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The Nanotoxicology of a Newly Developed Zero-Valent Iron

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The Nanotoxicology of a Newly Developed Zero-Valent Iron The nanotoxicology of a newly developed zero-valent iron nanomaterial for groundwater remediation and its remediation efficiency assessment combined with in vitro bioassays for detection of dioxin-like environmental pollutants Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Biologe Andreas Herbert Schiwy aus Tarnowitz (Polen) Berichter: Universitätsprofessor Dr. rer. nat. Henner Hollert Universitätsprofessor Dr. rer. nat. Andreas Schäffer Tag der mündlichen Prüfung 28. Juli 2016 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar. To my wife and my children Summary Summary The assessment of chemicals and new compounds is an important task of ecotoxicology. In this thesis a newly developed zero-valent iron material for nanoremediation of groundwater contaminations was investigated and in vitro bioassays for high throughput screening were developed. These two elements of the thesis were combined to assess the remediation efficiency of the nanomaterial on the groundwater contaminant acridine. The developed in vitro bioassays were evaluated for quantification of the remediation efficiency. Within the NAPASAN project developed iron based nanomaterial showed in a model field application its nanoremediation capabilities to reduce organic contaminants in a cost effective way. The ecotoxicological evaluation of the nanomaterial in its reduced and oxidized form was conducted with various ecotoxicological test systems. The effects of the reduced nanomaterial with field site resident dechlorinating microorganisms like Dehalococcoides sp., Desulfitobacterium sp., Desulfomonile tiedjei, Dehalobacter sp., Desulfuromonas sp. have been investigated in batch und column experiments. A short-term toxicity of the reduced nanomaterial was shown. However, in a prolonged investigation the NZVI did not show any chronic toxic effects to dechlorinating microorganisms in a time-frame up to 300 days. The contribution of this thesis was the toxicological assessment of the oxidized nanomaterial with ecotoxicological model organisms like Desmodesmus subspicatus, Daphnia magna, Danio rerio and Salmonella typhimurium. The oxidized NZVI showed a toxicity at elevated concentrations of >100 mg/L. The most sensitive test system was the Daphnia acute toxicity test with an EC50 value of 163 mg/L. All other test systems showed a lower or no toxicity of the nanomaterial. Therefore, the nanomaterial can be applied in nanoremediation applications without comprehensive constrains. Especially, as these elevated concentrations will only occur at the contaminated hot spots during the application of this technology as it has been shown that a transport away from the remediation site is not probable. As a second element of this thesis the development of in vitro bioassays to elucidate toxicity in high throughput applications have been conducted. Therefore, the micro-EROD bioassay to determine the CYP1A-inducing potential of samples was developed. Recently, its protocol to investigate environmental sample was presented in Nature protocols in detail. This protocol can be applied to a multitude of samples types (feed and food, chemicals, sediments etc.) und has the advantage of sensitivity and ready to use methodology. Evolving this bioassay new cell lines in a serum-free animal component-free chemically defined medium and suspension culturing conditions have been developed. The investigations with reference compounds like TCDD showed that these newly developed cell lines were highly comparable to the established adherent cell lines. The EC50 value for the newly developed H4IIE-S cell lines was 11 pM, which was comparable to the adherent H4IIE cell line. The adherent cell line was presented in Summary literature with EC50 values ranging from 5 pM to 47 pM. The newly developed system showed the feasibility of high throughput with clear financial benefits in comparison to the established technology, as the cultivation in suspension is more cost efficient. In this cultivation mode the whole volume is used for cultivation compared to adherent cell lines for which only the surface of a culturing vessel can be used. As synthesis the effect of the newly developed NZVI on a model compound in co-exposure has been investigated. The heterocyclic polyaromatic hydrocarbon acridine was applied as a model contaminant as it showed in literature mechanism specific toxicity. It was selected to evaluate the application of bioassays for the assessment of the remediation efficiency of the newly developed nanomaterial. At first the co-exposure of the iron nanomaterial with acridine was evaluated in the fish embryo toxicity test with accompanying instrumental analysis. The results showed a toxicity of acridine with an EC50 value of 1 mg/L, which was comparable to results obtained in literature. The newly developed NZVI did not show any effects on acridine in the fish embryo toxicity test and the instrumental analysis. Additionally, the micro-EROD bioassay did not indicate any dioxin-like potential for this compound. These results were not expected as acridine showed dioxin-like activity and NZVI were applied for remediation of heterocyclic polyaromatic hydrocarbons like acridine in literature. Various factors like passivation of the nanomaterial or a high stability of the model compound could have influenced the outcome of the investigations. For the in vitro investigations of the dioxin-like potential of acridine a substrate inhibition or a species related low receptor affinity could have caused this results. Both elements should be investigated in follow-up studies. The application of a dioxin-like activity monitoring in vitro bioassay to elucidate the remediation efficiency was not successful. Therefore, another compound should be applied as the model groundwater pollutant. Possible candidate substances are PAHs or β-naphthoflavone or TCDD as they are more potent CYP1- inducer. Nevertheless, in vitro bioassays can be applied as monitoring tools for remediation applications. Especially, for effect directed analysis in vitro bioassays are suitable to elucidate the fraction with a specific toxicity. For this application the newly developed cell lines in suspension are especially beneficial. They can be used to investigate extracts of environmental samples with reduced handling effort and thus improve the analytical throughput significantly Zusammenfassung Zusammenfassung Die Bewertung von Chemikalien und neuen Substanzen ist eine wichtige Aufgabe der Ökotoxikologie. Thema dieser Dissertation ist die Bewertung eines neu entwickelten eisenbasierten Nanomaterials zur Remediation von Grundwasserschadensfällen und die Entwicklung von bioanalytischen In vitro–Verfahren, um die Toxizität von Substanzen im Hochdurchsatz zu untersuchen. Diese beiden Untersuchungsschwerpunkte wurden in der vorliegenden Arbeit kombiniert. Anschließend sollte mit Hilfe des Schadstoffs Acridin die Anwendung von Bioassays zur Bewertung der Remediationsleistung erprobt werden. Das im NAPASAN Projekt neuentwickelte nullwertige Eisennanomaterial zeigte das Potential organische und anorganische Umweltgifte auf eine kostengünstige Art zu beseitigen. Dies wurde am Beispiel einer Nanoremediationsanwendung an einem Modellfeldstandort gezeigt. Die ökotoxikologische Untersuchung des Materials wurde mit unterschiedlichen ökotoxikologischen Tests in der reduzierten und oxidierten Form durchgeführt. Als Testorganismen für die Untersuchung des reduzierten Materials wurden Vertreter ausgewählt, die im Feldstandort heimisch sind. Zu diesen gehören die dechlorierenden Mikroorganismen Dehalococcoides sp., Desulfitobacterium sp., Desulfomonile tiedjei, Dehalobacter sp. und Desulfuromonas sp.. Diese wurden in Batch- und Säulenversuchen untersucht. Darüber hinaus wurden Modellorganismen wie Desmodesmus subspicatus, Daphnia magna, Danio rerio und Salmonella typhimurium zur ökotoxikologischen Bewertung des oxidierten Eisennanomaterials verwendet. Die Ergebnisse zeigten eine kurzzeitige Toxizität des reduzierten Nanomaterials gegenüber den Mikroorganismen. Jedoch wurde in Langzeitstudien über einen Zeitraum von 300 Tagen keine chronische Toxizität des Nanomaterials gegenüber den dechlorierenden Mikroorganismen beobachtet. Das oxidierte Material zeigte eine Toxizität nur bei Konzentrationen >100 mg/L. Die empfindlichsten Organismen waren die Daphnien mit einem EC50-Wert von 163 mg/L. Alle weiteren Testsysteme zeigten eine geringere Toxizität des oxidierten Nanomaterials. Aus diesem Grund kann das Nanomaterial ohne große Einschränkungen angewendet werden, besonders da solche erhöhten Konzentrationen nur in Rahmen einer gezielten Remediationsanwendung vorkommen und nur sehr lokal auftreten. Ein Transport des Materials in ökotoxikologisch relevanten Konzentrationen ist nicht wahrscheinlich. Im zweiten Teil der vorliegenden Arbeit lag der Fokus auf der Entwicklung eines In vitro- Verfahrens. Dazu wurde der Micro-EROD Bioassay zur Bewertung des CYP1A- Induktionspotentials entwickelt. Das in Nature Protocols veröffentlichte Protokoll des Mikro- EROD Bioassays bietet eine detaillierte Beschreibung der Methode. Mit dieser neu entwickelten Methode kann eine Vielzahl von Probentypen (Lebensmittel, Chemikalien, Sedimente etc.) untersucht werden. Zusätzlich bietet die Methode den Vorteil einer hohen Sensitivität und leichten Zugänglichkeit selbst für Personal
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