Intensificazione di processi biologici per la Bioremediation aerobica di suoli contaminati

Enzyveba, a partially characterized complex consortium of not-adapted microorganisms developed through prolonged stabilization of organic wastes, was found to markedly intensify the aerobic remediation of aged PAH- and PCB-contaminated soil by acting as a source of exogenous specialized microorganisms and nutrients. Thus, Enzyveba was tested in the bioremediation of Diesel (G1) and HiQ Diesel (G2) contaminated soils under aerobic slurry-phase conditions by means of a chemical, microbiological, ecotoxicological integrated analytical procedure. The addition of Enzyveba resulted in a higher availability of cultivable specialized bacteria and fungi but this resulted in a slight intensification of soil remediation, probably because of the high content of nutrients and specialized microorganisms of the soil. In many cases, the biotreatability of soils impacted by diesel fuel is limited by their poor content of autochthonous pollutant-degrading microorganisms. Thus, bioaugmentation with stable and reproducible cultures with the required broad substrate specificity might be the solution for a successful remediation. Two microbial consortia, ENZ-G1 and ENZ-G2, were enriched from Enzyveba on G1 and G2. Both consortia consist of a similar composition of bacterial and fungal species. They exhibited a comparable and significant biodegradation capability by removing about 90% of 1 g/l of diesel fuel under liquid culture conditions. Given their remarkable biodegradation potential, richness of quite diverse microbes, stability and resistance after cryopreservation at -20 °C for several months, both consortia appear very interesting candidates for bioaugmentation on site. The mycoflora of a soil historically contaminated by high concentration of PCBs was characterised before, at the beginning and at the end of the biotreatment mentioned above. Several mitosporic fungi isolated from soil grew in presence of a mixture of three PCBs congeners when also glucose was provided. This is the first study in which 5 strains of mitosporic species able to biodegrade PCB are reported in the literature.

Ricerca e applicazione di metodologie ecotossicologiche nel monitoraggio di ambienti marino-costieri: Sviluppo di nuovi bioassay e biomarker

Obiettivo del lavoro è stato lo sviluppo e la validazione di nuovi bioassay e biomarker quali strumenti da utilizzare in un approccio ecotossicologico integrato per il biomonitoraggio di ambienti marino-costieri interessati da impatto antropico negli organismi che vivono in tali ambienti. L’ambiente reale impiegato per l’applicazione in campo è la Rada di Augusta (Siracusa, Italia). Una batteria di bioassay in vivo e in vitro è stata indagata quale strumento di screening per la misura della tossicità dei sedimenti. La batteria selezionata ha dimostrato di possedere i requisiti necessari ad un applicazione di routine nel monitoraggio di ambienti marino costieri. L’approccio multimarker basato sull’impiego dell’organismo bioindicatore Mytilus galloprovincialis in esperimenti di traslocazione ha consentito di valutare il potenziale applicativo di nuovi biomarker citologici e molecolari di stress chimico parallelamente a biomarker standardizzati di danno genotossico ed esposizione a metalli pesanti. I mitili sono stati traslocati per 45 giorni nei siti di Brucoli (SR) e Rada di Augusta, rispettivamente sito di controllo e sito impattato. I risultati ottenuti supportano l’applicabilità delle alterazioni morfometriche dei granulociti quale biomarker di effetto, direttamente correlato allo stato di salute degli organismi che vivono in un dato ambiente. Il significativo incremento dell’area dei lisosomi osservato contestualmente potrebbe riflettere un incremento dei processi degradativi e dei processi autofagici. I dati sulla sensibilità in campo suggeriscono una valida applicazione della misura dell’attività di anidrasi carbonica in ghiandola digestiva come biomarker di stress in ambiente marino costiero. L’utilizzo delle due metodologie d’indagine (bioassay e biomarker) in un approccio ecotossicologico integrato al biomonitoraggio di ambienti marino-costieri offre uno strumento sensibile e specifico per la valutazione dell’esposizione ad inquinanti e del danno potenziale esercitato dagli inquinanti sugli organismi che vivono in un dato ambiente, permettendo interventi a breve termine e la messa a punto di adeguati programmi di gestione sostenibile dell’ambiente.

Impact of anthropogenic stressors on the population genetics of the common bioindicator Dreissena polymorpha (Pallas, 1771)

Toxicant inputs from agriculture, industry and human settlements have been shown to severely affect freshwater ecosystems. Pollution can lead to changes in population genetic patterns through various genetic and stochastic processes. In my thesis, I investigated the impact of anthropogenic stressors on the population genetics of the zebra mussel Dreissena polymorpha. In order to analyze the genetics of zebra mussel populations, I isolated five new highly polymorphic microsatellite loci. Out of those and other already existing microsatellite markers for this species, I established a robust marker set of six microsatellite loci for D. polymorpha. rnMonitoring the biogeographical background is an important requirement when integrating population genetic measures into ecotoxicological studies. I analyzed the biogeographical background of eleven populations in a section of the River Danube (in Hungary and Croatia) and some of its tributaries, and another population in the River Rhine as genetic outgroup. Moreover, I measured abiotic water parameters at the sampling sites and analyzed if they were correlated with the genetic parameters of the populations. The genetic differentiation was basically consistent with the overall biogeographical history of the populations in the study region. However, the genetic diversity of the populations was not influenced by the geographical distance between the populations, but by the environmental factors oxygen and temperature and also by other unidentified factors. I found strong evidence that genetic adaptation of zebra mussel populations to local habitat conditions had influenced the genetic constitution of the populations. Moreover, by establishing the biogeographical baseline of molecular variance in the study area, I laid the foundation for interpreting population genetic results in ecotoxicological experiments in this region.rnIn a cooperation project with the Department of Zoology of the University of Zagreb, I elaborated an integrated approach in biomonitoring with D. polymorpha by combining the analysis techniques of microsatellite analysis, Comet assay and micronucleus test (MNT). This approach was applied in a case study on freshwater contamination by an effluent of a wastewater treatment plant (WWTP) in the River Drava (Croatia) and a complementary laboratory experiment. I assessed and compared the genetic status of two zebra mussel populations from a contaminated and a reference site. Microsatellite analysis suggested that the contaminated population had undergone a genetic bottleneck, caused by random genetic drift and selection, whereas a bottleneck was not detected in the reference population. The Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that the contaminated population had an increased percentage of micronuclei in hemocytes in comparison to the reference population. The laboratory experiment with mussels exposed to municipal wastewater revealed that mussels from the contaminated site had a lower percentage of tail DNA and a higher percentage of micronuclei than the reference population. These differences between populations were probably caused by an overall decreased fitness of mussels from the contaminated site due to genetic drift and by an enhanced DNA repair mechanism due to adaptation to pollution in the source habitat. Overall, the combination of the three biomarkers provided sufficient information on the impact of both treated and non-treated municipal wastewater on the genetics of zebra mussels at different levels of biological organization.rnIn my thesis, I could show that the newly established marker set of six microsatellite loci provided reliable and informative data for population genetic analyses of D. polymorpha. The adaptation of the analyzed zebra mussel populations to the local conditions of their habitat had a strong influence on their genetic constitution. We found evidence that the different genetic constitutions of two populations had influenced the outcome of our ecotoxicological experiment. Overall, the integrated approach in biomonitoring gave comprehensive information about the impact of both treated and non-treated municipal wastewater on the genetics of zebra mussels at different levels of biological organization and was well practicable in a first case study.

Endocrine disrupting compounds: can they target the immune system of fish?

Endocrine disruption, in particular disruption by estrogen-active compounds, has been identified as an important ecotoxicological hazard in the aquatic environment. Research on the impact of endocrine disrupting compounds (EDCs) on wildlife has focused on disturbances of the reproductive system. However, there is increasing evidence that EDCs affect a variety of physiological systems other than the reproductive system. Here, we discuss if EDCs may be able to affect the immune system of fish, as this would have direct implications for individual fitness and population growth. Evidence suggesting an immunomodulatory role of estrogens in fish comes from the following findings: (a) estrogen receptors are expressed in piscine immune organs, (b) immune gene expression is modulated by estrogen exposure, and (c) pathogen susceptibility of fish increases under estrogen exposure.

Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

Immunotoxic effects of environmental toxicants in fish - how to assess them?

Numerous environmental chemicals, both long-known toxicants such as persistent organic pollutants as well as emerging contaminants such as pharmaceuticals, are known to modulate immune parameters of wildlife species, what can have adverse consequences for the fitness of individuals including their capability to resist pathogen infections. Despite frequent field observations of impaired immunocompetence and increased disease incidence in contaminant-exposed wildlife populations, the potential relevance of immunotoxic effects for the ecological impact of chemicals is rarely considered in ecotoxicological risk assessment. A limiting factor in the assessment of immunotoxic effects might be the complexity of the immune system what makes it difficult (1) to select appropriate exposure and effect parameters out of the many immune parameters which could be measured, and (2) to evaluate the significance of the selected parameters for the overall fitness and immunocompetence of the organism. Here, we present - on the example of teleost fishes - a brief discussion of how to assess chemical impact on the immune system using parameters at different levels of complexity and integration: immune mediators, humoral immune effectors, cellular immune defenses, macroscopical and microscopical responses of lymphoid tissues and organs, and host resistance to pathogens. Importantly, adverse effects of chemicals on immunocompetence may be detectable only after immune system activation, e.g., after pathogen challenge, but not in the resting immune system of non-infected fish. Current limitations to further development and implementation of immunotoxicity assays and parameters in ecotoxicological risk assessment are not primarily due to technological constraints, but are related from insufficient knowledge of (1) possible modes of action in the immune system, (2) the importance of intra- and inter-species immune system variability for the response against chemical stressors, and (3) deficits in conceptual and mechanistic assessment of combination effects of chemicals and pathogens.

Pathology working group review of histopathologic specimens from three laboratory studies of diclofenac in trout.

While the pathology peer review/pathology working group (PWG) model has long been used in mammalian toxicologic pathology to ensure the accuracy, consistency, and objectivity of histopathology data, application of this paradigm to ecotoxicological studies has thus far been limited. In the current project, the PWG approach was used to evaluate histopathologic sections of gills, liver, kidney, and/or intestines from three previously published studies of diclofenac in trout, among which there was substantial variation in the reported histopathologic findings. The main objectives of this review process were to investigate and potentially reconcile these interstudy differences, and based on the results, to establish an appropriate no observed effect concentration (NOEC). Following a complete examination of all histologic sections and original diagnoses by a single experienced fish pathologist (pathology peer review), a two-day PWG session was conducted to allow members of a four-person expert panel to determine the extent of treatment-related findings in each of the three trout studies. The PWG was performed according to the United States Environmental Protection Agency (US EPA) Pesticide Regulation (PR) 94-5 (EPA Pesticide Regulation, 1994). In accordance with standard procedures, the PWG review was conducted by the non-voting chairperson in a manner intended to minimize bias, and thus during the evaluation, the four voting panelists were unaware of the treatment group status of individual fish and the original diagnoses associated with the histologic sections. Based on the results of this review, findings related to diclofenac exposure included minimal to slightly increased thickening of the gill filament tips in fish exposed to the highest concentration tested (1,000 μg/L), plus a previously undiagnosed finding, decreased hepatic glycogen, which also occurred at the 1,000 μg/L dose level. The panel found little evidence to support other reported effects of diclofenac in trout, and thus the overall NOEC was determined to be >320 μg/L. By consensus, the PWG panel was able to identify diagnostic inconsistencies among and within the three prior studies; therefore this exercise demonstrated the value of the pathology peer review/PWG approach for assessing the reliability of histopathology results that may be used by regulatory agencies for risk assessment.

Into the abyss of Lake Geneva: the elemo interdisciplinary field investigation using the MIR submersibles

In summer 2011, the two Russian MIR sub- mersibles were brought to Switzerland to perform deep water dives in Lake Geneva. Research teams from several environmental science institutes, both national and inter- national, participated in this interdisciplinary effort to investigate the deeper parts of Lake Geneva. Using the MIRs allowed the scientists to see and precisely select the sites where they could extract specific sediment cores and carry out detailed in situ measurements at the sediment– water boundary. One focus site was the surrounding of the outlet of the wastewater treatment plant of the City of Lausanne, which discharges into the Vidy Bay. The investigations concentrated on the pollution of the local sediments, pollution-related ecotoxicological risks, micro- bial activity and spreading and removal of the effluents from the bay to the open waters of the lake. The other focus site was the Rhoˆne River delta and its subaquatic canyons, which formed as a result of the long-term interplay of the deposition of river-borne sediments and flood-triggered canyon erosion events.

Integrated testing strategy (ITS) for bioaccumulation assessment under REACH

REACH (registration, evaluation, authorisation and restriction of chemicals) regulation requires that all the chemicals produced or imported in Europe above 1 tonne/year are registered. To register a chemical, physicochemical, toxicological and ecotoxicological information needs to be reported in a dossier. REACH promotes the use of alternative methods to replace, refine and reduce the use of animal (eco)toxicity testing. Within the EU OSIRIS project, integrated testing strategies (ITSs) have been developed for the rational use of non-animal testing approaches in chemical hazard assessment. Here we present an ITS for evaluating the bioaccumulation potential of organic chemicals. The scheme includes the use of all available data (also the non-optimal ones), waiving schemes, analysis of physicochemical properties related to the end point and alternative methods (both in silico and in vitro). In vivo methods are used only as last resort. Using the ITS, in vivo testing could be waived for about 67% of the examined compounds, but bioaccumulation potential could be estimated on the basis of non-animal methods. The presented ITS is freely available through a web tool.

Linking Ah receptor mediated effects of sediments and impacts on fish to key pollutants in the Yangtze Three Gorges Reservoir, China – A comprehensive perspective

The Three Gorges Reservoir (TGR), created in consequence of the Yangtze River's impoundment by the Three Gorges Dam, faces numerous anthropogenic impacts that challenge its unique ecosystem. Organic pollutants, particularly aryl hydrocarbon receptor (AhR) agonists, have been widely detected in the Yangtze River, but only little research was yet done on AhR-mediated activities. Hence, in order to assess effects of organic pollution, with particular focus on AhR-mediated activities, several sites in the TGR area were examined applying the "triad approach". It combines chemical analysis, in vitro, in vivo and in situ investigations to a holistic assessment. Sediments and the benthic fish species Pelteobagrus vachellii were sampled in 2011/2012, respectively, to identify relevant endpoints. Sediment was tested in vitro with the ethoxyresorufin-O-deethylase (EROD) induction assay, and in vivo with the Fish Embryo Toxicity Test and Sediment Contact Assay with Danio rerio. Activities of phase I (EROD) and phase II (glutathione-S-transferase) biotransformation enzymes, pollutant metabolites and histopathological alterations were studied in situ in P. vachellii. EROD induction was tested in vitro and in situ to evaluate possible relationships. Two sites, near Chongqing and Kaixian city, were identified as regional hot-spots and further investigated in 2013. The sediments induced in the in vitro/in vivo bioassays AhR-mediated activities and embryotoxic/teratogenic effects - particularly on the cardiovascular system. These endpoints could be significantly correlated to each other and respective chemical data. However, particle-bound pollutants showed only low bioavailability. The in situ investigations suggested a rather poor condition of P. vachellii, with histopathological alterations in liver and excretory kidney. Fish from Chongqing city exhibited significant hepatic EROD induction and obvious parasitic infestations. The polycyclic aromatic hydrocarbon (PAH) metabolite 1-hydroxypyrene was detected in bile of fish from all sites. All endpoints in combination with the chemical data suggest a pivotal role of PAHs in the observed ecotoxicological impacts.

Comparative Cytotoxicity Study of Silver Nanoparticles (AgNPs) in a Variety of Rainbow Trout Cell Lines (RTL-W1, RTH-149, RTG-2) and Primary Hepatocytes.

Among all classes of nanomaterials, silver nanoparticles (AgNPs) have potentially an important ecotoxicological impact, especially in freshwater environments. Fish are particularly susceptible to the toxic effects of silver ions and, with knowledge gaps regarding the contribution of dissolution and unique particle effects to AgNP toxicity, they represent a group of vulnerable organisms. Using cell lines (RTL-W1, RTH-149, RTG-2) and primary hepatocytes of rainbow trout (Oncorhynchus mykiss) as in vitro test systems, we assessed the cytotoxicity of the representative AgNP, NM-300K, and AgNO3 as an Ag+ ion source. Lack of AgNP interference with the cytotoxicity assays (AlamarBlue, CFDA-AM, NRU assay) and their simultaneous application point to the compatibility and usefulness of such a battery of assays. The RTH-149 and RTL-W1 liver cell lines exhibited similar sensitivity as primary hepatocytes towards AgNP toxicity. Leibovitz's L-15 culture medium composition (high amino acid content) had an important influence on the behaviour and toxicity of AgNPs towards the RTL-W1 cell line. The obtained results demonstrate that, with careful consideration, such an in vitro approach can provide valuable toxicological data to be used in an integrated testing strategy for NM-300K risk assessment.

(Table 1) Body condition and plasma POP concentrations of glaucous gulls from Bjørnøya, Svalbard archipelago

Behavioural field observations are increasingly being used in ecotoxicological research to identify potential adverse effects of exposure to persistent organic pollutants (POPs). We investigated thermal conditions inside the nest and parental behaviour of glaucous gulls, Larus hyperboreus, breeding in the Norwegian Arctic in relation to the concentrations of major classes of POPs (organochlorines, brominated flame retardants and metabolically derived products) accumulated in their blood. Most notably, nest temperature was negatively correlated with the concentrations of the sum of DDT, sum of PCB and several quantitatively minor POP classes within the incubating parent. To investigate the relationship between incubation ability and parental POP exposure further, we experimentally increased the costs of incubation by artificially increasing the clutch size from two to four eggs. Clutch enlargement was followed by a decrease in nest temperature, but this drop in temperature was not associated with POP concentrations within the incubating parent. However, males, which had higher POP concentrations and lower white blood cell counts than females, seemed less able to maintain nest temperature. There was virtually no evidence to suggest that the sum of PCB or DDT were associated with changes in the time a bird spent incubating. However, there was some indication that nest site attendance by nonincubating males was negatively related to the sum of DDT, suggesting that nest protection may have been compromised. The results suggest that adverse effects of parental POP exposure may occur through suboptimal thermal conditions for embryo development and possibly increased egg predation risk.

Agricultural pesticide in sediment cores from the Lac de Saint-André, France

Agricultural pesticide use has increased worldwide during the last several decades, but the long-term fate, storage, and transfer dynamics of pesticides in a changing environment are poorly understood. Many pesticides have been progressively banned, but in numerous cases, these molecules are stable and may persist in soils, sediments, and ice. Many studies have addressed the question of their possible remobilization as a result of global change. In this article, we present a retro-observation approach based on lake sediment records to monitor micropollutants and to evaluate the long-term succession and diffuse transfer of herbicides, fungicides, and insecticide treatments in a vineyard catchment in France. The sediment allows for a reliable reconstruction of past pesticide use through time, validated by the historical introduction, use, and banning of these organic and inorganic pesticides in local vineyards. Our results also revealed how changes in these practices affect storage conditions and, consequently, the pesticides' transfer dynamics. For example, the use of postemergence herbicides (glyphosate), which induce an increase in soil erosion, led to a release of a banned remnant pesticide (dichlorodiphenyltrichloroethane, DDT), which had been previously stored in vineyard soil, back into the environment. Management strategies of ecotoxicological risk would be well served by recognition of the diversity of compounds stored in various environmental sinks, such as agriculture soil, and their capability to become sources when environmental conditions change.

Nematodes as indicators of the effect of soil fumigants on soil functioning

Debido al futuro incierto de la mayor parte de los fumigantes edáficos usados actualmente en la Unión Europea, que pueden implicar riesgos para la salud humana/animal y el medio ambiente, es necesario desarrollar programas de manejo integrado para el control de plagas de cultivos. Estos programas se incluyen como obligatorios en el Reglamento (EC) No. 1107/2009. De acuerdo con este Reglamento, es obligatoria la evaluación del riesgo asociado al uso de productos fitosanitarios sobre los organismos edáficos no diana y sus funciones, además de llevar a cabo ensayos con diferentes especies indicadoras para obtener datos de toxicidad que puedan ser usados posteriormente en la evaluación de riesgo. Sin embargo, la baja representatividad de algunas de estas especies indicadoras en el área Mediterránea supone una gran limitación. En esta situación, el Panel Científico de Productos Fitosanitarios y sus Residuos de la Autoridad Europea en Seguridad Alimentaria (EFSA), ha señalado la necesidad de modificar los datos ecotoxicológicos requeridos para evaluar los efectos adversos de los productos fitosanitarios de una manera más integrada, incluyendo criterios funcionales y estructurales mediante organismos como bacterias, hongos, protozoos y nematodos. De este modo, la EFSA ha recomendado el uso de los nematodos en la evaluación de la funcionalidad y estructura del suelo. Los nematodos están globalmente distribuidos y son morfológicamente diversos; esto junto con su gran abundancia y diversidad de respuestas a las perturbaciones edáficas, los convierte en indicadores adecuados del estado del suelo. Puesto que los nematodos interaccionan con muchos otros organismos que participan en diferentes eslabones de la red trófica edáfica, jugando papeles importantes en procesos edáficos esenciales en los agroescosistemas, la diversidad de nematodos es, a menudo, usada como indicador biológico de los efectos de las prácticas agrícolas en el estado del suelo. En los últimos años, diferentes índices basados en la comunidad nematológica han facilitado la interpretación de datos complejos sobre la ecología del suelo. Los índices de la red trófica edáfica, basados en la abundancia de grupos funcionales definidos como grupos C-P y grupos tróficos, permiten la evaluación de la funcionalidad de la red trófica edáfica. Por otra parte, la dificultad en la identificación taxonómica de nematodos para explicar su uso limitado como indicadores ecológicos, es ampliamente discutida, y existe cierta controversia en cuanto a la eficacia de los diferentes métodos de identificación de nematodos. Se argumenta que la identificación morfológica es difícil y puede llevar mucho tiempo debido a la falta de expertos especializados, y se afirma que las técnicas moleculares pueden resolver algunas limitaciones de las técnicas morfológicas como la identificación de juveniles. Sin embargo, los métodos de identificación molecular tienen también limitaciones; la mayoría de las bases de datos de secuencias de ADN están fuertemente orientadas hacia los nematodos fitoparásitos, los cuales representan sólo una parte de la comunidad edáfica de nematodos, mientras que hay poca información disponible de nematodos de vida libre a pesar de representar la mayoría de los nematodos edáficos. Este trabajo se centra en el estudio de los efectos de fumigantes edáficos en la funcionalidad del suelo a través del uso de diferentes indicadores basados en la comunidad de nematodos, como los índices de la red trófica, índices de diversidad, abundancia de los taxones más relevantes etc. También se han analizado otros indicadores funcionales relacionados con la supresividad edáfica, el ciclo de nutrientes o la actividad de la microfauna del suelo. En el capítulo 1, la diversidad de nematodos estudiada en una explotación comercial de fresa y sus alrededores durante dos campañas consecutivas en el suroeste español, fue baja en los suelos fumigados con fumigantes químicos ambas campañas y, aunque se observó una recuperación a lo largo de la campaña en la zona tratada, los suelos fumigados mostraron una condición perturbada permanente. La comunidad de nematodos estuvo más asociada al ciclo de nutrientes en la zona sin cultivar que en los suelos cultivados, y se observó poca relación entre la biomasa de las plantas y la estructura de la comunidad de nematodos. Los surcos sin tratar dentro de la zona de cultivo funcionaron como reservorio tanto de nematodos fitoparásitos como beneficiosos; sin embargo estas diferencias entre los surcos y los lomos de cultivo no fueron suficientes para mantener la supresividad edáfica en los surcos. Los suelos tratados fueron menos supresivos que los suelos sin tratar, y se observaron correlaciones positivas entre la supresividad edáfica y la estructura de la red trófica edáfica y la diversidad de nematodos. En el capítulo 2, se evaluaron los efectos de dos pesticidas orgánicos con efecto nematicida y dos nematicidas convencionales sobre las propiedades físico químicas del suelo, la diversidad de nematodos y la biomasa de las plantas en condiciones experimentales en dos tipos de suelo: suelos agrícolas poco diversos y suelos provenientes de una zona de vegetación natural muy diversos. El mayor efecto se observó en el tratamiento con neem, el cual indujo un gran incremento en el número de dauerlarvas en los suelos pobres en nutrientes, mientras que el mismo tratamiento indujo un incremento de poblaciones de nematodos bacterívoros, más estables y menos oportunistas, en los suelos del pinar ricos en materia orgánica. En el capítulo 3, se comparó la eficacia de métodos moleculares (TRFLP, Terminal Restriction Fragment Length Polymorphism) y morfológicos (microscopía de alta resolución) para la identificación de diferentes comunidades denematodos de España e Irlanda. Se compararon estadísticamente las diferencias y similitudes en la diversidad de nematodos, otros indicadores ecológicos y de la red trófica edáfica. Las identificaciones mediante el uso de TRFLP sólo detectó un porcentaje de los taxones presentes en las muestras de suelo identificadas morfológicamente, y los nematodos omnívoros y predadores no fueron detectados molecularmente en nuestro estudio. Los índices calculados en base a los nematodos micróboros mostraron más similitud cuando se identificaron morfológica y molecularmente que los índices basados en grupos tróficos más altos. Nuestros resultados muestran que, al menos con la técnica usada en este estudio, la identificación morfológica de nematodos es una herramienta fiable y más precisa que la identificación molecular, puesto que en general se obtiene una mayor resolución en la identificación de nematodos. En el capítulo 4, se estudiaron también los efectos de los nematicidas químicos sobre la comunidad de nematodos y la biomasa de las plantas en condiciones experimentales de campo, donde se aplicaron en una rotación de cultivo judía-col durante un ciclo de cultivo. Se aplicaron dos tipos de enmiendas orgánicas con el objetivo de mitigar el efecto negativo de los productos fitosanitarios sobre la diversidad edáfica. El efecto de los nematicidas sobre las propiedades del suelo y sobre la comunidad de nematodos fue más agudo que el efecto de las enmiendas. La incorporación de los restos de cosecha al final del ciclo de cultivo de la judía tuvo un gran efecto sobre la comunidad de nematodos, y aunque el número total de nematodos incrementó al final del experimento, se observó una condición perturbada permanente de la red trófica edáfica a lo largo del experimento. ABSTRACT Due to the uncertain future of the soil fumigants most commonly used in the EU, that might involve risks for human/animal health and the environment, there is a need to develop new integrated pest management programs, included as mandatory in the Regulation (EC) No. 1107/2009, to control crop diseases. According to this Regulation, evaluating the risk associated to the use of the plant production products (PPP) on non-target soil fauna and their function, and developing assays with different indicator species to obtain toxicity data to be used in the risk evaluation is mandatory. However, the low representativeness of some of these indicator species in the Mediterranean area is a relevant limitation. In this situation, the Scientific Panel of Plant Protection Products and their Residues of the European Food Safety Authority (EFSA) has pointed out the necessity of modifying the ecotoxicological data set required to evaluate non-target effects of PPP in a more integrated way, including structural and functional endpoints with organism such as bacteria, fungi, protists and nematodes. Thus, EFSA has recommended the use of nematodes in the assessment of the functional and structural features of the soil. Nematodes are globally distributed and morphologically diverse, and due to their high abundance and diversity of responses to soil disturbance, they are suitable indicators of the soil condition. Since nematodes interact with many other organisms as participants in several links of the soil food web, playing important roles in essential soil processes in agroecosystems, nematode diversity is often used as a biological indicator of the effects of agricultural practices on soil condition. In the last years, various indices based on soil nematode assemblages, have facilitated the interpretation of complex soil ecological data. Soil food web indices based on the abundances of functional guilds defined by C-P groups and trophic groups, permit evaluating soil food web functioning. On the other hand, the difficulty of nematode taxonomical identification is commonly argued to explain their limited used as ecological indicators, and there is a certain controversy in terms of the efficacy of various nematode identification methods. It is argued that the morphological identification is difficult and time consuming due to the lack of specialist knowledge, and it is claimed that molecular techniques can solve some limitations of morphological techniques such as the identification of juveniles. Nevertheless, molecular identification methods are limited too, since most of the DNA-based databases are strongly oriented towards plant-parasitic nematodes that represent only a fraction of the soil nematode community, while there is little information available on free-living nematodes, which represent most soil nematodes. This work focuses on the study of the effects of soil fumigants on soil functioning through the use of different indicators based on soil nematode community as soil food web indices, diversity indices, the abundance of more relevant taxa etc. Other functional indicators related to soil suppressiveness, nutrient cycling, or the activity of soil microfauna have been also studied. In chapter 1, nematode diversity assessed in a commercial strawberry farm and its surroundings for two consecutive growing seasons in southern Spain, was low in fumigated soils with chemical pesticides throughout both seasons and, although yearly recovery occurred within the treated fields, fumigated soils showed a permanent perturbed condition. The nematode community was more closely associated to nutrient cycling in the non-cropped than in the cropped soils, and the link between plant biomass and nematode community structure was weak. Non-treated furrows within the treated fields were a reservoir of both beneficial and plant-parasitic nematodes, but such difference between furrows and beds was not enough to maintain more suppressive soil assemblages in the furrows. Treated soils were less suppressive than unmanaged soils, and there was a positive and significant correlation between soil suppressiveness and soil food web structure and diversity. In chapter 2, the effects of two organic pesticides with nematicide effect and two chemical nematicides on soil physicalchemical properties, soil nematode diversity and plant biomass in experimental conditions were assessed in two types of soils: low diversity soils from an agricultural farm, and high diversity soils from a natural vegetation area. The larger effect was observed on the neem treatment, which induced a large boost of dauer juveniles in the nutrient-depleted soil, while the same treatment induced the increase of more stable, less opportunistic, populations of generalist bacterivore nematodes in the pine forest soil, rich in organic matter. In chapter 3, comparison of the efficiency of molecular (TRFLP, Terminal Restriction Fragment Length Polymorphism) and morphological (microscopy at high magnification) identification methods was carried out in different nematode communities from five sites of different land uses in Spain and Ireland. Differences and similarities on nematode diversity and other ecological and soil food web indices assessed by both methods, were statistically compared. Molecular identification with TRFLP only detected a percentage of the taxa present in the soil samples identified morphologically, and omnivores and predators were not detected molecularly in our study. Indices involving microbial feeding nematodes were more similar between identification methods than indices involving higher trophic links. Our results show that, at least with the technique used in this study, identifying nematodes morphologically is a reliable and more precise identification tool than molecular identification, since a higher taxonomic resolution is in general obtained compared to TRFLP. In chapter 4, the effect of chemical nematicides on nematode community descriptors and plant biomass was also studied in field conditions in an experimental area in which dazomet and dimethyl disulfide was applied in a bean-cabbage rotation system for a single season. Organic amendments were incorporated into the soil with the aim of mitigate the negative effect of the pesticides on soil diversity. The effect of the nematicides was much more noticeable than the effect of the amendments on soil properties and nematode community descriptors. The incorporation of bean crop residues into the soil at the end of bean crop cycle affected soil nematode community descriptors to a great extent, and although total number of nematodes increased at the end of the experiment, a permanent perturbed soil food web condition was observed along the experiment.

Caracterização de valores de referência de qualidade de sedimentos para compostos orgânicos no sistema estuarino da baixada santista, São Paulo

Em regiões altamente contaminadas como a região da Baixada Santista, é importante estabelecer metas para a recuperação do ambiente. Apesar da ausência da contaminação ser a meta ideal, as implicações e os custos associados a esse objetivo, demanda o estabelecimento de metas de recuperação realistas em relação aos contaminantes presentes na região. Com o objetivo de caracterizar valores de referência de qualidade de sedimentos para compostos orgânicos na região da Baixada Santista, o Canal de Bertioga foi escolhido como local de referência por ser uma região sem fontes industrais ou outras fontes pontuais relativas aos compostos analisados. Amostras de água, sedimento e ostras foram coletadas e os hidrocarbonetos policíclicos aromáticos (PAHs), bifenilos policlorados (PCBs) e pesticidas organoclorados (OCPs) foram determinados por técnicas cromatográficas. A avaliação dos resultados de análises de PAHs, permite afirmar com alguma segurança, que os valores da somatória de PAHs das amostras sedimentos são, na sua grande maioria, inferiores a 1.000 µg/kg, não superando 1.600 µg/kg, concentrações abaixo dos limites estabelecidos na Resolução CONAMA 344/04 e abaixo dos valores que possam causar algum efeito adverso à biota, conforme valores descritos na literatura. Resultados de análise de PCBs, OCPs, compostos fenólicos e compostos orgânicos voláteis (VOCs) em amostras de sedimento, indicaram concentrações destes compostos abaixo dos limites de quantificação, exceto DDE (5,30 g/kg) e HCB (2,34 g/kg), que foram detectados em apenas um ii sítio de amostragem. Não houve evidências de possíveis fontes de emissão próximas à região de referência para PCBs, OCPs, compostos fenólicos e VOCs. Finalizando, espera-se que os resultados obtidos neste estudo possam fornecer subsídios para futuramente estabelecer uma área de referência para qualidade de sedimento na região da Baixada Santista, ou ainda serem utilizados em conjunto com as avaliações de contaminantes inorgânicos, testes ecotoxicológicos e indicadores biológicos, como ferramenta para avaliação da qualidade de sedimento e/ou para a classificação de material a ser dragado na região da Baixada Santista.