Dissertations / Theses on the topic 'Enchytraeid'

Doutoramento em Biologia
Rapid climatic changes are taking place in Arctic, subarctic and cold temperate regions, where predictions point to an increase in freeze-thaw events, changes in precipitation, evaporation and salinity patterns. Climate change may therefore result in large impacts in ecosystem functioning and dynamics, especially in the presence of contaminants due to intense anthropogenic activities. Even though multiple stress approaches have received increasing interest in the last decades, the number of such studies is limited. In particular, knowledge on the effect of freezethaw events and salinity fluctuations on ecotoxicology of soil invertebrates is lacking, especially important when considering supralittoral species. Therefore, the aim of this thesis was to investigate the effects of low temperature and salinity fluctuations, singly and in combination with contaminants, in the freeze-tolerant and euryhaline enchytraeid Enchytraeus albidus. The assessment of population level endpoints (survival and reproduction), along with physiological and biochemical parameters such as levels of cryoprotectants, ice/water content, oxidative stress biomarkers, cellular energy allocation, and tissue concentration of chemicals (when applied), provided new and valuable knowledge on the effects of selected physical and chemical stressors in E. albidus, and allowed the understanding of adjustments in the primary response mechanisms that enable worms to maintain homeostasis and survival in harsh environments such as polar and temperate-cold regions. The presence of moderate levels of salinity significantly increased freeze-tolerance (mainly evaluated as survival, cryoprotection and ice fraction) and reproduction of E. albidus. Moreover, it contributed to the readjustments of cryoprotectant levels, restoration of antioxidant levels and changed singnificantly the effect and uptake of chemicals (copper cadmium, carbendazim and 4-nonylphenol). Temperature fluctuations (simulated as daily freeze-thaw cycles, between -2ºC and -4ºC) caused substancial negative effect on survival of worms previsouly exposed to non-lethal concentrations of 4-nonylphenol, as compared with constant freezing (-4ºC) and control temperature (2ºC). The decrease in cryoprotectants, increase in energy consumption and the highest concentration of 4-nonylphenol in the tissues have highlighted the high energy requirements and level of toxicity experienced by worms exposed to the combined effect of contaminants and freezing-thawing events. The findings reported on this thesis demonstrate that natural (physical) and chemical stressors, singly or in combination, may alter the dynamics of E. albidus, affecting not only their survival and reproduction (and consequent presence/distribution) but also their physiological and biochemical adaptations. These alterations may lead to severe consequences for the functioning of the ecosystems along the Arctic, subarctic and cold temperate regions, where they play an important role for decomposition of dead organic matter. This thesis provides a scientific basis for improving the setting of safety factors for natural soil ecosystems, and to underline the integration of similar investigations in ecotoxicology, and eventually in risk assessment of contaminants.
As alterações climáticas estão a atingir rapidamente as regiões do Ártico, SubÁrtico e as regiões temperadas, apontando as previsões para um aumento de eventos de congelamento-descongelamento, bem como mudanças nos padrões de precipitação, evaporação e de salinidade. Estas alterações climáticas poderão resultar em impactos francamente negativos no funcionamento e dinâmica de ecossistemas, especialmente quando associados à presença de contaminantes resultantes da intensa atividade antropogénica. Embora a incorporação de stressores múltiplos em estudos de ecotoxicidade tenha recebido um crescente interesse pela comunidade científica, o seu número é ainda reduzido. Particularizando, o conhecimento dos efeitos de eventos de congelamento-descongelamento e de flutuações de salinidade permanecem desconhecidos, especialmente quando se consideram espécies supra-litorais. Neste contexto, o objetivo geral da presente tese consistiu em investigar os efeitos das flutuações de temperaturas e salinidade, individualmente ou em combinação com contaminantes, no enquitraídeo tolerante ao frio e eurialino - o Enchytraeus albidus. A avaliação de parâmetros populacionais (sobrevivência, reprodução e bioacumulação), fisiológicos (níveis de crioprotetores, conteúdo em gelo / água, temperatura de fusão e sobrecongelamento) e bioquímicos (biomarcadores de stress oxidativo, alocação de energia celular) permitiu compilar novas e valiosas informações sobre os efeitos dos stressores físicos e químicos selecionados no enquitraídeo e compreender quais os reajustes nos mecanismos de resposta primários que lhes permitem manter a homeostasia e sobrevivência em ambientes inóspitos como as regiões Polares e temperadas-frias. A presença de níveis moderados de salinidade aumentou significativamente a tolerância a temperaturas congelantes (essencialmente avaliada como sobrevivência, crioprotecção e fracção de gelo extracelular) e a reprodução do E. albidus. Além disso, contribuiu para a regulação de crioprotectores, restauração dos níveis de antioxidantes nestes organismos e alterou significativamente o efeito e a incorporação/absorção de substâncias químicas (cádmio, cobre carbendazim e 4-nonilfenol). As flutuações de temperatura (simuladas como ciclos diários de congelamento-descongelamento, com temperaturas entre 2ºC e -4ºC) causaram um efeito substancialmente negativo na sobrevivência de organismos previamente expostos a concentrações não letais de 4-nonilfenol, quando comparados com organismos expostos a uma temperatura congelante constante (-4ºC) ou à temperatura controlo (2ºC). A diminuição na crioproteção, o aumento no consumo de energia e a maior concentração de 4-nonilfenol nos tecidos vieram sublinhar o elevado gasto energético e o nível de toxicidade sofrido pelos organismos expostos à combinação de contaminantes e eventos de congelamento e descongelamento. Os resultados apresentados nesta tese demonstram, assim, que a presença de stressores naturais (físicos) e químicos, isoladamente ou em combinação, podem alterar a dinâmica do E. albidus, afetando não só a sua sobrevivência e reprodução (e consequente presença / distribuição), mas também as suas adaptações fisiológicas e bioquímicas. Essas alterações podem levar a consequências graves para o funcionamento dos ecossistemas do Ártico, subÁrtico e regiões temperadas-frias, uma vez que estes organismos desempenham um papel importante para a decomposição de matéria orgânica morta. Esta tese fornece ainda uma base científica para melhorar a atribuição de coeficientes de segurança para os ecossistemas naturais do solo, alertando para a integração de investigações semelhantes em ecotoxicologia, e, eventualmente, para a avaliação de risco ecológico de contaminantes.

La contamination des sols est un enjeu environnemental majeur. Une grande partie des sols contaminés en Europe le sont par des éléments traces métalliques. Celle-ci peut affecter les organismes édaphiques, avec souvent comme conséquence une diminution des activités biologiques. Pourtant plusieurs études ont montré l’importance des relations dites « belowground-aboveground » liant les communautés souterraines et aériennes des sols. Parmi elles, l’une des plus connues est l’interaction sol – plante – ver de terre. Ces derniers sont connus pour impacter positivement les fonctions écologiques des sols non contaminés. En effet, ils peuvent augmenter la production primaire des plantes et jouent également un rôle dans la régulation des communautés du sol. Mais dans le cas de sols contaminés, leur rôle reste encore peu documenté. Les enchytréides sont également des annélides oligochètes auxquels on prête le même rôle écologique que les vers de terre. Toutefois, peu d’études permettent d’appuyer cette hypothèse.Dans ce travail nous avons fait l’hypothèse que les connaissances acquises sur les interactions sol – plante – annélides en sol fertile sont transférables en sols contaminés et donc que les annélides peuvent, en interaction avec les plantes, participer à une valorisation de ces sols. Les questions suivantes ont été soulevées : i) y-a-t 'il des modifications de biodisponibilité des contaminants par l’activité des organismes ? Quel est l’impact réciproque des organismes entre eux ? Peut-on mettre en évidence un effet court terme versus long terme sur plusieurs générations de vers ? Pour répondre à ces questions nous avons fait des expérimentations ex situ. Nous avons utilisé un sol considéré comme marginal par sa texture sableuse, et contaminé in situ par plusieurs éléments traces métalliques (teneurs sublétales en Cu, Zn, Cd). Trois espèces biologiques ont été étudiées seules ou en interaction : le ver de terre Aporrectodea caliginosa, un ver de terre endogé communément retrouvé dans les sols agricoles, Enchytraeus albidus, un enchytréide à large habitat approprié pour les tests écotoxicologiques et enfin Lolium perenne, une herbe fourragère.Nous avons montré une amélioration de la production primaire du sol contaminé par les deux groupes d’annélides, via une amélioration de la biomasse des plantes, lorsqu’il y a une interaction proche entre les racines et les annélides, en lien avec une augmentation de la biodisponibilité des nutriments. Cependant, lorsqu’ils s’éloignent des racines, cet effet bénéfique disparait. Concernant les éléments traces, les annélides du sol tendent à augmenter leur disponibilité. Toutefois, la plante possède un pouvoir fort de régulation limitant les transferts d’éléments trace. Les teneurs internes d'éléments mesurées dans les organismes varient d’un élément à l’autre et d’une espèce biologique à l’autre, ainsi qu’en présence d’interactions entre organismes. Nous avons ainsi pu montrer que les modifications de l’environnement induites affectent tous les organismes. Nous avons notamment montré que la présence de plantes sur le long terme augmente la capacité du sol à les héberger même en sol contaminé. En revanche, la présence de vers de terre dans le sol facilite l’enfouissement des enchytréides dans les couches plus profondes du sol et tend à réduire leur nombre retrouvé en surface. Cette étude montre que dans nos conditions expérimentales les connaissances acquises dans les sols non contaminés concernant la biofertilisation peuvent être appliquées aux sols contaminés. Cependant, les flux d’éléments majeurs s’accompagnant de flux d’éléments traces, l’impact résultant doit être examiné dans tous les compartiments biologiques du système. Nous avons de plus observé des effets de rétroactions de la plante sur les vers de terre, suggérant l’importance de les prendre en compte pour avancer dans la compréhension des mécanismes sous-jacents aux interactions sol-plante-annélides
Soil pollution is key environmental issue. A large part of European soils is contaminated by trace elements. Depending on the contamination level organisms exposed to degraded and contaminated soils can be affected in a variety of ways but generally contamination affects the biological activity. However, several researches have shown the importance of ecological linkages in soils, particularly the so-called "belowground-aboveground" relationships that connect the below-ground and above-ground soil communities. One of the most well-known of these relationships is the soil-plant-earthworm interactions. Earthworms are known to improve the ecological functions associated with fertile soils. For example, they can increase the primary production of both wild and cultivated plants and at the same time they have a key role in regulating soil communities. But in the case of contaminated soil their role is still poorly documented. Enchytraeids are oligochaete annelids that play the same ecological role as earthworms. However, there are few researches that confirm this hypothesis, whatever the soil contaminated or not. In this work, we hypothesised that the knowledge gained on soil-plant-annelid interactions in non-contaminated soils are transferable to polluted soils. In this case, annelids in interaction with plants could participate to the valorisation of these soils. We raised several questions: Do activities of annelids in contaminated soils affect the bioavailability of trace elements? Is there a reciprocity of influence between the soil organisms or plants? Are the annelids effects on the short or the long-term following several annelid life cycles? To answer these questions, we performed ex-situ experiments in cosmes. We used a soil classified as marginal because of its sandy texture, and field contaminated by several metallic trace elements (sublethal contents in Cu, Zn, Cd). Three different biological species were studied, alone or in interaction: Aporrectodea caliginosa, an endogeic earthworm found in agricultural soils, Enchytraeus albidus, an enchytraeid with a large habitat suitable for ecotoxicological testing, and finally Lolium perenne, a forage grass.We have seen an increase in the primary output of the contaminated soil via an increase in plant biomass, when a close connection between the roots and the annelids was assessed. We also showed that both annelid species have the ability to increase biomass production. However, when they move away from the roots, this positive effect decreases. This favourable effect on plants is due to an increase in nutrient bioavailability, linked to the annelid activities. However, annelids also tend to increase trace metal fluxes, and then their environmental availability with high contents in soil solution. But the plant has in turn a powerful regulatory power that limits metal transfers to the soil pore water. Furthermore, the trace element values observed in organisms differ from one chemical species to the other, as well as from one biological species to the other. Finally, the presence of species and the changes in their habitat have an impact on other organisms. We have shown that the presence of plants is favourable to the development of earthworms, boosting the soil's capacity to host them over time. The presence of earthworms in the soil, on the other hand, accelerates the burial of enchytraeids in the deeper layers of the soil and tends to diminish the amount of enchytraeids detected in soil surface. In our experimental conditions, we confirm that knowledge acquired for uncontaminated soils can be applied also for contaminated soils. However, fluxes of major elements are accompanied by fluxes of trace elements whose impact have to be checked in all the biological compartments of the system. Finally, we detected feedback from the plant to the earthworms, indicating intricate soil-plant-annelid connections that has to be taken into account for their better understanding

Doutoramento em Biologia
Nowadays, a systems biology approach is both a challenge as well as believed to be the ideal form of understanding the organisms’ mechanisms of response. Responses at different levels of biological organization should be integrated to better understand the mechanisms, and hence predict the effects of stress agents, usable in broader contexts. The main aim of this thesis was to evaluate the underlying mechanisms of Enchytraeus albidus responses to chemical stressors. Therefore, there was a large investment on the gene library enrichment for this species, as explained ahead. Overall, effects of chemicals from two different groups (metals and pesticides) were assessed at different levels of biological organization: from genes and biochemical biomarkers to population endpoints. Selected chemicals were: 1) the metals cadmium and zinc; 2) the insecticide dimethoate, the herbicide atrazine and the fungicide carbendazim. At the gene and sub-cellular level, the effects of time and dosage were also adressed. Traditional ecotoxicological tests - survival, reproduction and avoidance behavior - indicated that pesticides were more toxic than metals. Avoidance behaviour is extremely important from an ecological point of view, but not recommended to use for risk assessment purposes. The oxidative stress related experiment showed that metals induced significant effects on several antioxidant enzyme activities and substrate levels, as well as oxidative damage on the membrane cells. To increase the potential of our molecular tool to assess transcriptional responses, the existing cDNA library was enriched with metal and pesticide responding genes, using Suppression Subtractive Hybridization (SSH). With the sequencing information obtained, an improved Agilent custom oligonucleotide microarray was developed and an EST database, including all existing molecular data on E. albidus, was made publicly available as an interactive tool to access information. With this microarray tool, most interesting and novel information on the mechanisms of chemical toxicity was obtained, with the identification of common and specific key pathways affected by each compound. The obtained results allowed the identification of mechanisms of action for the tested compounds in E. albidus, some of which are in line with the ones known for mammals, suggesting across species conserved modes of action and underlining the usefulness of this soil invertebrate as a model species. In general, biochemical and molecular responses were influenced by time of exposure and chemical dosage and these allowed to see the evolution of events. Cellular energy allocation results confirmed the gene expression evidences of an increased energetic expenditure, which can partially explain the decrease on the reproductive output, verified at a later stage. Correlations found throughout this thesis between effects at the different levels of biological organization have further improved our knowledge on the toxicity of metals and pesticides in this species.
Actualmente, em ecotoxicologia, uma abordagem ao nível da biologia de sistemas representa não só um desafio, como se acredita ser a forma ideal para a compreensão dos mecanismos de resposta dos organismos. A integração de respostas de diferentes níveis de organização biológica, permite a melhor percepção dos mecanismos envolvidos, e assim possibilita a previsão de efeitos de agentes tóxicos num contexto alargado. O objectivo principal desta tese foi a avaliação dos mecanismos de resposta de Enchytraeus albidus a stressores químicos. Desta forma, um grande investimento foi feito de forma a enriquecer a biblioteca genómica desta espécie, como explicado mais adiante. Em suma, os efeitos de compostos químicos pertencentes a duas classes diferentes (metais e pesticidas) foram avaliados a níveis de organização biológica distintos: desde parâmetros populacionais até bioquímicos e moleculares com a alteração de expressão genética. Os compostos seleccionados foram: 1) os metais cádmio e zinco; 2) o insecticida dimetoato, herbicida atrazina e fungicida carbendazim. Ao nível sub-cellular e genético, os efeitos do tempo de exposição e da dose aplicada também foram avaliados. Os testes de ecotoxicologia tradicionais – sobrevivência, reprodução e evitamento – mostraram uma maior toxicidade dos pesticidas em relação aos metais. O evitamento é um parâmetro de extrema importância do ponto de vista ecológico, apesar de não recomendado para avaliação de risco. O estudo dos indicadores de stress oxidativo revelou que o Zn e o Cd induziram efeitos significativos na actividade de várias enzimas antioxidantes e níveis de substratos, provocando igualmente danos oxidativos nas membranas celulares. De forma a aumentar as potencialidades da nossa ferramenta molecular disponível para avaliar respostas ao nível da transcrição, a biblioteca de cDNA existente foi enriquecida com genes de resposta a metais e a pesticidas, usando o método de SSH. A partir das sequências obtidas foi desenvolvido um microarray de oligonucleótidos (Agilent), assim como uma base de dados de utilização gratuita na internet que reúne todos os dados moleculares disponíveis para E. albidus e constitui uma ferramenta interactiva de acesso a informação. Com a aplicação do novo microarray, foram obtidos dados novos e relevantes acerca dos mecanismos de toxicidade, tendo sido possível a identificação de diferentes vias metabólicas afectadas por cada composto químico. Os resultados obtidos permitiram a identificação de mecanismos de acção destes compostos em E.albidus que, em grande parte, coincidem com mecanismos descritos para mamíferos, sugerindo que os modos de acção são conservados em várias espécies e sublinhando a utilidade deste invertebrado como espécie modelo. De um modo geral, as respostas bioquímicas e moleculares foram influenciadas pelo tempo de exposição e concentração do composto tóxico, permitindo seguir a evolução dos eventos. Os resultados da determinação da alocação energética confirmaram as evidências de um aumento de gastos energéticos, sugeridas pelo microarray, e que podem explicar parcialmente o decréscimo na reprodução verificado numa fase posterior. As correlações encontradas no decorrer desta tese entre parâmetros de vários níveis de organização biológica, contribuíram para uma melhor compreensão da toxicidade de metais e pesticidas nesta espécie.

Mestrado em Biologia Molecular e Celular
A maioria dos estudos ecotoxicológicos têm investigado os efeitos de curto prazo dos contaminantes. Comparativamente, tem sido desenvolvido pouco trabalho na avaliação dos efeitos a longo prazo dos químicos existindo portanto uma necessidade de preencher esta lacuna. De entre as espécies de solo usadas em ecotoxicologia estão os Enquitraídeos (Oligochaeta), membros importantes da mesofauna terrestre com diretrizes padrão para testar os efeitos ao nível da sobrevivência, reprodução e bioacumulação (ISO, 2004; OECD, 2010, 2004). Para a espécie Enchytraeus crypticus, existe também disponível o cDNA microarray com mais de 40 000 transcritos (Castro-Ferreira et al., 2014) sendo uma vantagem competitiva em relação a outras espécies padrão. O principal objetivo desta pesquisa foi desenvolver novos testes de curto e longo prazo, abrangendo novos/diferentes endpoints para E. crypticus. A habilidade de regeneração do E. crypticus foi descoberta e o processo de regeneração foi descrito; este pode ser adicionalmente utilizado para avaliar os efeitos de tóxicos em testes de curto prazo. O desenvolvimento embrionário de E. crypticus foi investigado e um teste de embriotoxicidade foi desenvolvido (pela primeira vez em um invertebrados do solo). O cádmio (Cd) foi usado como substância de teste para validar o teste embriotoxicidade dados os seus conhecidos efeitos embriotóxicos. Os resultados mostraram que o Cd causou uma diminuição no sucesso da eclosão devido a um atraso ou interrupção na formação de estruturas embrionárias. Um teste de longevidade (com avaliação da sobrevivência e reprodução ao longo do tempo) foi desenvolvido para E. crypticus. Este ensaio de exposição de longo prazo foi utilizado para avaliar os efeitos de nanopartículas de óxido de cobre (CuO-NPs) em comparação com CuCl2 revelando que CuO-NPs causou efeitos superiores (diminuindo a longevidade e reduzindo a reprodução) do que CuCl2, a uma concentração de efeito semelhante. Este ensaio traz um novo conceito em ecotoxicidade, a longevidade. Este é um especto particularmente importante quando o assunto é a toxicidade de nanomateriais (NMs), onde se espera que o tempo de exposição a longo prazo revele efeitos imprevisíveis através dos testes correntes de curto/longo prazo. O uso dos novos ensaios desenvolvidos podem melhorar a avaliação dos perigos dos produtos químicos.
Most of the ecotoxicity studies have investigated the short-term effects of chemicals. Comparatively, little work has been done in the assessment of the long-term effects of chemicals and there is a need to fill this gap. Among soil species used in ecotoxicology are Enchytraeids (Oligochaeta), important members of the terrestrial mesofauna with standard guidelines for testing effects at survival, reproduction and bioaccumulation level (ISO, 2004; OECD, 2010, 2004). For the species Enchytraeus crypticus, there is also available the cDNA microarray with more than 40 000 transcripts (Castro-Ferreira et al., 2014) being a competitive advantage in comparison to other standard species. The main goal of this research was to develop novel short and long-term tests, covering new/different endpoints, for E. crypticus. Regeneration ability of E. crypticus was discovered and the regeneration process was described; this can be further used as endpoint to assess the effects of toxicants in short-term studies. The embryonic development of E. crypticus was investigated and an embryotoxicity test was developed (for the first time in a soil invertebrate). Cadmium (Cd) was used as a test substance to validate the embryotoxicity test given its known embryotoxic effects. Results showed that Cd caused a decrease in the hatching success due to a delay or disruption in formation of embryonic structures. A lifespan test (with assessment of survival and reproduction over time) was developed for E. crypticus. This long-term exposure assay was used to assess the effects of copper oxide nanoparticles (CuO-NPs) in comparison with CuCl2 revealing that CuO-NPs caused higher effects (shortening lifespan and reducing reproduction) than CuCl2, at similar effect concentration. This lifespan assay brings a novel concept in ecotoxicity, the longevity. This is a particularly important aspect when the subject is nanomaterials (NMs) toxicity, where longer term exposure time is expected to reveal unpredicted effects via the current short/long-term tests. The use of the new assays developed can improve the hazard assessment of chemicals.

A agricultura ocupa uma grande área do território brasileiro, interagindo com praticamente todos os grandes biomas. O manejo do solo, aliado ao uso de agrotóxicos e de fertilizantes, pode contribuir para o desequilíbrio da fauna edáfica, trazendo impactos que podem influenciar na harmonia do ecossistema do solo. O revolvimento do solo o expõe à radiação solar, que pode influenciar na abundância de espécies e os fertilizantes de base sintética desequilibram os níveis de nutrientes do solo, impactando na dinâmica dos organismos. Os agrotóxicos contaminam e causam mortalidade de diversos organismos. Na tentativa de melhor analisar esses impactos, foram escolhidos quatro diferentes sistemas de uso do solo: Olericultura Orgânica (OO), Olericultura Convencional (OC), Lavoura Convencional (LC) e uma região de fragmento de Floresta Nativa (FN), com repetições verdadeiras, na região metropolitana de Curitiba, no município de Quitandinha-PR, com os objetivos: avaliar o potencial dos enquitreídeos como bioindicadores em áreas de diferentes sistemas de uso do solo (SUS); identificar os fatores que influenciam a abundância e diversidade desses organismos nestes diferentes sistemas; avaliou-se o potencial reprodutivo destes organismos expondo-os em amostras de solos provenientes das áreas dos sistemas de uso do solo (SUS) estudados, em condições laboratoriais; foi também avaliado em ensaios laboratoriais com uso de solo artificial tropical (SAT) o impacto do glifosato na reprodução dos enquitreídeos sob concentrações baseadas nas recomendações do fabricante e das quantidades comumente utilizadas na região. Nossos resultados mostraram que houve diferença significativa na abundância entre FN e LC, e a riqueza de gêneros sofreu redução de seis para dois, respectivamente. Dentre os atributos do solo encontrados nas análises físico-químicas, as áreas de florestas (FN) apresentaram pH mais baixo, matéria orgânica e potássio mais altos que nos outros sistemas estudados, e menor quantidade de fósforo do que as áreas de plantio convencional (LC e OC). Os ensaios ecotoxicológicos em condições laboratoriais mostraram que o número de juvenis de Enchytraeus sp produzidos em solo das áreas estudadas foi maior em solo de FN do que LC, em concordância com os dados de abundância. Os ensaios ecotoxicológicos com glifosato em solo artificial tropical reduziu significativamente a reprodução, mesmo em concentrações no solo equivalentes a doses recomendadas, sugerindo que esse herbicida popular pode ser deletério aos enquitreídeos e, portanto, deve ser usado com cautela. Os resultados sugerem que a abundância e riqueza de enquitreídeos são bons indicadores do uso do solo na região estudada e que a espécie autóctone Enchytraeus sp respondeu satisfatoriamente em ensaios laboratoriais para avaliar a qualidade do solo.
Agriculture occupies a large area of the Brazilian territory, interacting with the major biomes. Soil management, the use of pesticides and fertilizers can contribute to the imbalance of the biota, the soil fauna, impacting the harmony of the whole soil ecosystem. The soil disturbance in plowing and disking exposes the soil fauna to the sun' radiation, which can be harmful to of species in this environment. Synthetic fertilizers can unbalance levels of soil nutrients, directly impacting the dynamics of organisms. The pesticides can be deleterious to the living organisms in the soil. This study aims to evaluate potential of the enchytraeids as soil management indicators in areas of horticulture and grain farming and to identify which factors influence the abundance and diversity of these worms in different land use systems (SUS); It also aims to assess the potential of the enchytraeids to respond to the natural soil quality of the areas of study and the toxicity of recommended doses of the most used pesticide (glyphosate) in laboratory tests; Four land use systems (SUS) in Quitandinha – PR were chosen for the present study: Organic Vegetable Crops (OO), Conventional Vegetable Crops (OC) and Conventional tillage (LC), Native Forest (FN) with true repetitions. The abundance of enchytraeids was statistically higher in FN than LC, while the richness of genera reduced from six to two, respectively. Among the soil attributes, forest areas had lower pH, organic matter, nitrogen and higher potassium than other SUS in the other SUS and lower amount of phosphorus than the areas of conventional tillage. In ecotoxicological tests under laboratory conditions the number of juveniles of Enchytraeus sp produced in soil of the areas studied for 21 days of incubation were higher in soils from FN than LC. in agreement to field abundance data. Ecotoxicological tests with gliphosate in concentrations based on doses used by the agricultures in the area sudied showed that in artficial soil, even concentrations equivalent to recommended doses may reduce reproduction significantly, suggesting this popular herbicide may be harmful to enchytraeids and should be used with caution. The results suggest the abundance and richness of genera are good indicators of soil use systems in the area studied and that the autochthonous enchytraeid Enchytraeus sp responded well to laboratory tests to evaluate soil quality.

Made available in DSpace on 2014-08-20T14:33:01Z (GMT). No. of bitstreams: 1 dissertacao_greice_de_almeida_schiavon.pdf: 2595058 bytes, checksum: b92d92d5c319dfe3fef953ccaf262aed (MD5) Previous issue date: 2012-12-12
This study was conducted on Estação Experimental Cascata, Embrapa Clima Temperado, in Pelotas, and on one family farm, in Morro Redondo. The objective of the study was to evaluate the scientific and local knowledge about the influences of management practices on soil fauna in ecological production systems. The methodology for samples of soil fauna, in each season, was done using the TSBF (Tropical Soil Biology and Fertility). Local knowledge was assessed using semi-structured interviews. The data analysis was done using th ecological indices as explanatory tools, and also the multivariate analysis (Principal Component Analysis - PCA) to evaluate the influence of management practices on soil fauna. In the first chapter, the ecological horticultural production systems cultivated under laminar composting with manure (CE) and mulch on the soil (CM), at Embrapa, showed the best values of soil fauna diversity, while treatment with frequent soil mechanization (MF) obtained a strong correlation with enchytraeids in all seasons. In the second chapter, results from interviews showed that the studied family recognize the role of earthworms in soil and understand the influence of the soil practices on soil fauna. It s possible to conclude that local knowledge is a valuable contribution to scientific knowledge to assess the influence of management practices on soil fauna. Moreover, the treatments CE and CM demonstrated their importance for to maintaining the soil diversity and for decreasing the hand labor in ecological horticultural production systems.
Este trabalho foi desenvolvido na Estação Experimental Cascata, Embrapa Clima Temperado no município de Pelotas e em uma propriedade agrícola familiar no município de Morro Redondo. O objetivo geral do estudo foi avaliar o conhecimento local e acadêmico sobre as influências das práticas de manejo na fauna edáfica nos sistemas de produção de base ecológica. A metodologia para a coleta da fauna edáfica, a cada estação do ano, foi baseada no programa TSBF (Tropical Soil Biology and Fertility). O conhecimento local foi avaliado a partir de entrevista semiestruturada com a família agricultora. Para avaliação dos dados foram utilizados como ferramentas explicativas, os índices ecológicos, e a análise multivariada, conhecida como Análise de Componentes Principais (ACP) para correlação das influências das práticas de manejo na fauna edáfica. No primeiro capítulo foi possível observar no sistema de produção de base ecológica de hortaliças da Estação Experimental Cascata, que os tratamentos com compostagem laminar com esterco bovino (CE) e cobertura morta sobre o solo (CM) apresentaram os melhores valores de diversidade da fauna edáfica, enquanto o tratamento com mecanização frequente do solo (MF) obteve uma forte correlação com os enquitreídeos em todas as estações do ano. No segundo capítulo, realizado através de entrevista com a família agricultora, foi ressaltada a função das minhocas no solo, além da compreensão que as práticas de manejo influenciam a presença da fauna edáfica. Com isso, foi possível concluir que o conhecimento local é um importante aliado ao conhecimento acadêmico para avaliar a influência das práticas de manejo na fauna edáfica. Além disso, os tratamentos CE e CM demonstraram sua importância para a manutenção da diversidade do solo e diminuição da mão-de-obra nos sistemas de produção de base ecológica de hortaliças.

Mestrado em Toxicologia e Ecotoxicologia
A nanotecnologia é uma área em constante crescimento, com cada vez mais produtos contendo nanopartículas (NPs) a serem introduzidos no mercado e no nosso dia-a-dia, desde cosméticos a baterias e dispositivos médicos. As características das NPs (composição química, tamanho, forma, reatividade) prometem novas aplicações e tornam estes materiais altamente desejáveis. Devido ao aumento de produção, as NPs irão eventualmente chegar ao meio ambiente de forma direta ou indireta, onde podem causar efeitos negativos nos diferentes compartimentos dos ecossistemas (solo, ar, água). Apesar de haver estudos ecotoxicológicos que se focam em nanomateriais, diferentes resultados (e por vezes contraditórios) podem ser encontrados mesmo para NPs com a mesma composição química, o que se pode dever a influência de fatores bióticos e abióticos que podem alterar a biodisponibilidade e portanto a toxicidade desses materiais. NPs metálicas, como o níquel (Ni) são atualmente utilizadas em diversas aplicações, no entanto os efeitos das nanopartículas de níquel (NiNPs) em organismos de solo ainda são pouco conhecidos. No presente estudo, a toxicidade de NiNPs (em comparação com NiNO3) para o organismo modelo de solo Enchytraeus crypticus (Oligochaeta) foi investigada com base no teste padrão Teste de Reprodução em Enquitreídeos (em inglês Enchytraeid Reprodução test - ERT) e no recentemente desenvolvido teste de ciclo de vida completo (FLC), que adiciona ao teste padrão, parâmetros como a eclosão, o crescimento e o tempo para atingir a maturidade. No geral, NiNO3 foi mais tóxico para o E. crypticus do que as NiNPs e a toxicidade parece ocorrer através de mecanismos diferenciados. Na exposição a NiNO3, os efeitos foram visíveis na redução do número de juvenis eclodidos ao nível da eclosão e mantiveram-se ao longo de todos os parâmetros avaliados no ciclo de vida (crescimento, estado de maturação, sobrevivência e reprodução). Relativamente à exposição a NiNPs, a eclosão foi o parâmetro mais sensível (CE10 = 47 mg NiNPs/kg), mas os organismos sobreviveram e reproduziram-se a concentrações até 1800 mg NiNPs/kg, mostrando que o efeito observado na eclosão foi um atraso. Exposição a 100 mg Ni/kg causou efeitos similares a concentrações mais altas (1000 e 1800 mg NiNPs/kg) indicando um maior efeito associado ao tamanho nano. Os atuais resultados realçam a potencial falta de uma dose-resposta monótona (com base na massa) para a avaliação de perigo de NPs e consequentemente a exigência da revisão dos procedimentos para a avaliação de risco.
Nanotechnology is an area of increasing growth, with more and more products containing nanoparticles (NPs) being introduced in the market and used in our daily life, from cosmetics to batteries and medical devices. NPs characteristics (chemical composition, size, shape, reactivity) promises new applications and make these materials highly desirable. Due to the increase of production NPs will eventually reach the environment either directly or indirectly, where they may cause negative effects in different compartments of ecosystems (soil, air, water). Although there are ecotoxicological studies that focus on nanomaterials, different (and sometimes contradictory) results can be found even with NPs of the same chemical composition, which may be due to the influence of abiotic and biotic factors that can affect the bioavailability and hence the toxicity of those materials Metallic NPs, such as nickel (Ni) are currently used in many applications, however the effects of nickel nanoparticles (NiNPs) in soil organisms are still poorly known. In the present work, the aim was to evaluate the toxicity of NiNPs (compared to NiNO3) for soil model organism Enchytraeus crypticus (Oligochaeta) using the standard Enchytraeid reproduction test (ERT) and the newly developed full life cycle test (FLC ), which adds to the standard the parameters hatching, growth and time to reach maturity. NiNO3 was more toxic to E. crypticus than NiNPs and toxicity seems to occur via differentiated mechanisms. For NiNO3 exposure effects where visible in the reduced number of hatched juveniles and then remained throughout all the endpoints evaluated in the life cycle (growth, maturity status, survival and reproduction). Regarding exposure to NiNPs, hatching was the most sensitive endpoint (EC10 = 47 mg NiNPs/kg), but organisms survived and reproduced at concentrations up to 1800 mg NiNPs/kg, revealing that the effect in the hatching was a delay. Exposure to 100 mg NiNPs/kg caused similar effects to the higher concentrations (1000 and 1800 mg NiNPs/kg), indicating higher nano-particulate effect. These results highlights the potential lack of monotone dose-response (based on mass) for hazard assessment of NPs and hence the requirement of a revised procedure for Risk Assessment.

The Enchytraeidae (Annelida, Oligochaeta) are small whitish worms especially prevalent in moorland and coniferous forest soils. They are less abundant but show varying densities in agricultural soils. They are important in soil decomposition processes and soil structure formation. This thesis reports the results of a study into the population sizes of the enchytraeids in agricultural plots at Craibstone Farm, five miles west of Aberdeen, Scotland, and the reasons for the variations in population size. It also describes the effects of the enchytraeids on soil structure, as determined from careful study of soil thin sections by light microscope and image analysis. The agricultural plots at Craibstone Farm grew an eight-year crop rotation at seven different pH levels. The average enchytraeid population size was found to be 3,5000 m^-2 in the first year and 11,000 m^-2 in the second. Populations tended to be larger at lower pH (4.2 to 5.2) and under pasture rather than annual crops. Fertilizer was seen to reduce numbers in the short-term but increases were seen in the long-term. In nearby grassland and mixed woodland, populations were considerably larger due to higher soil organic matter and moisture contents. At all sites the enchytraeids concentrated in the top 5cm of the soil, again correlating with moisture content. During the two years of sampling populations were seen to be smallest in late summer, coinciding with low rainfall. Soil thin sections showed that at low pH enchytraeids produced abundant granular faecal pellets which eventually fused to form a soil matrix. At higher pH (5.2 and above) small clusters of faecal pellets were more commonly seen partially or totally infilling soil channels and earthworm vermiform fragments were seen to be broken down into smaller granules.

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.

Os nematóides do gênero Meloidogyne (Goeldi, 1887) causam grandes prejuízos nas principais culturas no mundo. Com a sensibilização da população sobre os problemas causados pelos nematicidas químicos, uma nova alternativa de controle para os fitonematóides precisa ser encontrada para que se possa otimizar o manejo sustentável das lavouras. O controle biológico surgiu como uma alternativa potencial de controle e muitos estudos estão sendo desenvolvidos visando avaliar a aplicação do fungo Paecilomyces lilacinus (Thom., 1910) Samson, 1974, em campo para obter uma forma de controle ecologicamente correta. Porém, faltam trabalhos para entender como esta aplicação massal de conídios do fungo P. lilacinus afeta os organismos bioindicadores de qualidade do solo, que são organismos padronizados pelos testes do ISO e que respondem rapidamente aos estresses causados ao ambiente. Este trabalho objetivou avaliar os isolados do fungo P. lilacinus UELpae 18, UELpae 22 e UELpae Mix na formulação em pó molhável e suspensão concentrada em plantas de cafeeiro (Coffea arabica L. cv Iapar 59) em casa de vegetação no Centro de Ciências Agrárias da Universidade Estadual de Londrina, Londrina- Brasil. Foi avaliada a aplicação de diferentes concentrações de conídios do isolado UELpae 18 na formulação pó molhável em testes de reprodução com os organismos bioindicadores de qualidade de solo, um teste utilizando Folsomia candida (Willem, 1902) e outro utilizando Enchytraeus crypticus (Westheide & Graefe, 1992) em laboratório. Avaliou-se também o efeito desta aplicação deste fungo em microcosmos e as interações entre os organismos Meloidogyne paranaensis, P. lilacinus, F. candida e E. crypticus em plantas de tomateiro (Lycopersicon esculentum Mill. cv. Easypeel) nas dependências do Instituto do Mar (IMAR), Universidade de Coimbra, Coimbra-Portugal. Os resultados indicaram que os isolados UELpae 18, UELpae 22 e UELpae Mix nas duas formulações foram eficazes na redução da população de nematóide M. paranaensis em plantas de cafeeiro. No efeito da aplicação de diferentes concentrações do fungo P. lilacinus na reprodução dos dois organismos bioindicadores F. candida e E. crypticus, verificou-se que o aumento da concentração dos conídios do fungo P. lilacinus diminuiu a reprodução dos colêmbolos, o fungo não influenciou na reprodução de enquitreídeos e houve indícios de que os dois organismos bioindicadores se alimentaram do fungo P. lilacinus. Em microcosmos foi verificado que o tratamento que continha apenas o fungo nematófago e os tratamentos que continham o fungo em conjunto com os dois bioindicadores juntos ou sozinhos, reduziram a população de M. paranaensis em raízes de tomateiro, inclusive na presença de E. crypticus e F. candida, ambos na ausência do fungo indicando que são organismos com potencial biocontrole de nematóides. Mais estudos devem ser feitos sobre medidas conjuntas de manejo sustentável permitindo um manejo associado ecologicamente correto e eficaz.
Nematodes of the genus Meloidogyne (Goeldi, 1887) cause losses in major crops grown worldwide. With the population`s awareness about the problems caused by chemical nematicides, a new alternative for nematode control inorder one can optimize the sustainable management of plantations. Biological control has emerged as a potential alternative control and many studies are being developed to evaluate the application of the fungus Paecilomyces lilacinus (Thom., 1910) Samson, 1974, in field and thus obtaining a form of environmentally friendly control. However, there are not enough studies to understand how this mass application of the fungus P. lilacinus affects bioindicators organisms of soil quality, which are standardized by ISO testing methodologies and instantly respond to the aggressions caused to the environment. This study aimed the evaluation of the isolates of the fungus P. lilacinus UELpae 18, UELpae 22 and UELpae Mix in wettable powder and concentrated suspension formulation in coffee plants (Coffea arabica L. cv Iapar 59) in greenhouse in the Center for Agricultural Sciences, State University of Londrina, Londrina-PR, Brazil. It was evaluated the application of different concentrations of conidia of isolate UELpae 18 wettable powder formulation in reproduction tests of bioindicators organisms of soil quality, a test using Folsomia candida (Willem, 1902) and Enchytraeus crypticus (Westheide & Graefe, 1992) in laboratory. It was also evaluated the effect of this application of the fungus in microcosms with all the interactions of organisms Meloidogyne paranaensis, P. lilacinus, F. candida and E. crypticus on tomato plants (Lycopersicon esculentum Mill. cv. Easypeel) on the premises of the Institute of Marine Research (IMAR), University of Coimbra, Coimbra, PT. The results indicated that the isolates UELpae 18, UELpae 22 and UELpae Mix the two formulations were effective in reducing the population of nematode M. paranaensis in coffee plants. When it was evaluated the effect of different concentrations of the fungus P. lilacinus on the reproduction of the two bioindicators organisms F. candida and E. crypticus it was observed that the concentration of conidia of the fungus P. lilacinus influences the reproduction of Collembola and there was evidence the two bioindicators organisms fed on the fungus P. lilacinus. And when the work was assessed in microcosms it was found that the treatment with only the nematophagous fungus and the treatments containing the fungus together with the two bioindicators alone or together reduced the population of M. paranaensis in tomato roots, even when containing E. crypticus and F. candida without the presence of the fungus, indicating that they are organisms for potencial biocontrol of nematodes. More studies to be made on joint measures for sustainable management in order to get a friendly and efficient associated management.

As modificações ambientais provocadas pela humanidade, em especial pela agricultura, têm sido motivo de preocupação e estudos por todo o mundo. Nesse sentido, os solos figuram como um dos compartimentos ambientais mais impactados pelo uso de agrotóxicos, produtos amplamente utilizados na agricultura moderna. O objetivo deste trabalho foi avaliar se e como os agrotóxicos Score® 250 EC (i.a. difenoconazol) e Kraft® 36 EC (i.a. abamectina), isolados e em mistura, afetam a flora (germinação e crescimento do tomate - Lycopersicon lycopersicum), as comunidades microbianas do solo (biomassa microbiana em carbono, respiração, quociente metabólico e enzima β-glucosidase) e os enquitreídeos (reprodução de Enchytraeus crypticus em diferentes densidades e a relação trófica entre estes e os microrganismos), ao longo do tempo após a contaminação do solo. Em relação aos parâmetros microbianos, a despeito de algumas variações em decorrência da presença dos agrotóxicos, os indicadores das funções gerais do sistema edáfico não foram grandemente prejudicados, expressando uma redundância funcional. Não obstante, o solo-controle apresentou maior estabilidade em relação às variações dos parâmetros, sugerindo que os microrganismos presentes neste solo apresentaram uma maior robustez e estabilidade ao longo do tempo. Em relação aos testes com E. crypticus, os índices de reprodução não foram significativamente alterados em função da ação dos agrotóxicos, mas ratificou-se a importância dos microrganismos na dieta dos mesmos, haja vista as correlações entre o tamanho das comunidades microbianas e as taxas de crescimento dos enquitreídeos. Com relação ao crescimento e germinação do tomate, puderam ser observados valores maiores de altura e massa seca das plântulas em relação ao controle. Em contrapartida, observou-se uma possível melhor tendência de crescimento das plântulas no solo controle ao longo do tempo. Por fim, considerando a complexa problemática brasileira em relação ao uso de agrotóxicos, reiterou-se a importância do cumprimento das recomendações de aplicação de ambos os agrotóxicos avaliados por parte dos agricultores e recomendou-se a iniciativa de diminuição progressiva da sua utilização.
Environmental changes caused by humanity, especially by agriculture, have been a cause for concern throughout the world. The soils are one of the environmental compartments most impacted by the use of pesticides, which are widely used in modern agriculture. This research aimed at evaluating whether and how the insecticide Kraft® 36 EC (a.i. abamectin) and the fungicide Score® 250 EC (a.i. difenoconazole), isolated and in mixture, affect: a) the soil microbial communities (microbial biomass carbon, respiration, metabolic quotient and β-glucosidase enzyme); b) enchytraeid worms (reproduction of Enchytraeus crypticus in different densities and their trophic relation with microorganisms); and c) the flora (emergence and growth of tomato - Lycopersicon lycopersicum), over 84 days after contamination. The microbial indicators were not greatly impaired and expressed a functional redundancy, despite some variations due to the presence of pesticides. Nevertheless, the control group presented a greater stability than the contaminated ones, suggesting a greater robustness and stability of microorganisms over time, which are important factors for a better quality of the soil. In relation to E. crypticus tests, the importance of microorganisms on their diet was ratified, since positive correlations between the size of microbial communities and the enchytraeids rate of increase were observed. The different densities of E. crypticus were not significantly influenced by the effects of pesticides on the microorganisms. With respect to the growth and germination of tomato, higher values of height and dry mass of the seedlings were observed in contaminated groups. On the other hand, a higher growth tendency of the seedlings in the control soil was observed over time. Finally, considering the complexity of the Brazilian pesticide issue, we emphasize the importance of compliance of the recommendations on pesticides application and reinforce the initiative of decreasing their use.

The effects of reafforestation with seedlings of spruce, birch, mountain-ash, beech and larch, partially in combination with liming and removal of the topsoil layer before planting, on the community of saprophagous invertebrates (lumbricids, enchytraeids, oribatids, collembolans) were examined. The study area was located on plateau sites of the eastern Ore Mountains (Saxonia, Germany). Investigations were carried out on study sites exposed to high sulphur dioxide immissions and reforested 3 respectively 10 years ago. Invertebrates were sampled by the extraction of soil cores and by the ?electro-octett-method? (lumbricids). Additionally, the ?minicontainer method? was used to investigate decomposition rates of different types of litter. On the control area, a diedback spruce stand, the decomposer community showed high adaptations to the acidic soil substrate and was dominated by few enchytraeid species. Long-termed loss of tree shelter, liming and topsoil removal before planting resulted in a severe decline of most saprophagous species. However, changes in invertebrate community pattern due to different plantations were not found. A comparison of leaf and needle litter decomposition rates resulted in the following order: birch (k = 0.50) &gt; mountain-ash (0.40) &gt; spruce (0.30) &gt;&gt; larch (0.12). Considering all investigated taxa, the metabolic decomposing capacity of the larch litter reached 39 %, that the spruce litter 54 % of the foliage litter results.
Anläßlich des kompletten Ausfalls der Fichte in den extrem immissionsbelasteten Kammlagen des Osterzgebirges (Sachsen) wurden im Rahmen eines interdisziplinären Verbundprojektes verschiedene Waldbaukonzepte hinsichtlich ihrer Einflüsse auf die saprophage Invertebratenfauna (Enchytraeidae, Lumbricidae, Oribatida, Collembola) untersucht. Zur Disposition standen Verfahren zur Waldneugründung (Kulturparzellen mit Reihenpflanzung von Birke, Eberesche oder Fichte; zusätzlich waldbaulich unbeeinflußte Sukzessionsparzellen) und zum Waldumbau (Kulturparzellen mit Reihenpflanzung von Buche oder Lärche nach Abtrieb und flächigem Oberbodenabschub). Ein absterbender Fichtenreinbestand in fortgeschrittener Auflösung diente als Referenz- bzw. Korrelationsmaßstab. Die Erfassung der Mesofauna erfolgte über die Extraktion von Stechrohrproben. Lumbriciden wurden mit der Elektrooktett-Methode aufgenommen. Neben der flächenbezogenen Datenerfassung wurden 1.200 mit Blattstreu von Birke, Eberesche, Fichte und Lärche befüllte Minicontainer über 19 Monate auf einer Sukzessionsparzelle exponiert, um substratspezifische Abbaugeschwindigkeiten zu ermitteln und faunistische Sukzessionsverläufe zu studieren. Die Referenzfläche zeichnete sich durch eine stark enchytraeendominierte Zersetzergemeinschaft aus, die deutliche Anzeichen einer kalkungsbedingten Überprägung erkennen ließ. Der Enchytraeenanteil an den potentiellen Umsatzleistungen der untersuchten Destruententaxa (berechnet über metabolische Äquivalenzwerte) lag bei über 90 %. Hiervon ausgehend zeigten die Zersetzergemeinschaften der Versuchsanlagen &quot;Waldneugründung&quot; und &quot;Waldumbau&quot; stark divergierende Entwicklungsrichtungen. Als ausschlaggebende Faktoren konnten Schirmverlust, Kalkung und Oberbodenbeseitigung wahrscheinlich gemacht werden. Dagegen waren Einflüsse der unterschiedlichen Kulturbaumarten kaum nachzuweisen. Die streuspezifischen Dekompositionsgeschwindigkeiten unterschieden sich wie folgt: Birke (k = 0,50) &gt; Eberesche (0,40) &gt; Fichte (0,30) &gt;&gt; Lärche (0,12). Diese vergleichsweise geringen Abbauraten stehen überwiegend mit dem rauhen Montanklima in Zusammenhang. Enchytraeen besiedelten alle Streutypen zügig, während Collembolen vor allem die Ebereschenstreu verzögert aufsuchten und mit geringster Dichte bevölkerten. Oribatiden zeigten die geringste Besiedlungsgeschwindigkeit und hatten wie die Enchytaeen in der Laubstreu signifikant höhere Wohndichten. Nur Steganacarus spinosus zeigte eine besondere Affinität für Nadelstreu. Das metabolische Leistungspotential des Destruentenbesatzes der Lärchenstreu erreichte 39 %, das der Fichtenstreu 54 % der Laubstreuresultate.

The aim of the present work was to improve the ecological risk assessment (ERA) of plant protection products (PPPs) to in-soil fauna in European Union based on the new Scientific Opinion of European Food Safety Authority (EFSA) (2017) and to execute the same approach in Brazilian scenarios. The tiered approach, which modes from more protective to higher complexity tests was used. Bravonil 500® (500 g.L-1 chlorothalonil) and Lorsban 480® (480 g.L-1 chlorpyrifos) were used as model PPPs. In the lower tier, reproduction laboratory tests with eleven in-soil organisms (five collembolans and six oligochaete species) were performed using ISO and OECD standard protocols with adaptations when necessary. Effect concentrations to 10% (EC10) and 50% (EC50) of the tested species were estimated and has been used on the first intermediate tier to elaborate the species sensitivity distribution (SSD) curves. These allowed to estimate the hazard concentrations (HC) assumed to protect 95% (HC5) or 50% (HC50) of the Collembola and Oligochaeta species. In a second intermediate tier, microarthropod community tests in Mediterranean soil using native organisms under laboratory conditions were performed. Besides of toxicity data, exposure was also estimated though PPPs predicted environmental concentrations (PECs) in soils considering European and Brazilian scenarios. Toxicity-exposure ratios (TER) were determined and compared with the trigger values to estimate the potential risk for these organisms. Finally, mesocosms tests in terrestrial model ecosystem (TME) using subtropical soil from Brazil were executed as a surrogate higher tier. For these experiments two different application scenarios were considered: 1) continued application (2x) based on the general agricultural procedures for soybean crop application; 2) single application, estimated based on Europe’s ERA instructions for higher tier testing. Lower tier chlorothalonil results to collembolans indicates the higher EC10 to the standard Folsomia candida (2.44 mg a.i. kg-1). However, there were overlaps on confidence interval to all tested species, indicating similar sensitivity to this fungicide. Among oligochaetes EC10 data, Eisenia andrei did not present a protective value for all tested species (22.69 mg a.i. kg-1). Chlorpyrifos was highly toxic to all collembolans tested (EC10 < 0.004 mg a.i. kg-1). Among oligochaetes, E. andrei was the least sensitive species in EC10 data (5.2 mg a.i. kg-1). The non-protectiveness of E. andrei when estimating risk to all oligochaetes species is clear in the SSDs approach (chlorothalonil: HC5: 2.98 and chlorpyrifos: HC5: 0.084 mg a.i.kg-1). The lower tier must be the most protective step in ERA, which was not observed to oligochaetes in the present work. Microarthropod community tests pointed to similar results to those found in the SSDs approach for collembolans (chlorothalonil: EC20: 1.90 – 9.36 and chlorpyrifos: EC20: 0.0020 - 0.024 mg a.i. kg-1), corroborating risk prediction in laboratory tests to Collembola species. Results indicates that the used approach to test for microarthropod communities could be useful as an intermediate tier. However, standardization is still necessary, due the high variability in dataset, mainly due to mites. Higher tier though TMEs tests showed a population reduction of microarthropods due to both products, even at the lowest concentrations tested, regardless the exposure scenario. Results also indicated an absence of recovery eight weeks after application (Bray-Curtis dissimilarity > 35%). Effects on earthworms in TMEs were not observed due the low number of organisms. No effects has been observed in enchytraeids either. The previous tiers were capable of predicting risks, which were still detected at higher tier, mostly for collembolans. EFSA (2017) suggestions to estimate PPPs risks to in-soil fauna are suitable on ERA and the proposed approaches for intermediate tiers could help risk assessors and management. At Brazil, it is suggested to change the current regulation shifting from acute testing towards the adoption of chronic reproduction tests and the addition of more species than just E. andrei. Not only the acute test have been highlighted as inefficient in predicting risks, this earthworm species was not the most sensitive species to both products in lower and intermediate tiers with SSDs. Furthermore, research in dissipation time (DT) of PPPs in Brazilian soils are immediately necessary to estimate accurate PEC values under different scenarios to better predict PPPs risks.
Os agrotóxicos são moléculas utilizadas para proteger os cultivos agrícolas de pragas e doenças. Contudo, esses produtos podem apresentar efeitos adversos indesejados em organismos não-alvo. O objetivo deste trabalho foi contribuir para aprimorar a análise de risco ecológica (ARE) de agrotóxicos para a fauna do solo na União Europeia (EU) com base na nova Opinião Científica da European Food Safety Authority (EFSA) (2017) e executar a mesma abordagem de ARE para cenários brasileiros, verificando a possibilidade de utilização do modelo Europeu para o Brasil. Foi utilizada a abordagem de tiers, etapas que avançam de testes mais protetivos para ensaios de maior complexidade, com dois produtos comerciais: o fungicida Bravonil 500 (500 g.L-1 clorotalonil) e o inseticida Lorsban 450 (450 g.L-1 clorpirifós). No lower tier foram elaborados ensaios laboratoriais de reprodução com onze espécies de invertebrados de solo (cinco colêmbolos e seis oligoquetas) utilizando protocolos ISO e OECD com adaptações quando necessário. Os ensaios resultaram em concentrações de efeito (CE) para 10% e 50% das populações que foram utilizadas em um primeiro tier intermediário, na elaboração de species sensitivity distribution (SSD) curves. Em seguida, também foram calculados valores de hazard concentration (HC) para proteção de 95% (HC5) ou de 50% (HC50) das espécies de colêmbolos e oligoquetas. Para um segundo tier intermediário foram conduzidos ensaios de comunidade de microartrópodes com solo natural de clima temperado (Portugal) e fauna nativa em condições de laboratório. Além dos dados de toxicidade, a exposição dos organismos também foi estimada por meio das PECs (predicted environmental concentrations) dos agrotóxicos em solos para cenários Europeus e Brasileiros. Os dados de toxicidade e de exposição permitiram estimar as TERs (toxicity-exposure ratio). Quando esses valores são comparados a um nível de proteção (trigger value) o risco pode ser, enfim, estimado. Finalmente, foram executados ensaios em mesocosmos, utilizando terrestrial model ecosystem (TMEs) com solo subtropical (Brasil) como proposta de higher tier. Para esses experimentos, concentrações de contaminação foram estimadas por meio de diferentes cenários: 1) aplicação continuada (2x) estimado conforme aplicação dos produtos em campo na cultura da soja; 2) aplicação única, estimada por modelagem matemática conforme o executado na análise de risco da União Europeia para o higher tier. Resultados do clorotalonil em lower tier com colêmbolos indicaram que a espécie padrão mais utilizada, Folsomia candida, apresentou o maior CE10 (2.44 mg i.a. kg-1), no entanto, os intervalos de confiança de todas as espécies se sobrepuseram, indicando uma sensibilidade similar. Quanto aos oligochaetas, Eisenia andrei, não apresentou valores de CE que fossem suficientemente protetivos aos outros organismos testados (CE10: 22.69 mg i.a. kg-1). O clorpirifós apresentou uma alta toxicidade a todos os colêmbolos (CE10 < 0.004 mg i.a. kg-1) e entre as oligoquetas, E. andrei foi o organismo com menor sensibilidade quanto aos dados de CE10 (5.2 mg i.a. kg-1). A falha da espécie de oligoqueta utilizada atualmente na ARE em proteger o grupo taxonômico ao qual pertence é evidenciada pelas SSDs (clorotalonil: HC5: 2.98 e clorpirifós: HC5: 0.084 mg i.a. kg-1), levando em consideração que uma etapa intermediária não deveria ser mais sensível que a etapa preliminar (lower tier). Os ensaios de comunidades tiveram resultados semelhantes aos das SSDs para os colêmbolos (clorotalonil: CE20: 1.90 – 9.36 e clorpirifós: CE20: 0.0020 - 0.024 mg i.a.kg-1) confirmando a previsão de risco dos ensaios laboratoriais para esse grupo. Os resultados indicam que essa metodologia pode ser utilizada como um tier intermediário, mas uma padronização ainda é necessária devido à alta variabilidade verificada, especialmente para os ácaros. Os resultados de higher tier apontam reduções das populações nativas na presença dos dois agrotóxicos, mesmo na concentração mais baixa testada em diferentes cenários de exposição, indicando riscos e ausência de recuperação, especialmente para os colêmbolos, mesmo oito semanas após a contaminação (dissimilaridade de Bray-Curtis > 35%). Possíveis riscos para as minhocas em TMEs não puderam ser verificados devido ao baixo número de indivíduos presentes. Não houve efeitos dos produtos para os enquitreideos de acordo com a metodologia utilizada. Verificou-se ainda que os tiers anteriores foram capazes de prever o risco, que não foi reduzido avançando nas etapas da ARE. As sugestões da opinião científica da EFSA (2017) para mensurar o risco dos pesticidas à fauna do solo são pertinentes, e a adoção de etapas intermediárias pode auxiliar legisladores e reguladores de risco na Europa. Para o Brasil, sugere-se a adoção legislativa de ensaios crônicos de reprodução ao invés de ensaios agudos de letalidade com outras espécies além de E. andrei. Além dessa estimativa ser apontada como ineficiente, a espécie não foi o melhor indicador de toxicidade dos dois produtos aos oligoquetas em lower tier e no tier intermediário com SSDs. Também se sugere o estudo de tempo de dissipação (DT) de agrotóxicos em solos brasileiros para melhores estimativas do risco dos produtos. Sem esses dados torna-se inviável a análise de risco por meio da abordagem em tiers utilizada na Europa, uma vez que as estimativas de exposição para os cenários brasileiros com os dados oficiais existentes são irreais.

The first paper describes the ecotoxicological effects of TNT on enchytraeids. TNT was shown to be transformed in the artificial soil used as an exposure matrix, and resulted in enchytraeid mortality and reductions in the number of juveniles as determined by standard tests. The toxicity of TNT and its biotransformation by enchytraeids at the organismal and biochemical levels are described in the second manuscript. Adult enchytraeids were analysed after exposure to TNT in both liquid and soil milieu. TNT-related metabolites were found in enchytraeids after exposure to TNT-spiked artificial (OECD) soil, as well as TNT in liquid. The in vitro experiments showed that biotransformation of TNT in enchytraeids was dependent on the protein concentration and the period of incubation. In addition, TNT biotransformation in vitro was dependent on the sub-cellular fraction, with the 8,000 x g pellet containing the highest activity. Furthermore, the observed TNT-transforming activity in vitro was related to increased bacterial growth found in the potworm homogenate after prolonged periods of incubation. When the antibiotics, tetracycline or streptomycin-penicillin, were added to incubation mixtures, the biotransformation of TNT was inhibited and the bacterial numbers in the potworm homogenate after the exposure time period did not increase significantly. Based on the studies reported in the second manuscript, it is concluded that the transformation of TNT to selected amino-derivatives by enchytraeids is mainly due to bacteria associated with the potworms. A literature review on enchytraeid species, and the biotransformation of TNT by bacteria, is also presented

碩士
國立臺灣大學
生命科學系
105
Hedgehog, one of the arthropod segmental polarity genes, is crucial for embryogenesis and regeneration in many metazoan species. Previous studies in annelids showed that hedgehog signaling is involved in segmentation in a polychaete, Platynereis and that hedgehog signaling regulates gut formation but not segmentation in the leech (one clitellate). To understand the evolution of hedgehog expression in annelid segmentation and regeneration, we have isolated a hedgehog homolog (Enc-hh) from Enchytraeus sp., a small transparent microdrile oligochaete reproducing asexually by fragmentation, and studied segment formation during its regeneration. This worm was isolated from the local soil sample, and DNA barcoding suggested that it belongs to a novel, previously undescribed species. It can completely regenerate its tail in 3~4 days and head in 6~8 days after amputation or fragmentation. Enc-hh is expressed in the brain, coelom wall of the clitellum segments, gut, and a site in the posterior growth zone, where the new body segments are formed, in intact worms. The pharmacological disruption experiments suggested that the major function of the hedgehog pathway in the intact worm may be in regulating the morphological homeostasis of the gut tissue. In contrast to the constitutive expression of hedgehog throughout the entire life cycle of Enchytraeus, hedgehog expression is significantly reduced in the post-embryonic stage of the leech. During regeneration, Enc-hh begins to be expressed in blastema two days after amputation. Enc-hh expression patterns differ between anterior and posterior regeneration. During posterior regeneration, Enc-hh expression pattern is similar to an intact worm. On the other hand, Enc-hh expression pattern during anterior regeneration suggests that Enc-hh plays a role in anterior patterning during anterior regeneration. This study revealed different roles of hedgehog during anterior and posterior regeneration in Enchytraeus. This is the first report of hedgehog gene expression pattern in an oligochaete annelid.

The objective of the thesis is the model-based analysis of spatial patterns of decomposition properties on the forested slopes of the montane level (ca. 1200-2200 m a.s.l.) in a study area in the Italian Alps (Val di Sole / Val di Rabbi, Autonomous Province of Trento). The analysis includes humus forms and enchytraeid assemblages as well as pH values, activities of extracellular enzymes and C/N ratios of the topsoil. The first aim is to develop, test and apply data-based techniques for spatial modelling of soil ecological parameters. This methodological approach is based on the concept of digital soil mapping. The second aim is to reveal the relationships between humus forms, soil organisms and soil microbiological parameters in the study area. The third aim is to analyze if the spatial patterns of indicators of decomposition differ between the landscape scale and the slope scale. At the landscape scale, sample data from six sites are used, covering three elevation levels at both north- and south-facing slopes. A knowledge-based approach that combines a decision tree analysis with the construction of fuzzy membership functions is introduced for spatial modelling. According to the sampling design, elevation and slope exposure are the explanatory variables. The investigations at the slope scale refer to one north-facing and one south-facing slope, with 30 sites occurring on each slope. These sites have been derived using conditioned Latin Hypercube Sampling, and thus reasonably represent the environmental conditions within the study area. Predictive maps have been produced in a purely data-based approach with random forests. At both scales, the models indicate a high variability of spatial decomposition patterns depending on the elevation and the slope exposure. In general, sites at high elevation on north-facing slopes almost exclusively exhibit the humus forms Moder and Mor. Sites on south-facing slopes and at low elevation exhibit also Mull and Amphimull. The predictions of those enchytraeid species characterized as Mull and Moder indicators match the occurrence of the corresponding humus forms well. Furthermore, referencing the mineral topsoil, the predictive models show increasing pH values, an increasing leucine-aminopeptidase activity, an increasing ratio alkaline/acid phosphomonoesterase activity and a decreasing C/N ratio from north-facing to south-facing slopes and from high to low elevation. The predicted spatial patterns of indicators of decomposition are basically similar at both scales. However, the patterns are predicted in more detail at the slope scale because of a larger data basis and a higher spatial precision of the environmental covariates. These factors enable the observation of additional correlations between the spatial patterns of indicators of decomposition and environmental influences, for example slope angle and curvature. Both the corresponding results and broad model evaluations have shown that the applied methods are generally suitable for modelling spatial patterns of indicators of decomposition in a heterogeneous high mountain environment. The overall results suggest that the humus form can be used as indicator of organic matter decomposition processes in the investigated high mountain area.