Toxicity and mutagenicity of Upper Danube River sediments determined by chemical fractionation, the Danio rerio embryo assay, the Ames fluctuation test and the H295R assay.
Higley, Eric Bertram
Declines in some fish populations in the Upper Danube River, Germany, have been reported during the past decades despite extensive stocking efforts. Many theories exist for why such declines have occurred including habitat change, dams, invasive species, disease and pollution. One of the factors of concern in the Upper Danube River is pollution because a number of studies have shown that sediments collected from this area were acutely and/or chronically toxic to fish. Although it can be difficult to link bioassay results to direct effects on the population level, bioassays can give us insight into the potential of exposure of wildlife including fish to sediment. In combination with other researchers a large battery of sediment testing on the Upper Danube River is being performed. Testing includes sediment testing of estrogen receptor mediated processes, dioxin-like responses and genotoxic effects. In this study, four sediment extracts from the Upper Danube River in Germany were used with a novel fractionation technique to characterize the sediment extracts and fractions for their ability to disrupt steroidogenesis, for their mutagenic activities and their teratogenic effects. Fractionation of each of the four sediment samples was performed by separating compounds according to their polarity, planarity, and the size of the aromatic ring system in an on-line fractionation procedure on coupled high performance liquid chromatography columns. Mutagenic activity was measured in the raw sediment extracts and all 18 fractions using the Ames fluctuation assay and the Danio rerio embryo assay was used to assess lethal endpoints. Furthermore, disruptions of steroidogenesis were assessed by first establishing methods and a proof of concept of the H295R assay by exposing H295R cells to 7 model chemicals and measuring changes from a control in estradiol, testosterone and aromatase activity. Once methods were established all sediments and their fractions were analyzed using the Assay. Specifically, in the Danio rerio assay, two raw sediment extracts killed 100% of Danio rerio embryos at a concentration of 33.3 mg sediment equivalents (SEQ)/ml, but none of the 18 fractions of these samples produced any measured toxicity at a concentration of 100 mg SEQ/ml. In the Ames fluctuation assay, significant mutagenic activity was measured in raw sediment extracts and in the fractions. Fraction 10 produced a significant mutagenic response in all sediment samples measured only in S9 bio-activated samples. Furthermore, fraction 15 produced a significant mutagenic response in all sediment samples measured only in non bio-activated samples. All raw extracts tested in the H295R assay caused an increase in estradiol production up to 4-fold from controls. Testosterone production increased slightly from controls in only two of the raw extract samples. Of the 18 fractions, fractions 7, 10 and 15 increased estradiol in at least three of the samples studied (Sigmaringen2006, Opfingen2006, Lauchert2006 and Lauchert2004). Furthermore, fraction 7 significantly decreased testosterone production compared to controls in three of the four sediment samples. Taken as a whole, these results show the value of using multiple bioassays and fractionation to characterize sediments that covers a variety of different biological endpoints. This study also demonstrates the usefulness of the H295R assay when combined with a new fraction technique to assess endocrine disrupting chemicals in sediment samples.
DegreeMaster of Science (M.Sc.)
SupervisorHecker, Markus; Giesy, John P.
CommitteeHoldway, Douglas A.; Pietrock, Michael
Copyright DateNovember 2009
Upper Danube River