Methylierung von anorganischem Quecksilber im Intestinaltrakt des Kompostwurms Eisenia foetida

Im Rahmen dieser Arbeit wurde die Methylierung von Quecksilber in Intestinaltrakt des Kompostwurms Eisenia foetida untersucht. Des Weiteren wurden aerobe und anaerobe Mikroorganismen aus dem Darmtrakt von Eisenia fotida isoliert, identifiziert und auf ihr Potential zur Methylierung von Quecksilber getestet. Die Bestimmung von Methylquecksilber erfolgte mittels GC-ICPMS (Gaschromatographie mit induktiv gekoppelter Plasma-Massenspektrometrie) und GC-AFS (Gaschromatographie- Atomfluoreszenzspektrometrie). Für die GC-ICPMS erfolgte die Quantifizierung des Methylquecksilbers mittels der Isotopenverdünnungsmethode. Die Extraktion des Methylquecksilbers aus dem Wurmgewebe erfolgte durch einen alkalischen Aufschluss mit TMAH (Tetramethylammoniumhydroxid) und anschließender Derivatisierung des Methylquecksilbers durch Natriumtetrapropylborat. Für die Extraktion des gebildeten Methylquecksilbers aus Bakterienkulturen wurde eine Extraktion mit einer methanolischen Kaliumhydroxidlösung verwendet. Wie bei dem Wurmgewebe wurde das Methylqueckilsber ebenfalls mit Natriumtetrapropylborat derivatisiert.rnrnFür die Untersuchung einer in vivo Methylquecksilberbildung in bodenlebenden Invertebraten wurde der Kompostwurm Eisenia foetida als Modellorganismus verwendet. Die Tiere wurden aus einer Kultur in einen Boden überführt, der mit anorganischem Quecksilber versetzt wurde. Nach zehn Tagen Inkubationszeit wurden die Würmer entnommen und das Methylquecksilber extrahiert. Um eine mögliche Methylierung von Quecksilber durch Bodenorganismen auszuschließen wurde sowohl steriles als auch unsteriles Bodenmaterial verwendet. In den Wurmproben aus dem unsterilen Bodenmaterial konnte eine Konzentration an Methylquecksilber von 17,4 ng/g Trockengewicht (Boden ohne Zugabe von Quecksilber) und 62,4 ng/g Trockengewicht (Boden mit Quecksilberzugabe). Bei den Wurmproben aus sterilem Bodenmaterial lag die Konzentration an Methylquecksilber bei 17,2 ng/g Trockengewicht (Boden ohne Zugabe von Quecksilber) und 51,9 ng/g Trockengewicht (Boden mit Quecksilberzugabe).rnrnBei den Bakterienkulturen konnte in Reinkulturen keine Methylierung von Quecksilber nachgewiesen werden. In einer fakultativ anaeroben Mischkultur konnte eine Methylierung von Quecksilber beobachtet werden. Für die Identifizierung der Mikroorganismen wurde die 16s rDNA mittels PCR amplifiziert und anschließend über eine DGGE aufgetrennt. Die Banden wurden ausgeschnitten und sequenziert. Dabei konnten drei Enterobacteriaceen identifiziert werden.rnIn the scope of this thesis the methylation of mercury in the intestine of the worm Eisenia foetida was investigated. Furthermore aerobic and anaerobic microorganisms were isolated from the intestine of Eisenia fotida identified and screened for their potential to methylate mercury. . The analysis of methyl mercury was carried out by GC-ICPMS (gas chromatography-inductivly coupled plasma mass specrometry) and GC-AFS (gas chromatography-atomic fluorescence spectrometry). The quantification of methylmercury by GC-ICPMS was done with the isotope dilution method. rnExtraction of methylmercury in worm tissue was carried out by an alkaline digestion with TMAH and derivatisation with Sodiumtetrapropylborate. For bacterial culture the extraction was done with a methanolic potassium hydroxide solution followed by derivatisation with Sodiumtetrapropylborate.rnrnThe experiments for the methylation by soil living invertebrates were done with the compost worm Eisenia foetida. The organisms were transferred from a in house culture into soil to which inorganic mercury was added. After an incubation of ten days the worms were taken and prepared for the measurements. To look for a possible methylation by soil living microorganisms sterilized and non sterilized soil were used. In the worm tissue from non sterilized soil a methylmercury concentration of 17.4 ng per g dry weight (soil without addition of inorganic mercury) and 62.4 ng per g dry weight (soil with addition of inorganic mercury). In the worm tissue from sterilized soil a methylmercury concentration of 17.2 ng per g dry weight (soil without addition of inorganic mercury) and 51.9 ng per g dry weight (soil with addition of inorganic mercury).rnrnIn pure cultures of isolated intestine microorganisms no methylation of mercury could be observed. In contrast a mix facultative anaerobic culture showed a methylation of mercury. Identification of microorganisms in the mix culture by 16s rDNS and DGGE results in three members of the Family of the Enterobacteriaceae.rn147 S

A microcosm study to support aquatic risk assessment of nickel : community-level effects and comparison with bioavailability-normalized species sensitivity distributions

The aquatic risk assessment for nickel (Ni) in the European Union is based on chronic species sensitivity distributions and the use of bioavailability models. To test whether a bioavailability-based safe threshold of Ni (the hazardous concentration for 5% of species [HC5]) is protective for aquatic communities, microcosms were exposed to 5 stable Ni treatments (6-96 mu g/L) and a control for 4mo to assess bioaccumulation and effects on phytoplankton, periphyton, zooplankton, and snails. Concentrations of Ni in the periphyton, macrophytes, and snails measured at the end of the exposure period increased in a dose-dependent manner but did not indicate biomagnification. Abundance of phytoplankton and snails decreased in 48 mu g Ni/L and 96 mu g Ni/L treatments, which may have indirectly affected the abundance of zooplankton and periphyton. Exposure up to 24 mu g Ni/L had no adverse effects on algae and zooplankton, whereas the rate of population decline of the snails at 24 mu g Ni/L was significantly higher than in the controls. Therefore, the study-specific overall no-observed-adverse-effect concentration (NOAEC) is 12 mu g Ni/L. This NOAEC is approximately twice the HC5 derived from a chronic species sensitivity distribution considering the specific water chemistry of the microcosm by means of bioavailability models. Thus, the present study provides support to the protectiveness of the bioavailability-normalized HC5 for freshwater communities

Determination of low environmental free cyanide concentrations in freshwaters

Cyanide compounds are naturally emitted into the environment in low levels by degradation processes or emitted from anthropogenic sources. In surface water, complex cyanide compounds as well as 'free cyanide' are present. The latter term covers hydrogen cyanide and cyanide compounds which easily liberate hydrogen cyanide under slightly acidic conditions. Especially free cyanide may cause adverse effects in the environment. To exclude negative impacts on freshwater systems, in the context of the European Water Framework Directive (WFD), preventive regulatory activities for free cyanide are currently under discussion. However, established analytical methods for quantification of free cyanide only obtain limits of quantification (LOQs) in the range of 1 μg 

Development of a method to determine the bioaccumulation of manufactured nanomaterials in filtering organisms (Bivalvia). Final Report

Entwicklung einer Methode zur Bestimmung der Bioakkumulation von synthetischen Nanomaterialien in filtrierenden Organismen (Bivalvia) Immer größer werdende Mengen an synthetischen Nanomaterialien (MNMs) werden für den industriellen Einsatz produziert und können während der Produktion, dem Einsatz der Produkte, sowie bei deren Entsorgung in die Umwelt gelangen. MNMs mit hohen Produktionsvolumina unterliegen einer Bioakkumulationsbewertung im Rahmen der EU REACH Verordnung, um potentielle Umweltbelastungen abschätzen zu können. Die hierbei für die Chemikalienbewertung klassischerweise verwendeten Methoden, etwa Durchflussstudien mit Fischen gemäß OECD TG 305, sind für das Testen von MNMs in aquatischen Medien jedoch nur bedingt geeignet. So neigen die meisten MNMs dazu, in aquatischen Systemen nur metastabile Suspensionen zu bilden und direkt nach dem Eintreten in das Medium oder im zeitlichen Verlauf zu sedimentieren. Eine konstante homogene Exposition im Testsystem wird somit stark erschwert. Für Corbicula fluminea, eine weit verbreitete Süßwassermuschel, wurde bereits in früheren Studien gezeigt, dass sie MNMs aus der Wasserphase durch Filtration aufnehmen kann. Im Rahmen dieses Projekts wurde die Eignung von C. fluminea für Bioakkumulationsstudien mit MNMs geprüft. Hierzu wurde ein neues Durchflusssystem entwickelt, welches eine konstante und homogene Exposition von MNMs ermöglicht. Zur Überprüfung wurden synthetische Nanomaterialien gewählt, welche jeweils MNMs mit bestimmten Eigenschaften repräsentieren. Das Silbernanopartikel NM 300K (Ag NP) wurde als Repräsentant der Gruppe der gutdispergierbaren und ionenfreisetzenden MNMs getestet und mit AgNO3 als nichtnanopartikuläre Form desselben Elements verglichen. NM 105, ein Titandioxid NP, wurde für die Gruppe der nicht ionenfreisetzenden MNMs getestet. Für die Gruppe der MNMs, welche auf organischen Polymeren basieren, wurde das Polystyrol NP Fluoro-Max™ getestet, welches mit einem Fluoreszenzfarbstoff markiert war. Somit konnte die Aufnahme und Verteilung des NPsim Weichkörper der Muscheln u.a. mittels Fluoreszenzmikroskop untersucht werden. Für die Ag und TiO2 Behandlungen konnten nach Messung der Gewebekonzentrationen BAF bzw. BCFWerte im Konzentrationsgleichgewicht ermittelt werden. BAFss Werte von 31 und 128 für die beiden NM 300K Konzentrationen (0,624 und 6,177 μg Ag/L) und 6150 und 9022 für die beiden NM 105 Konzentrationen (0,099 und 0,589 μg TiO2/L) zeigten, dass BAFss Werte für die untersuchten MNMs abhängig von der jeweiligen Expositionskonzentration sind. Für die AgNO3Behandlung wurden ebenso konzentrationsabhängige BCFss Werte von 31 und 711 für die höhere und niedrigere Konzentrationen ermittelt. Die Kinetik der gemessenen Partikelkonzentrationen in den Muschelgeweben (sp-ICP-MS) wie auch die ermittelten Distributionsfaktoren für einzelne Kompartimente lieferten Hinweise, dass die untersuchten MNMs zwar aufgenommen, aber nicht inkorporiert wurden

Retrospective monitoring of mercury in fish from selected European freshwater and estuary sites

International audienc

Detoxification and sensing mechanisms are of similar importance for Cd resistance in Caenorhabditis elegans

The present study employed mass spectrometry (ICP-MS) to measure the internal cadmium concentrations (Cdint) in Caenorhabditis elegans to determine Cd uptake from a Cd-containing environment as well as Cd release under Cd-free conditions. To analyze the functional role of several ATP binding cassette (ABC) transporters (e.g., HMT-1 and MRP-1) and phytochelatin synthase (PCS), we compared wild-type (WT) and different mutant strains of C. elegans. As a pre-test on selected mutant strains, several time-resolved experiments were performed to determine the survival rate and avoidance behavior of C. elegans under Cd stress, which confirmed the already known Cd sensitivity of the deletion mutants mrp-1Δ, pcs-1Δ, and hmt-1Δ. In addition, these experiments revealed flight reactions under Cd stress to be almost completely absent in mrp-1Δ mutants. The ICP-MS studies showed Cd uptake to be significantly higher in mrp-1Δ and WT than in hmt-1Δ. As Cd is ingested with food, food refusal due to very early Cd stress and its perception was likely the reason for the reduced Cd uptake of hmt-1Δ. Cd release (detoxification) was found to be maximal in mrp-1Δ, minimal in hmt-1Δ, and intermediate in WT. High mortality under Cd stress, food refusal, and minimal Cd release in the case of hmt-1Δ suggest a vital importance of the HMT-1/PCS-1 detoxification system for the survival of C. elegans under Cd stress. High mortality under Cd stress, absence of an avoidance behavior, missing food refusal, and maximal Cd release in the case of mrp-1Δ indicate that MRP-1 is less important for Cd detoxification under severe stress, but is probably important for Cd perception. Accordingly, our results suggest that the survival of WT under Cd stress (or possibly other forms of metal stress) primarily depends on the function of the HMT-1/PCS-1 detoxification system and the presence of a sensing mechanism to control the uptake of Cd (or other metals), which keeps internal Cd (or metal) concentrations under control, to some extent, for the timely mobilization of protection and repair systems

Long-term effects of sulfidized silver nanoparticles in sewage sludge on soil microflora

The use of silver nanoparticles (AgNPs) in consumer products such as textiles leads to their discharge into wastewater and consequently to a transfer of the AgNPs to soil ecosystems via biosolids used as fertilizer. In urban wastewater systems (e.g., sewer, wastewater treatment plant [WWTP], anaerobic digesters) AgNPs are efficiently converted into sparingly soluble silver sulfides (Ag2S), mitigating the toxicity of the AgNPs. However, long-term studies on the bioavailability and effects of sulfidized AgNPs on soil microorganisms are lacking. Thus we investigated the bioavailability and long-term effects of AgNPs (spiked in a laboratory WWTP) on soil microorganisms. Before mixing the biosolids into soil, the sludges were either anaerobically digested or directly dewatered. The effects on the ammonium oxidation process were investigated over 140 d. Transmission electron microscopy (TEM) suggested an almost complete sulfidation of the AgNPs analyzed in all biosolid samples and in soil, with Ag2S predominantly detected in long-term incubation experiments. However, despite the sulfidation of the AgNPs, soil ammonium oxidation was significantly inhibited, and the degree of inhibition was independent of the sludge treatment. The results revealed that AgNPs sulfidized under environmentally relevant conditions were still bioavailable to soil microorganisms. Consequently, Ag2S may exhibit toxic effects over the long term rather than the short term

Testing the bioaccumulation of manufactured nanomaterials in the freshwater bivalve Corbicula fluminea using a new test method

Increasing amounts of manufactured nanomaterials (MNMs) are produced for their industrial use and released to the environment by the usage or disposal of the products. As depending on their annual production rate, substances are subjected to PBT assessment, the availability of reliable methods to evaluate these endpoints for (corresponding) nanoforms/MNMs becomes relevant. The classical method to elucidate the bioaccumulation potential of chemicals has been the flow-through study with fish, which has limitations as regards meeting the requirements of MNMs. Most MNMs tend to sediment in the aquatic environment. Thus, maintenance of stable exposure conditions for bioaccumulation testing with fish is nearly impossible to achieve when using MNMs. Corbicula fluminea, a freshwater filter-feeding bivalve distributed worldwide, has been previously shown to ingest and accumulate MNMs present in the water phase. To investigate the suitability of C. fluminea for bioaccumulation testing we developed a new flow-through system to expose mussels under constant exposure conditions. Two nanoparticles (NPs), the AgNP NM 300K and the TiO2NP NM 105, were applied. In addition, C. fluminea was exposed to AgNO3 as a source of dissolved Ag+ to compare the bioaccumulation of Ag in dissolved and nanoparticulate forms. For each MNM exposure scenario we were able to determine steady-state bioaccumulation factors. BAFss values of 31 and 128 for two NM 300K concentrations (0.624 and 6.177 μg Ag per L) and 6150 and 9022 for TiO2 (0.099 and 0.589 μg TiO2 per L) showed the exposure dependence of the BAFss estimates. The progression of metal uptake and elimination in the soft tissue provided clear indications that the uptake and thus accumulation is mainly driven by the uptake of NPs and less of dissolved ions

Assessment of seafood contamination under the marine strategy framework directive: Contributions of the German environmental specimen bank

Descriptor 9 (D9) of the European Union Marine Strategy Framework Directive refers to the compliance of contaminant levels in fish and other seafood of a defined marine region or subregion with human health threshold values. This requires georeferenced samples that are often difficult to obtain when relying on commercial fisheries or programs designed for monitoring human exposure. The present study examines whether georeferenced samples of blue mussel (Mytilus edulis) and eelpout (Zoarces viviparus) fillet of the German environmental specimen bank (ESB) can be used in this context. The suitability of the ESB samples, procedures, and analytical methods is evaluated with respect to D9 requirements. Based on ESB data for the D9 relevant contaminants Pb, Cd, Hg, Σ4 PAHs, PCDD/Fs, dioxin-like (dl)-PCBs, and indicator non-dl-PCBs and the potentially relevant substances TBT, PFOS, PBDE, and HBCDD, the Good Environmental Status for D9 is assessed at the ESB sites in the North and Baltic Seas. The overall evaluation indicates that ESB samples are suitable for D9 assessment with the limitation that only coastal areas of the North and Baltic Seasare covered. Over a period of up to 30 years, concentrations of the D9 relevant contaminants were well below the maximum levels allowed for human consumption