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.

This thesis is concerned with the fate of hydrophobic organic pollutants (HOCs) in soil. The theoretical part summarizes the current knowledge about bioavailability, factors influencing the bioavailability and methods for its measurement. Attention is paid to the organic carbon content, which is considered to be one of the key factors and to the influence of microorganisms that can degrade or interact with the substances, thus changing their bioavailable ratio. The practical part of the thesis uses bioaccumulation test with earthworm Eisenia andrei for evaluating the uptake kinetics HOCs in three soils with different organic carbon content. Soils were laboratory contaminated with mixtures of selected HOCs (phenanthrene, pyrene, lindane, p,p'-DDT and PCB 153) and the concentrations of substances in these soils were followed for 217 days. Subsequently, bioaccumulation test was conducted; earthworms were exposed from one to twenty-one days. The collected data were used to model uptake kinetics curves for individual substances. Peak-shaped bioaccumulation were found for pyrene, for lindane a steady-state concentration during exposure was observed, whereas for p,p'-DDT and PCB 153 growing concentrations after twenty days of exposure were detected. The reason for the different behavior of the substances was probably their diverse sorption on the organic material, which together with the different speed of elimination influenced final concentration in earthworms. Phenanthrene was not detected in some samples and therefore the modeling of uptake kinetics was not possible.

Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The terrestrial Oligochaete species Eisenia andrei and Enchytraeus doerjesi were exposed to different concentration series of Cd and Zn, both separately and in mixtures for 28 days in artificial OECD soil at 15, 20 and 25°C. At the end of the four week exposure period, survival and reproduction were assessed in E. doerjesi and survival, reproduction, biomass change, metal uptake and biomarker responses (MTT and comet assays) in E. andrei. Survival results for both E. andrei and E. doerjesi indicated that the lethality of Cd increased at higher temperatures, whereas the opposite was observed for Zn.Cadmium LC50 values were the highest at 15°C and the lowest at 25°C. In the Zn exposures LC50 increased with increasing temperature. Mixture results in both test organisms indicated that mixtures were less lethal than the metals separately. Effects of mixtures on survival, nonetheless, increased with increasing temperature. In the Cd experiment, reproduction in E. andrei was only recorded in the control treatments at the three temperatures investigated. However, the deleterious effect of Zn on the reproduction of both E. andrei and E. doerjesi decreased with increasing temperature and Zn EC50 for reproduction increased with increasing temperature. Results for exposures to mixtures indicated in both test organisms that the interaction between Cd and Zn were antagonistic. In both E. andrei and E. doerjesi, the effect of mixture exposures on reproduction decreased with increasing temperatures. The highest mixture EC50 values for reproduction were found at higher temperature. In E. doerjesi (using reproduction results in MixToxModules) Cd and Zn interactions were dose level dependent at the three temperatures investigated. Antagonism was the predominant interaction at lower mixture concentrations whereas synergism occurred at mixture concentrations equal to or higher than the mixtures’ EC50 values. Biomass loss increased with increasing temperature in the Cd exposures (p 9 0.05) but not in the Zn exposures in E. andrei. In this species mixture results indicated antagonistic interactions between Cd and Zn at all temperatures investigated. The deleterious effect of mixtures on the biomass of E. andrei increased with increasing temperature. When Cd and Zn interactions were further investigated in E. andrei (using biomass results in MixToxModules) it was found that they were dose level dependent at the three temperatures investigated. Antagonism was the predominant interaction at lower mixture concentrations whereas synergism occurred at mixture concentrations higher than the mixtures EC50 values. The assessment of metal uptake in E. andrei revealed a temperature dependent Cd uptake with higher Cd body burdens occurring at higher exposure concentrations and temperatures (p ≤ 0.05). In the case of Zn, although uptake was lower at higher temperature, there was no statistical difference in uptake between exposure concentrations and between temperatures. Mixture results however indicated that in mixture exposures less Cd was accumulated by E. andrei than in single Cd exposures (p ≤ 0.05). Inversely, in mixture exposures more Zn was accumulated by E. andrei than in single Zn exposures (p ≤ 0.05). Biomarker studies revealed that Cd and Zn were both cytotoxic and genotoxic whether in single or mixture exposures. Factorial ANOVA analyses of the effects of temperature and metals on the reduction of MTT by E. andrei indicated that temperature rather than the metals was the most important factor controlling mitochondrial activity (p < 0.001). In both Cd and Zn exposures significant deleterious metal effects on mitochondrial processes were found to increase with temperature (p ≤ 0.01). Mixture exposures indicated decreasing cytotoxicity with increasing temperature (p ≤ 0.05) and possible antagonism between Cd and Zn at cellular level. Results of the comet assay showed that the genotoxic profile of Cd was the opposite of the genotoxic profile of Zn. Cd was less genotoxic at lower temperature and increasingly deleterious at higher temperature while Zn was more genotoxic at lower than higher temperature (p ≤ 0.05). The results of mixture exposures indicated decreasing mixture genotoxicity with increasing temperature and suggested that the interactions between Cd and Zn at molecular level were probably antagonistic. / AFRIKAANSE OPSOMMING: Eisenia andrei en Enchytraeus doerjesi is aan verskillende konsentrasiereekse van Cd en Zn, afsonderlik en in mengsels, vir 28 dae in OECD kunsmatige grond onderskeidelik by 15, 20 en 25°C blootgestel Die volgende eindpunte is aan die einde van die vier weke blootstellingsperiode gemeet: oorlewing en voortplanting (by E. doerjesi) en oorlewing, voortplanting, biomassaverandering, metaalopname, MTT en komeettoetse (by E. andrei). Oorlewingsresultate by beide E. andrei en E. doerjesi het getoon dat toenemende temperatuur die letale toksisiteit van Cd laat toeneem terwyl die teenoorgestelde waar was vir Zn. By die Cd blootstellings was die LK50 waardes die hoogste by 15°C en die laagste by 25°C. By die blootstellings aan Zn het die LK50 waardes toegeneem by hoër temperatuur. Resultate by die mengsels by beide toestspesies het aangetoon dat die mengsels minder letaal was as die afsonderlike metale. Effekte van mengsels op oorlewing het nietemin toegeneem met toenemende temperatuur. By die Cd blootstellings is voortplanting slegs by die kontroles en in die geval van E. andrei by die drie onderskeie temperature ondersoek. Die nadelige uitwerking van Zn op voortplanting by beide E. andrei en E. doerjesi het afgeneem met stygende temperatuur en die Zn LK50 vir voortplanting het toegeneem met toenemende temperatuur. Resultate van die blootstellings aan mengsels het getoon dat die wisselwerking tussen Cd en Zn by beide spesies antagonisties was. By beide spesies het die invloed van die mengsels op voortplanting afgeneem met stygende temperatuur. Die hoogste mengsel LK50 waardes vir voortplanting is by hoër temperature gevind. By E. doerjesi was Cd en Zn wisselwerkings by blootstelling aan mengsels (voortplantingsresultate ondersoek deur van MixToxModules gebruik te maak) dosisvlak verwant by die drie temperature wat ondersoek is. Antagonisme was die oorwegende wisselwerking by laer mengsel konsentrasies terwyl sinergisme voorgekom het by mengsel konsentrasies gelyk aan of hoër as die LK50 waardes van die mengsels. In die geval van die Cd blootstellings by E. andrei het biomassaverlies toegeneem met toenemende temperatuur (p ≤ 0.05) maar nie by die Zn blootstellings nie. Resultate van blootstellings aan mengsels het getoon dat die uitwerking van mengsels op die biomassa van E. fetida toegeneem het met toenemende temperatuur. By die verdere ondersoek van Cd en Zn wisselwerkings, waar gekyk is na dosis verhouding of dosisvlak antagonisme (deur van biomassa resultate in MixToxModules gebruik te maak), is gevind dat Cd en Zn wisselwerkings dosisvlak afhanklik was by die drie temperature wat ondersoek is. Antagonisme was die oorwegende wisselwerking by laer mengselkonsentrasies terwyl sinergisme voorgekom het by mengselkonsentrasies hoër as die mengsel EK50 konsentrasies. Die bepaling van metaalopname deur E. andrei het ’n temperatuurafhanklike opname van Cd getoon met hoër Cd liggaamskonsentrasies by hoër blootstellingskonsentrasies en temperature (p ≤ 0.05). Alhoewel Zn opname laer was by hoër temperatuur was daar geen statisties betekenisvolle verskille in opname tussen blootstellingskonsentrasies of temperature nie. Die bepaling van metaalopname by wurms wat aan mengsels blootgestel is, het getoon dat minder Cd deur E. andrei opgeneem is as waneer die wurms aan Cd as enkelmetaal blootgestel is (p ≤ 0.05), Daarteenoor het die teenoorgestelde gebeur in die geval van Zn, Meer van die metaal is opgeneem wanneer E. andrei aan mengsels blootgestel is as aan die enkelmetaal. Biomerkerstudie het getoon dat Cd en Zn beide sito- en genotoksies kan wees ongeag of dit as enkelmetale of in mengsels toegedien is. Faktoriale ANOVA analises van die effekte van temperatuur en metale op die verlaging van MTT by E. andrei het getoon dat temperatuur ’n belangriker faktor was as metaalbesoedeling by die kontrole van mitochondriale aktiwiteit. (p ≤ 0.001). By beide Cd en Zn blootstellings was daar in elk geval statisties betekenisvolle metaaleffekte op mitochondriale prosesse met toename in temperatuur (P ≤ 0.01). By blootstellings aan mengsels is gevind dat sitotoksisiteit afgeneem het met toenemende temperatuur (p ≤ 0.05) asook ’n moontlike antagonisme tussen Cd en Zn op sellulêre vlak. Resultate van die komeettoets het getoon dat die genotoksiese profiel van Cd die teenoorgestelde was as die van Zn. Cd was minder genotoksies by laer temperature en meer en meer skadelik by hoër temperature terwyl Zn meer genotoksies was by laer as by hoër temperature (p ≤ 0.05). Die resultate van blootstlling aan mengsels het laer genotoksisiteit getoon met toename in temperatuur. Dit dui daarop dat wisselwerkings tussen Cd en Zn op molekulêre vlak moontlik antagonisties was.

El objetivo de esta tesis es estudiar las características y propiedades de tres materiales -un compost y dos vermicomposts- obtenidos mediante procesos de compostaje y vermicompostaje, aplicados separadamente o en combinación (precompostaje seguido de vermicompostaje) a una mezcla de residuos hortícolas, con vistas a la aplicación de dichos materiales como componentes de sustratos para la producción de plantas en contenedor. Las propiedades y características físicas, químicas y biológicas de la mezcla de residuos experimentaron cambios significativos durante los procesos de tratamiento y estabilización aplicados a ésta. La magnitud de los cambios dependió de los hechos diferenciales de los procesos estudiados y fue, además, cuantitativamente superior durante las etapas con mayor actividad biológica. La caracterización del compost y los dos vermicomposts obtenidos como sustratos para el sultivo sin suelo reveló diferencias marcadas en los parámetros estudiados entre estos tres materiales así como con la turba utilizada como control. La evaluación agronómica realizada puso de manifiesto que los materiales estudiados, especialmente los dos vermicomposts, pueden utilizarse con éxito como sustitutivos parciales de los materiales usados como sustratos en viveros comerciales. Los vermicomposts pueden emplearse en proporciones del 50% al 75% del volumen total del sustrato, mientras que el compost puede representar del 25% al 50% de éste, proporcionando estas mezclas, además, resultados similares o superiores a los controles comerciales. La respuesta vegetal a los sustratos preparados con los tres materiales estudiados dependió del uso de dichos sustratos, observándose que el más sensible a las características y propiedades de éstos fue la germinación de semillas y posterior emergencia de las plántulas, seguido del estaquillado, que presentó una sensibilidad moderada, y, finalmente, del desarrollo de la planta desde el final de la etapa inicial hasta su madurez comercial. / Mendoza Hernandez, DDJ. (2010). Vermicompost y compost de residuos hortícolas como componentes de sustratos para la producción de planta ornamental y aromática. Caracterización de los materiales y respuesta vegetal [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8685