La production et l’utilisation des nanomatériaux manufacturés est en plein essor, ce qui entraîne leur déversement dans l’environnement sous forme de nanoparticules ou de sous produits d’altération. Les nanoparticules de dioxyde de titane sont utilisées dans un grand nombre de produits manufacturés du fait de leurs propriétés photocatalytiques et d’absorption des UV. Les risques écotoxicologiques liés aux nanoparticules de TiO2 et aux sous produits d’altération de TiO2 sur les organismes terrestres sont encore peu connus. A cette fin, des vers de terre ont été utilisés car ce sont des indicateurs clés de la qualité des écosystèmes terrestres. L’espèce Eisenia fetida employée présente l’avantage d’être facile à manipuler et est utilisée dans des tests normalisés d’écotoxicité. La toxicité potentielle des nanoparticules de TiO2 et des sous produits d’altération de TiO2 sur Eisenia fetida a été étudiée selon différentes approches: un test in vitro sur les nanoparticules de TiO2 (1 à 25 µg/ml), une exposition in vivo en milieu liquide contaminé par des SPA de TiO2 (0,1 à 10 mg/l) et une exposition in vivo en milieu solide dans des sols artificiels contaminés par des nanoparticules de TiO2 (2 à 200 mg/kg) ou par des sous produits d’altération de TiO2 (2-20 mg/kg). La toxicité des nanomatériaux de TiO2 a été évaluée en utilisant une batterie de biomarqueurs à différentes échelles biologiques : la survie et la reproduction à l’échelle de l’individu, la viabilité cellulaire (test MTT et LDH), la phagocytose et l’apoptose à l’échelle cellulaire et l’expression des ARNm des gènes des systèmes de détoxication (métallothionéine, GST), antioxydant (SOD, CAT) et immunitaire (CCF et fétidines) par RT-qPCR à l’échelle moléculaire. Nos résultats ont montré que les nanoparticules de TiO2 sont internalisées par les cellules immunitaires des vers de terre (in vitro) et que le titane est bioaccumulé par les vers exposés aux sous-produits d’altération de TiO2 dans des conditions d’exposition artificielle (milieu liquide). Seuls les biomarqueurs moléculaires (métallothionéine, CCF et fétidines) sont sensibles aux nanoparticules de TiO2 dans le test in vitro alors que les biomarqueurs moléculaire (métallothionéine et SOD) et cellulaire (apoptose et phagocytose) répondent aux sous-produits d’altération de TiO2 en milieu liquide après seulement 24h d’exposition. En revanche, dans des conditions d’exposition plus réalistes, aucun effet biologique sur la survie, la reproduction et l’expression des ARNm des gènes de détoxication, antioxydant et immunitaire n’a été observé sur les vers exposés aux sols contaminés. La faible biodisponibilité des nanomatériaux de TiO2 dans les matrices solides expliquerait l’absence d’effet biologique aux concentrations testées. Ces travaux soulignent la nécessité d’étudier le comportement, la mobilité, et la biodisponibilité des nanomatériaux avant d’évaluer leur potentielle écotoxicité. / The amount of nanomaterials produced annually is constantly increasing and this trend may result in a strong input of manufactured nanoparticles in air, water, soils, and therefore in contact with organisms. Among the manufactured nanoparticles, titanium dioxide (TiO2) nanoparticles have been produced massively for their photocatalytic properties and UV absorption ability. Surprisingly, ecotoxicological risks associated with TiO2 nanoparticles and TiO2 byproducts resulting from the alteration and aging of nanomaterials are poorly documented on terrestrial organisms. The potential toxicity of TiO2 nanoparticles and TiO2 byproducts on the earthworm Eisenia fetida has been evaluated, using different approaches: the first with in vitro assay to determine toxicity of TiO2 nanoparticles (1 to 25 µg/ml), the second with in vivo exposure of earthworm to liquid medium contaminated by TiO2 byproducts (0.1 to 10 mg/l) and the third with in vivo exposure to artificial soil contaminated by TiO2 nanoparticles (2 to 200 mg/kg) or TiO2 byproducts (2-20 mg/kg). Ecotoxicity of TiO2 nanomaterials was determined using a battery of biomarkers at different biological levels: survival and reproduction at individual level, cell viability (MTT and LDH assay), phagocytosis and apoptosis at cellular level, mRNA expression of detoxification (metallothioneins, GST), antioxidant (SOD, CAT) and immunes genes (CCF and fetidins) by RT-qPCR at molecular level. Our results showed that TiO2 nanoparticles were internalized by immune cells (in vitro assay) and titanium was bioaccumulated by worms exposed to TiO2 byproducts in artificial exposure assay (liquid medium). Molecular biomarkers (metallothioneins, CCF and fetidins) were sensitive to TiO2 nanoparticles after in vitro exposures, while both molecular (metallothioneins and SOD) and cellular (phagocytosis, apoptosis) biomarkers were sensitive to TiO2 byproducts in liquid medium after only 24 hours of exposure. In contrast, in more realistic conditions of exposure, no effect on life-history traits or molecular biomarker were noticed on earthworms exposed to soils contaminated by TiO2 nanoparticles or byproducts. A low bioavailability of TiO2 nanomaterials in soil matrixes could explain the lack of effect at the concentrations tested. These results underline the necessity to evaluate mobility, behaviour and bioavailability of nanomaterials in soil matrixes before their ecotoxicity assessment.

Les activités métallurgiques entrainent l'accumulation d'éléments traces métalliques dans les couches . superficielles des sols, où l'on peut observer des concentrations largement supérieures au fond pédogéochimique et aux normes en vigueur. La contamination des sols est une menace pour la santé publique et la présence de grandes quantités d'éléments traces métalliques peut générer un stress susceptible d'affecter les organismes exposés à des sols pollués. Les Annélides Oligochètes vivent en contact étroit avec ces sols pollués et sont parmi les organismes vivants présentant une sensibilité exacerbée aux métaux. Très peu de choses sont connues quant à l'identification et la mise en place des mécanismes de réponse à ces métaux au niveau moléculaire. En exploitant, la conservation phylogénique observée entre espèces nous avons été capables de cloner et de caractériser un ensemble de biomarqueurs de pollution potentiels à partir des cœlomocytes de l'Annélide Oligochètes Eisenia fetida. Deux approches ont été mises en place. Premièrement, une approche qualifiée de ciblée, consistant à identifier tous les effecteurs parmi des protéines fortement conservées pour lesquels une variation lors d'une exposition métallique était reportée dans la littérature. Deuxièmement, nous avons entrepris une approche qualifiée de globale consistant en la construction de banques soustractives pour identifier chez Eisenia fetida, les gènes dont l'expression est affectée lors d'une exposition à un mélange complexe de métaux, représentatif d'un site naturel fortement contaminé. Ces deux approches ont permis l'identification de 4 candidats biomarqueurs de pollution métallique. / Metallurgical activities lead to accumulation of metal trace elements in the topsoils, where one typically observes concentrations largely exceeding the natural background. Contaminations of soils are a threat for public health and the presence of high amount of metals can generate an environmental stress likely to affect the organisms exposed to polluted soils. Earthworms living in close contact with polluted soils were studied in order to better understand the physiological changes, the mechanisms of acclimation and of detoxification caused by metals. By exploiting the conservation observed between species and using molecular biology techniques, we have been able to clone and transcriptionally characterise potential biomarkers form the immune ceIls of the ecotoxicologically important earthworm species Eisenia fetida. During this Ph. D. we developed two approaches. First, a targeted approach based on bibliographie work, enabled us to identify and select effectors among highly preserved proteins for which variations following metal exposure were reported in invertebrates. Second, an open strategy was undertaken by performing a Suppression Subtractive Hybridization in order to identify genes which are differentially expressed following exposure to a complex mixture of metals representative of a strongly polluted soil. These approaches allowed the description of a group of genes which expression varies following metallic exposure as weIl as the analysis of their expression profiles may be used to analyse mechanistically the immune response to a metallic stress. Moreover, expression profiles of selected genes may constitute a signature of changes due to MTE.

L’objectif du travail de recherche était de mettre en relation dans des sols de cokeries anciennement contaminés, la biodisponibilité de polluants (HAP et/ou métaux lourds), leur génotoxicité et leurs effets populationnels chez le ver de terre Eisenia fetida. Cette étude a montré que les sols étudiés, malgré une contamination similaire, présentaient une toxicité vis-à-vis des vers de terre et d’autres organismes terrestres (plantes, collemboles) totalement différente. Ces différences d’écotoxicité seraient imputables à la biodisponibilité des polluants, différente entre les sols. Cette étude a également montré que le traitement de thermodésorption, appliqué à l’un des sols contaminés, augmentait la biodisponibilité des métaux lourds. Le traitement de thermodésorption modifierait à la fois 1) la nature et la composition de la matière organique du sol, 2) la spéciation des métaux lourds, 3) les liens entre la matière organique et les métaux, les rendant plus biodisponibles et génotoxiques vis-à-vis des vers de terre. L’approche biologique-écotoxicologique, qui prend en considération la biodisponibilité des polluants, se révèle donc essentielle en complément de l’approche physico-chimique dans l’évaluation 1) des risques et 2) de l’efficacité de remédiation des sols contaminés. Cette étude a également montré que la mesure des dommages à l’ADN des coelomocytes de vers de terre est un biomarqueur pertinent dans l’évaluation de la génotoxicité des polluants des sols. Ce biomarqueur de génotoxicité peut être utilisé en tant qu’indicateur de biodisponibilité des polluants. Il s’est révélé plus sensible que la survie et au moins aussi sensible que la reproduction, qui sont deux paramètres étudiés classiquement chez les vers de terre. Ce biomarqueur de génotoxicité pourrait être utilisé en tant qu’indicateur précoce de perturbations physiologiques, même si le lien mécanistique entre les dommages à l’ADN et les répercussions sur la reproduction des vers de terre nécessite des recherches ultérieures / The aim of this work research was to show in formerly-contaminated coking plant soils a relationship between the bioavailability of soil pollutants (PAH and/or heavy metals), their genotoxicity and their populational effects in the Eisenia fetida earthworm. This study showed that despite a similar contamination studied soils exhibited a great difference in ecotoxicity to earthworms and other terrestrial organisms (plants, springtails). Differences in ecotoxicity of soils would be attributable to bioavailability of soil pollutants which is different between soils. This study also showed that thermal desorption applied on one of the contaminated soils increased bioavailability of heavy metals. Thermal desorption would modify 1) the nature and composition of soil organic matter, 2) the speciation of heavy metals, 3) links between soil organic matter and heavy metals, rendering them more bioavailable and genotoxic to earthworms. The biological-ecotoxicological approach, which takes into consideration the bioavailability of soil pollutants, reveals to be necessary in addition of the physico-chemical approach in the evaluation of 1) risks and 2) remediation efficiency of contaminated soils. This study also showed that the measure of DNA damage in coelomocytes of earthworms is a relevant biomarker in the evaluation of genotoxicity of soil pollutants. This biomarker of genotoxicity can be used as indicator of bioavailability of soil pollutants. It revealed more sensitive than survival and as sensitive as reproduction, which are classical endpoints measured in earthworms. This biomarker of genotoxicity could be used as early indicator of physiological disturbances, even if the mechanistic link between DNA damage and effects on reproduction require further studies

The landfilling of biodegradable waste is proven to contribute to environmental degradation. Much wood and lumber is discharged as waste from the cleared fields. These woody wastes are subsequently disposed of by burning. However, it would be preferable to dispose of them without combustion to avoid the release of carbon dioxide, one of the critical greenhouse gases. Instead of burning these woody wastes, we should recycle them as future resources. One solution to this problem is to make compost from the waste. Compost use in agriculture is increasing as both an alternative to landfilling for the management of biodegradable waste, as well as means of increasing or preserving soil organic matter. This research aimed to contribute to the identification of a system for managing the production and utilization of wood waste (Quercus rubra and Pinus sylvestris) compost for sustainable agriculture, with particular regards to carbon dioxide produced from both compost and combustion of wood. Compost of wood was implemented in two consecutive trials. The first was carried out in greenhouse experiment in 4 liter pot of Quercus rubra and Pinus sylvestris (QR and PS) moisted by compost and tap water and infected by tiger worm (Eisenia fetida, EF) and European night crawlers (Dendrobaena veneta, DV) at different mixed ratios with lake mud (LM). The second was conducted in greenhouse experiment in 40 liter pot of the successful wood and worm from the first compost trial (QR and EF respectively). The tested wood (QR) was mixed separately by lake mud and horse manure and irrigated by compost and tap water. The final product, successful wood compost (QR) produced from the first trial (4 liter pot) was utilized in different mixed ratios with coal mine tailings (tertiary sand) in greenhouse pot trial to study his effects on improving soil physical and chemical properties and some plant growth parameters of RSM 7.2.1 grass. The wood compost produced from the second compost experiment (40 liter pot) and other artificial component named Arkadolith® were used as soil amelioration in field experiments of different selected sites with extremely unsuitable characteristics (tertiary and quaternary sand in Lusatia lignite region, Germany). The soil in each site was sowed by RSM 7.2.1 and autochthonous grasses. Also, some vermicompost samples were selected to study its thermal stability which compared with a soil sample (Niedermoorgleys) by using thermogravimetric analysis technique. Further investigation was achieved to evaluate the effect of charcoal as a source of carbon on vermicompost stability. Moreover some selected vermicompost samples were used to examine its microstructure under scanning electron microscope which compared also with the same soil used in thermal analysis. The obtained results under all studied experiments can be arranged as follow: First compost trial, Cumulative amount of carbon dioxide produced during composting period was lower than that evolved by combustion of wood. The results showed composting of wood can reduce the emitted CO2 up to 50 % when compared with the amount of CO2 produced from combustion of wood. The effect of different studied factors on different studied parameters show that, QR wood compost have more responsive to decomposition processes and humification rate in comparison with PS wood compost. Under different infection worms, Eisenia fetida (EF) was better than Dendrobaena veneta (DV) in biodegradation rate. Compost water has had a better impact of tap water in all studied decomposition parameters. For example, The compost content of OM and total OC was decreased with the increase of the decomposition period in the treatments of compost water and EF worm, where this decrease was higher at mixed ratio of 1:3 (wood: mud, w/w). The total content of N in the final products takes reversible trend regarding to OM and C content. The high content of N was found in QR wood compost moisted by compost water and infected by EF worm. The content of both macro-and micro-nutrients was clearly positive affected by the studied factors. The content of these nutrients in QR wood compost was higher than that found in the compost of PS wood. Second compost trial, The observed data show that, the amount of CO2 produced by composting was lower than that evolved by combustion of wood. Composting of wood reduced CO2 emission up to 40 % of the combustion wood CO2. Cumulative amount of CO2 produced from wood compost treated by horse manure was higher than that fount in the other treated by LM. The compost of wood treated by horse manure has had a high decomposition rate in comparison with that treated by lake mud. The treatments left without worms during all composting period and moisted by compost water have a responsive effects but it was lower than that infected by worms. Total and available contents of N, P, K, Mg, Zn, and Cu in the compost treated by HM were higher than that found in compost treated by LM. The other nutrients (Ca, Fe, Cu, and Mn) take reversible trend, which it was higher in LM than HM treatments. Thermal and microstructure analysis, The selected vermicompost samples from both first and second compost experiments showed, up to 200° C temperature the mass loss was due to free water and bound water (It was in vermicompost samples higher than soil sample). Mass loss from 200 to 550° C is due to easily oxidizable organic forms and it was higher in vermicompost than soil. In this stage the soil OM seems to be more stable than vermicompost which can be explained by a more intensive bond between the organic and inorganic components. At higher temperatures (T> 550° C) no significant detectable was appeared of soil organic matter. In contrast, the vermicompost treatments showed a high proportion of stable groups, especially aromatic compounds. These statements seem to be importance particularly for the practical application of the wood compost in terms of their long-term effect in the soil. The application of charcoal, showed no additional stabilizing effect of vermicompost. Also, the data show that, vermicompost structure characterized with high homogeneity and ratio of surface area to volume compared to those in soil structure. First plant trials (greenhouse), Different compost mixed ratios had positive impact on different extreme soil physiochemical properties. At the end of experiment (42 days) compost increased soil water holding capacity, decreased soil bulk and particle density and increased total porosity. The used wood compost modified soil buffering capacity and soil acidity. The availability of soil macro and micro nutrients were increased after adding wood compost. The wood compost had a positive effect in some growth parameters like fresh and dry matter yield of the selected grass. High dry matter yield and nutrients uptake was achieved with higher rates of compost application (25.0% > 12.5% > 3.0% > 0.0 %, w/w). Second plant trials (field experiment), Regarding to the effect of wood compost (QR) and Arkadolith® component on tertiary and quaternary sand, at the end of grown season (6 month) most soil and plant characteristics of tertiary sand were improved and it was better than that in quaternary sand. This trend reveals to, physical and chemical properties of tertiary sand was better than that in quaternary sand, like organic matter content, CEC, WHC, TOC, available nutrients. In the both sites, the effects of different type of soil conditioners arranged as follow: the treatments treated with wood compost is the better followed by the other treated with both wood compost and Arkadolith. Wood compost increased soil pH, CEC, soil buffering capacity, OM content, and soil WHC in comparison with Arkadolith which make a small improvement of these properties in both sites. Finally, Different growth parameters (height, covering, fresh and dry matter yield) of the used grasses were clearly positive affected by wood compost, with the highest production inherent to the treatments treated by the high amount of wood compost. / Entsprechend der Zielstellung wurden zunächst verschiedene Varianten der Kompostierung von Holzsubstanz getestet, um eine optimale Technologie, die auch für Entwicklungsländer realisierbar ist, herauszufinden. Hierzu sind in Pflanztöpfe Holzspäne (Woodchips) von zwei verschieden Holzarten (Laub- und Nadelholz) gefüllt und mit verschiedenen natürlichen Stickstoffquellen gemischt worden. Diese Ansätze wurden regelmäßig mit Kompostwasser appliziert. Nach vier Wochen sind zwei verschiedene Wurmarten (Dendrobaena veneta und Eisenia fetida) hinzugegeben worden. Die Feuchthaltung erfolgte ab diesem Zeitpunkt durch Frischwasser. Die qualitativ beste Versuchsvariante ist im nächsten Schritt mit weiteren natürlichen Stickstoffquellen, die in Entwicklungsländern zur Verfügung gestellt werden könnten, getestet worden. Von allen Kompostvarianten sind im Labor eine Vielzahl von bodenphysikalischen (z.B. Dichte, Wasserhaltekapazität) und bodenchemischen Zustandsgrößen (z.B. Elektrische Leitfähigkeit, Totalgehalte biophiler Elemente, Bodenreaktion, organische Substanzgehalte, Kationenaustauschkapazität) bestimmt worden. Die Wiederum qualitativ beste Mischung ist in einer weiteren Versuchsreihe in verschiedenen Mengenverhältnissen mit tertiärerem Abraumsand des Braunkohlebergbaus gemischt worden. In diese Versuchsmischungen wurde die Grasmischung RSM 7.2.1 eingesät und regelmäßig bewässert sowie die Wuchshöhe gemessen. Nach 42 Tagen wurden das Gras geerntet und die biometrischen Parameter, die Nährstoffgehalte (pflanzenverfügbare Fraktionen), die Bodenreaktion, die effektive bzw. potentielle Kationenaustauschkapazität sowie die Pufferkapazitäten der Mischsubstrate bestimmt. Die nächsten Versuchsvarianten sind als Feldversuche in der Niederlausitz durchgeführt worden. Für ihre Realisierung wurde als weiterer Zuschlagsstoff Arkadolith® zugemischt. Die Plotflächen sind sowohl auf Abraumsanden des Tertiärs als auch Quartärs angelegt worden. In jeweils eine Subvariante ist RSM 7.2.1, in die andere eine autochthone Grasmischung eingesät worden. Diese Experimente wurden nach 6 Monaten beendet, die Bestimmung aller Parameter erfolgte in gleicher Weise wie bei den Gewächshausversuchen. Auf Basis aller Versuchsreihen konnten die besten Kompostqualitäten und ihre optimalen Herstellungsvarianten ermittelt werden. Eine weitere Aufgabe war es zu untersuchen, wie im Vergleich zur Verbrennung von Holzmasse die CO2-Emission in die Atmosphäre durch Holzkompostierung verringert werden kann. Hierzu wurde während der verschiedenen Kompostierungsvarianten die CO2-Freisetzung gemessen. Im Vergleich dazu ist jeweils die gleiche Masse an Holzsubstanz verbrannt worden. Die Ergebnisse zeigten, dass im Vergleich zu der thermischen Verwertung von Holsubstanz die CO2-Emission bis zu 50 % verringert werden kann. Dem Boden kann darüber hinaus energiereiche organische Substanz zugeführt werden, die eine Entwicklung der Bodenorganismen ermöglicht. Ein weiteres Experiment zielte darauf ab, die Stabilität der Holzkomposte zu bestimmen. Darüber hinaus sollte untersucht werden, ob durch die Zufuhr von pyrogenem Kohlenstoff eine Vergrößerung der Stabilität zu erreichen ist. Diese Untersuchungen wurden mit Hilfe der Thermogravimetrie vorgenommen. Alle wichtigen Kompostierungsvarianten sind sowohl mit verschiedenen Zusatzmengen als auch ohne Zusatz von pyrogenem Kohlenstoff vermessen worden. Als Vergleichssubstanz diente der Oberboden eines Niedermoorgleys, der naturgemäß einen relativ hohen Anteil an organischer Substanz aufweist. Die Ergebnisse zeigten, dass im Bereich niedriger Temperaturen die Wasserbindung im Naturboden fester ist. In der Fraktion der oxidierbaren organischen Substanz, im mittleren Temperaturbereich gemessen, ist die natürliche Bodensubstanz ebenfalls stabiler, was auf eine intensivere Bindung zwischen den organischen und anorganischen Bestandteilen, also auf stabilere organisch-mineralische Komplexe, schlussfolgern lässt. Im Bereich höherer Temperaturen (T> 550° C) waren im Naturboden keine nennenswerten organischen Bestandteile mehr nachweisbar. Hingegen wiesen die Kompostvarianten einen hohen Anteil stabiler Fraktionen, vor allem aromatische Verbindungen, auf. Diese Aussagen erscheinen vor allem für die praktische Anwendung der Holzkomposte in Hinblick auf ihre Langzeitwirkung bedeutsam. Der Zusatz von pyrogenem Kohlenstoff zeigte keine zusätzliche Stabilisierungswirkung.

Många antropogena ämnen är toxiska och har lång halveringstid och återfinns därför lång tid efter att de hamnat i miljön. Ett exempel är polycykliska aromatiska kolväten (PAH), som bildas vid ofullständig förbränning av organiskt material och som kan förekomma i höga halter vid före detta gasverk och gamla träimpregneringsplatser där kreosotimpregnering förekommit. I marken på dessa områden kan halterna av PAH vara mycket höga, och det kan påverka markekosystemet negativt. En viktig markorganism är daggmask, som har stor betydelse i många markekosystem. Därför är det viktigt att veta om PAHer i mark är tillgängliga för upptag i mask, eftersom det kan påverka masken negativt. Dessutom skulle eventuella PAHer i mask kunna transporteras vidare i näringskedjor. Syftet med studien var därför att undersöka upptaget av polycykliska aromatiska kolväten (PAH) i Eisenia Fetida från förorenad jord. E. Fetida exponerades under två veckor för nio olika PAH-förorenade jordar som samlats in från två kreosotförorenade områden, en före detta gasverkstomt och en bangård. Sammanlagt 24 PAHer inklusive PAH16 analyserades i jord och mask med GC/MS. Resultaten visade upptag av PAHer i E. Fetida från samtliga jordprov, men upptaget varierade beroende på prov. Ett jordprov från en före detta gasverkstomt, gasverk c hade den högsta uppmätta totala bioackumuleringsfaktorn (BAF) för PAHer från respektive prov. De enskilda BAF-värdena i jorden var högre för de högmolekylära PAHerna, medan jordprov insamlade på en bangård visade på ett högre upptag i mask av de låg- och mellan molekylära PAHerna. Ett av de kreosotförorenade jordproverna, kreosot a hade det högsta enskilda BAF-värdet för PAHer, vilket var trifenylen som hade en BAF 18.Det var inget signifikant samband mellan PAH-halten i jordarna och PAH-halten i mask, vilket tyder på att upptaget i mask inte enbart beror på halten PAHer i jorden. Det var heller inget signifikant samband mellan halten organiskt material och upptaget av PAHer i E. Fetida, men resultaten tyder på att upptaget i E. Fetida beror mer på jordegenskaper som organiskt material än halterna av PAHer i jorden. Det är därför viktigt att fortsätta forska kring PAHer och biotillgänglighet i mark.

The aim of this diploma thesis was to determine the dehydrogenase activity of matrix samples that can enter the environment and the use of ecotoxicological tests to assess the quality of the affected soil. The effects of sludge, biochar samples, two landfill samples and a sediment sample were monitored. Ecotoxicology included tests on decomposers - tests of avoidance behavior (Eisenia fetida and Folsomia candida), acute, chronic and reproductive toxicity tests (Eisenia fetida) and Lactuca sativa root growth inhibition tests. The results indicate that dehydrogenase activity assays serve as a suitable complement to ecotoxicological tests. They provide valuable additional information on soil quality and sample effect after application to soil. The sediment sample had the least favorable effect on the activity of the enzyme and the vitality of the organisms, both in the determination of dehydrogenase activity and in ecotoxicological tests. Furthermore, it was found that biochar as a product of sewage sludge treatment is less toxic to organisms than the original sludge.

Une étude approfondie a été menée dans deux zones distinctes (Estarreja au Portugal, noté EST, et Casier Peyraud 6 en France, noté PEY) pour évaluer les niveaux de contamination, ainsi que leurs comportements dans des anthroposols et les risques potentiels de ces contaminants pour des organismes vivants du sol. L’objectif principal étant d’évaluer la mobilité environnementale des retardateurs de flamme bromés (RFBs) dans un écosystème terrestre, notamment vers les vers de terre (Eisenia fetida) et les plants (luzerne (Medicago sativa), cresson (Nasturtium officinale), moutarde blanche (Sinapsis alba), dans une prospective d’évaluation du risque de transferts de ces molécules.Le facteur de bioaccumulation (BAF) ainsi que les indices SET et ERITME ont été calculés. Le BAF permet de déterminer si une substance est accumulée dans un organisme donné et s’il existe un risque d’entrée et de diffusion tout au long de la chaine alimentaire. Les indices SET et ERITME permettent de classer les sites testés en fonction du transfert efficace des COs par les anthroposols aux organismes testés, et d’avoir une idée du risque potentiel pour l’écosystème. L’indice SET donne une idée globale de l’excès de transfert pour tous les contaminants dans les matrices étudiées. L’indice ERITME permet d’évaluer le risque environnemental global inhérent associé à l’excès de transfert des contaminants considérés. Compte tenu des valeurs ERITME, les matrices étudiées peuvent être classées dans un ordre de toxicité qui est aussi conforme pour l’ensemble des organismes testés (E. fetida, luzerne, cresson et moutarde). / A comprehensive study was conducted in two distinct areas (Estarreja in Portugal, noted EST, and Casier Peyraud 6 in France, noted PEY) to assess the contamination levels as well as their behavior in soils and the potential risks posed by these contaminants to soil organisms. The principal aim was to evaluate the environmental mobility of brominated flame retardants (BFRs) in a terrestrial ecosystem, namely to earthworms (Eisenia fetida) and plants (alfalfa (Medicago sativa), watercress (Nasturtium officinale) and white mustard (Sinapis alba)), concerning the BFRs transfer risk evaluation. The Bioaccumulation factor (BAF) as well as the SET and ERITME indexes were calculated. The BAF allow to determine if a substance is accumulated in a certain organism and if there exists the risk of entry and diffusion along the food chain. SET and ERITME indexes allow to rank the tested sites according to the effective OCs transfer from anthroposoils to the tested organisms and have an idea of the potential risk to the ec system. The SET index gives a global idea of the excess of transfer for all the contaminants in the studied matrices. The ERITME index allows to evaluate the possible inherent global environmental risk associated with the excess of transfer on the considered contaminants. Considering the ERITME values, the studied matrices can be classified in an apparent increasing order of toxicity that it is also in accordance with the levels of OCs found in the all the tested organisms (E. fetida, alfalfa, cress and mustard).

The extensive agricultural usage of organohalogenated pesticides has raised many concerns about their potential hazards especially in the soil environment. Environmental metabolomics is an emerging field that investigates the changes in the metabolic profile of native organisms in their environment due to the presence of an environmental stressor. Research presented here explores the potential of Nuclear Magnetic Resonance (NMR)-based metabolomics to examine the sub-lethal exposure of the earthworm, Eisenia fetida to sub-lethal concentrations of organohalogenated pesticides. Various one-dimensional (1-D) and two dimensional (2-D) NMR techniques were compared in a contact filter paper test earthworm metabolomic study using endosulfan, a prevalent pesticide in the environment. The results determined that both the 1H Presaturation Utilizing Gradients and Echos (PURGE) and the 1H-13C Heteronuclear Single Quantum Coherence (HSQC) NMR techniques were most effective in discriminating and identifying significant metabolites in earthworms due to contaminant exposure. These two NMR techniques were further explored in another metabolomic study using various sub-lethal concentrations of endosulfan and an organofluorine pesticide, trifluralin to E. fetida. Principal component analysis (PCA) tests showed increasing separation between the exposed and unexposed earthworms as the concentrations for both contaminants increased. A neurotoxic mode of action (MOA) for endosulfan and a non-polar narcotic MOA for trifluralin were delineated as many significant metabolites, arising from exposure, were identified. The earthworm tissue extract is commonly used as the biological medium for metabolomic studies. However, many overlapping resonances are apparent in an earthworm tissue extract NMR spectrum due to the abundance of metabolites present. To mitigate this spectral overlap, the earthworm’s coelomic fluid (CF) was tested as a complementary biological medium to the tissue extract in an endosulfan exposure metabolomic study to identify additional metabolites of stress. Compared to tests on the tissue extract, a plethora of different metabolites were identified in the earthworm CF using 1-D PURGE and 2-D HSQC NMR techniques. In addition to the neurotoxic MOA identified previously, an apoptotic MOA was also postulated due to endosulfan exposure. This thesis also explored the application of 1-D and 2-D NMR techniques in a soil metabolomic study to understand the exposure of E. fetida to sub-lethal concentrations of endosulfan and its main degradation product, endosulfan sulfate. The earthworm’s CF and tissue extract were both analyzed to maximize the significant metabolites identified due to contaminant exposure. The PCA results identified similar toxicity for both organochlorine contaminants as the same separation, between exposed to the unexposed earthworms, were detected at various concentrations. Both neurotoxic and apopotic MOAs were observed as identical fluctuations of significant metabolites were found. This research demonstrates the potential of NMR-based metabolomics as a powerful environmental monitoring tool to understand sub-lethal organohalogenated pesticide exposure in soil using earthworms as living probes.

The extensive agricultural usage of organohalogenated pesticides has raised many concerns about their potential hazards especially in the soil environment. Environmental metabolomics is an emerging field that investigates the changes in the metabolic profile of native organisms in their environment due to the presence of an environmental stressor. Research presented here explores the potential of Nuclear Magnetic Resonance (NMR)-based metabolomics to examine the sub-lethal exposure of the earthworm, Eisenia fetida to sub-lethal concentrations of organohalogenated pesticides. Various one-dimensional (1-D) and two dimensional (2-D) NMR techniques were compared in a contact filter paper test earthworm metabolomic study using endosulfan, a prevalent pesticide in the environment. The results determined that both the 1H Presaturation Utilizing Gradients and Echos (PURGE) and the 1H-13C Heteronuclear Single Quantum Coherence (HSQC) NMR techniques were most effective in discriminating and identifying significant metabolites in earthworms due to contaminant exposure. These two NMR techniques were further explored in another metabolomic study using various sub-lethal concentrations of endosulfan and an organofluorine pesticide, trifluralin to E. fetida. Principal component analysis (PCA) tests showed increasing separation between the exposed and unexposed earthworms as the concentrations for both contaminants increased. A neurotoxic mode of action (MOA) for endosulfan and a non-polar narcotic MOA for trifluralin were delineated as many significant metabolites, arising from exposure, were identified. The earthworm tissue extract is commonly used as the biological medium for metabolomic studies. However, many overlapping resonances are apparent in an earthworm tissue extract NMR spectrum due to the abundance of metabolites present. To mitigate this spectral overlap, the earthworm’s coelomic fluid (CF) was tested as a complementary biological medium to the tissue extract in an endosulfan exposure metabolomic study to identify additional metabolites of stress. Compared to tests on the tissue extract, a plethora of different metabolites were identified in the earthworm CF using 1-D PURGE and 2-D HSQC NMR techniques. In addition to the neurotoxic MOA identified previously, an apoptotic MOA was also postulated due to endosulfan exposure. This thesis also explored the application of 1-D and 2-D NMR techniques in a soil metabolomic study to understand the exposure of E. fetida to sub-lethal concentrations of endosulfan and its main degradation product, endosulfan sulfate. The earthworm’s CF and tissue extract were both analyzed to maximize the significant metabolites identified due to contaminant exposure. The PCA results identified similar toxicity for both organochlorine contaminants as the same separation, between exposed to the unexposed earthworms, were detected at various concentrations. Both neurotoxic and apopotic MOAs were observed as identical fluctuations of significant metabolites were found. This research demonstrates the potential of NMR-based metabolomics as a powerful environmental monitoring tool to understand sub-lethal organohalogenated pesticide exposure in soil using earthworms as living probes.

Earthworm-microbial interactions with emphasis on the passage effects of Eisenia spp. on microbial community were investigated. The study was focused on earthworm potential to regulate functional microbiota in cattle-impacted soils. Microbial communities were studied through a combination of polar lipid analyses, molecular, and culturing methods.