred in 11-year-old A. sturio (110 to 140 cm total length) kept in freshwater at 20°C under a natural photoperiod; (2) during vitellogenesis the growth factor insulin-growth-like factor (IGF-I) plays an important role as a paracrine modulator, as observed in the model species A. ruthenus; (3) analysis of recent and historical material revealed the presence of the A. oxyrinchus mitochondrial haplotype A in the Baltic. Investigation of the MHC (Major Histocompatibility Complex) nuclear gene, however, showed that these fish carrying this haplotype represent a hybrid population (A. oxyrinchus × A. sturio). The current restoration strategy concerning the Baltic (restocking with A. oxyrinchus) therefore needs to be reconsidered. The mtDNA studies in addition demonstrated the genetic similarity of the Gironde and the North Sea population; (4) A. sturio-specific microsatellites were established for brood stock management of the German and French brood stocks; (5) Evaluation of historical spawning grounds in the River Oder drainage in collaboration with Polish scientists showed intact spawning grounds in the Drawa River.

Screening of multiple hormonal activities in water and sediment from the river Nile, Egypt, using in vitro bioassay and gonadal histology

In Egypt, until yet no records are available regarding possible multiple hormonal activities in the aquatic systems and especially in the river Nile. In this paper, in vitro yeast estrogen screen (YES) and yeast androgen screen (YAS) were used to assess (for the first time) the multiple hormonal activities in surface waters and sediments of the river Nile. This study aimed to determine whether river Nile water can cause changes in gonadal histology of Nile tilapia (Oreochromis niloticus niloticus). All water samples exhibited extremely low levels of estrogenicity. Estrogenicity was nearly not detected in any of the sediment samples. Unlike the estrogenicity, significant androgenic activities were recorded in the water and sediment samples along the course of the river Nile. The present study reports for the first time quantification anti-estrogenic and anti-androgenic activities with high levels in both water and sediment of the river Nile. The greatest anti-estrogenic and anti-androgenic activities were observed in samples from downstream river Nile. These results indicated that the anti-estrogenic and anti-androgenic activities along the Nile course were great and the pollution of the sites at downstream was more serious than the upstream sites due to industrial and anthropogenic activities at these sites. Good correlations were observed among some hormonal activities, suggesting coexistence of these contaminants in the environmental matrices. There were no signs of sexual disruption in any of the gonads analyzed from either male or female Nile tilapia, demonstrating that no hormonal activity present along the Nile course was sufficient to induce adverse effects on reproductive development. Further investigation is necessary to identify the compounds responsible for the hormonal activities in the river Nile and to examine effects of very low levels of hormonally active compounds on gonadal histology, as well as in the development of more sensitive biomarkers.

Wie beeinflussen technische Parameter von Schwebstoffzentrifugen die Analyseergebnisse

Zur Bewertung der Schadstoffbelastung von Gewassern mus auch der mit den Schwebstoffen sorptiv transportierte Anteil von Schadstoffen bestimmt werden. Eine fur die Spurenanalytik ausreichende Menge an partikularem Material kann mit einer sogenannten Schwebstoffzentrifuge (mobile Kombination von Pumpe, Schlauchzuleitung und Durchfluszentrifuge) gewonnen werden (Kruger und Bungartz 1996, Breitung 1997). Der Beantwortung der Frage, inwieweit technische Parameter bei der Probenahme das Analysenresultat beeinflussen, d.h. wie reprasentativ Schwebstoffe gewonnen werden konnen, diente ein gemeinsamer Feldversuch des Institutes fur Gewasserokologie und Binnenfischerei (IGB) und der Bundesanstalt fur Gewasserkunde, Ausenstelle Berlin (BfG) bei Ratzdorf (Oder-km 542) im Mai 1997. Es kamen zwei Zentrifugen unterschiedlicher Bauart zum Einsatz (Tab.1).

The progestin norethisterone affects thyroid hormone-dependent metamorphosis of Xenopus laevis tadpoles at environmentally relevant concentrations

Previously, levonorgestrel (LNG) has been shown to be an endocrine disruptor of the amphibian thyroid system. In the present study, we investigated whether anti-thyroidal effects are a common property of progestins other than LNG. Premetamorphic Xenopus laevis tadpoles were exposed to norethisterone (NET) and dienogest DIE (each at 0.1-10nM) and LNG (10nM) until completion of metamorphosis. LNG and NET at all concentrations caused a significant developmental retardation whereas DIE did not impair time to metamorphosis. In LNG and 10nM NET exposed animals, tsh mRNA levels increased considerably later than the developmental delay occurred and thyroid histopathology showed no signs of TSH-hyperstimulation. Instead, thyroid glands from these treatments appeared inactive in producing thyroid hormones. Thyroidal transcript levels of dio2 and dio3 were increased by treatments with LNG and NET at 1nM and 10nM, whereas iyd mRNA was reduced by LNG and 10nM NET. Expression of slc5α5 was not changed by any treatment. Effects of DIE differed from those induced by LNG and NET. No developmental delay was measurable; however, tshβ and dio2 mRNAs were increased in pituitary glands of tadpoles exposed to 1.0nM and 10nM DIE. Thyroid histopathology displayed no abnormalities and thyroidal mRNA expression of the genes analyzed (slc5α5, iyd, dio2, dio3) was not changed by DIE. Overall, our results provide evidence that the anti-thyroidal effects already known from LNG are also present in another progestin, namely NET, even at environmentally relevant concentrations. In conclusion we suggest that progestins do not only pose an environmental risk in terms of their impact on reproductive success of aquatic vertebrates, but also with respect to their anti-thyroidal properties affecting amphibian metamorphosis.