Dissertations / Theses on the topic 'Ecotoxicology'

The estuarine tube dwelling amphipod Paracorophium excavatum was investigated for its suitability as a bio-indicator and bio-monitor. Distribution patterns of P. excavatum were determined at 13 sites in the Canterbury region that differed in particle size distribution ranging from sandy to muddy sediment, with overall low organic content. Low tide salinity ranged from 5 to 33 0/00 between sites and sediment moisture content ranged between 23 to 41% moisture. Amphipods were absent from most sites within the Avon-Heathcote Estuary. The availability, life history and fecundity of P. excavatum were compared from intertidal mudflat sites in Brooklands Lagoon and Kairaki over a period of thirteen months. Four sediment core samples were collected at monthly intervals and P. Excavatum’s population structure and life history pattern studied. The life history of P. excavatum can be characterised by fast-growing, annual, iteroporous, bivoltine, females ovigerous throughout the year and thelygenous (female biased) population. P. excavatum showed relative consistency in abundance throughout the year with monthly densities ranging from 875.79 per 0.1 m⁻² (July) to 1754.77 per 0.1 m⁻² (December) at Brooklands Lagoon and 1031.83 per 0.1 m² (November) to 1780.24 per 0.1 m² (December) at Kairaki. There was a linear relationship between numbers of eggs per female and female length. P. excavatum show no statistically significant difference in brood mortality between the early and later development stages. P. excavatum was investigated for its potential use in short-term (acute) sediment toxicity tests. In 10-days sediment tolerance tests using sediment from nine sites (8 + control) in the Avon-Heathcote Estuary, amphipod survival was overall high in most sites (above 70%). However, low survival was found for site 7 and 9, indicating that P. excavatum may be a good discriminator of sediment quality. However, P. Exc avatum mortality was not significantly correlated to sediment type (p = 0.99, ƒ = 0.06) nor copper content of sediment (p = 0.99, ƒ = 0.01). The effects of copper concentrations on the survival, emergence and reburial of P. excavatum were investigated. Amphipods were exposed to 10-days to 7 sub-lethal copper concentrations (2.7, 15.96, 20.45, 23.92, 32.02, 42.99 and 84.62 μg g-¹ wet weight sediment). The results show clear dose-dependent effects of copper on survival and behaviour on P. excavatum. Copper toxicity test indicates P. excavatum high sensitivity to the heavy metal copper. Median lethal concentration (LC₅₀) was 53.026 μg g⁻¹ Cu (wet weight) and edian effect concentration (EC₅₀) was 47.89 μg g⁻¹ Cu (wet weight). Emergence increased with increasing metal concentrations indicating that the ontaminants had a sublethal behavioural effect. Concentration of copper in P. excavatum tissues increased significantly with increasing metal sediment concentration. The ability of P. excavatum to accumulate heavy metals into their body tissues makes this species suitable for use used as a copper bio-indicator. Further work should concentrate on the response of P. excavatum to known contaminants and the influence of non-contaminant variables to better characterises P. excavatum's relative sensitivity. It is concluded that P. excavatum can be used as an effective tool for use in ecotoxicological studies. Natural behaviour, laboratory experiments, and sediment bioassays and its availability throughout the year has demonstrated that P. excavatum can be used as a bio-monitor.

Levando-se em consideração a grande demanda por água e por fim a poluição emergente nos dias de hoje, o gerenciamento dos corpos hídricos deve ser algo constante, com o propósito preventivo e corretivo, sendo o objetivo deste trabalho a análise das condições ecotoxicológicas e limnológicas dos reservatórios Guarapiranga, Billings e Paiva Castro, na Região Metropolitana de São Paulo, que é abastecida por tais reservatórios. Foram realizadas duas coletas, sedo uma em Maio de 2011, na estação seca, e outra em Janeiro de 2012, na estação chuvosa. Em cada reservatório foram analisados cinco pontos próximos à captação de água pela SABESP. A água e sedimento foram avaliados quanto à toxicidade aguda e crônica através de bioensaios com os cladóceros Daphnia similis e Ceriodaphnia dubia para a água e D. similis e Chironomus xanthus para o sedimento. O tratamento dos dados ocorreu através dos testes de Dunnett\'s ou Steel\'s Many-one Hank e Kruskal-Wallis (fecundidade / sobrevivência) e Fisher Exact Test (mortalidade). Os parâmetros limnológicos analisados na água foram: pH, temperatura, condutividade elétrica, material em suspensão (orgânico e inorgânico), sólidos totais, oxigênio dissolvido, DQO, DBO, nitrogênio total, nitrato, nitrito, amônio, fósforo total, ortofosfato e clorofila a. Os parâmetros físico-químicos do sedimento analisados foram: pH, temperatura, granulometria, teor de Matéria Orgânica e metais (Cd, Cu, Ni, Zn, Pb e Mn). O grau de associação entre as variáveis limnológicas da água, sedimento, e testes ecotoxicológicos, foi avaliado em teste não-paramétrico, através do Coeficiente de Correlação de Spearman. As mesmas variáveis também foram analisadas através da Análise dos Componentes Principais (ACP). O Índice de Estado Trófico mostrou serem os reservatórios Guarapiranga e Billings os mais eutrofizados dentre os corpos hídricos estudados. Conforme os parâmetros analisados na água, alguns parâmetros de certos pontos amostrais estiveram fora dos Padrões estabelecidos pela Resolução CONAMA nº 357/05. Houve influência das chuvas em relação aos parâmetros limnológicos da água, mas o mesmo não foi evidenciado em relação aos resultados dos testes ecotoxicológicos. No geral os dados sugerem ser o sedimento dos reservatórios com maior potencial tóxico do que a água. Os dados ainda sugerem um gradiente de toxicidade, começando pelo reservatório Billings, Guarapiranga e por último Paiva Castro, sendo este o menos tóxico. Houve uma possível correlação entre os metais encontrados no sedimento dos reservatórios mais eutrofizados e a toxicidade obtida nos testes ecotoxicológicos. Os dados sugerem que maiores esforços preventivos e corretivos devem ser alocados para os reservatórios mais eutrofizados e com maiores ocorrências de toxicidade, como é o caso de Guarapiranga e Billings, enquanto que no terceiro reservatório, Paiva Castro, o monitoramento e esforços preventivos serão satisfatórios, mantendo os padrões de qualidade de suas águas em relação aos parâmetros analisados neste trabalho.
Considering the great water demand and the increase pollution in the present-day, the management of the hydric bodies must be something constant, with the preventive and corrective purpose, being the objective of this work to analyze the ecotoxicological and limnological conditions of Guarapiranga, Billings and Paiva Castro reservoirs, in the Metropolitan Region of São Paulo, that\'s supplied by these reservoirs. It was made 2 collections, one in May 2011, in the dry season, and the other in January 2012, in the wet season. In each reservoir it was analyzed 5 points next to the water captation station by the SABESP. The water and sediment were analyzed as for acute and chronic toxicity through bioEssays with the cladocerans Daphnia similis and Ceriodaphnia dubia to the water and D. similis and Chironomus Xanthus to the sediment. The data treatment occurred through the Dunnett\'s or Steel\'s Many-one Hank and Kruskal-Wallis (fecundity/ survival) and Fisher Exact Test (mortality). The limnological water parameters analyzed were: pH, temperature, electrical conductivity, suspended solids (organic and inorganic), total solids, dissolved oxygen, COD, BOD, total nitrogen, nitrate, nitrite, ammonium, total phosphorus, orthophosphate and chlorophyll a. The physical-chemical analyzed parameters in sediment were: pH, temperature, granulometry, organic matter, and metals (Cd, Cu, Ni, Zn, Pb and Mn). The association level between the limnological variables in water, sediment, and ecotoxicological tests, was available in non-parametric tests, through the Spearman correlation coefficient. The same variables were analyzed through the Principal Component Analysis. The Trophic State Index show the Guarapiranga and Billings reservoir as the most eutrophicated between the hydric bodies studied. According to the analyzed parameters in the water, some parameters of certain points were out of the standards established by CONAMA Resolution nº 357/05. There was influence from the rain in relation to the limnological parameters from water, but the same standard was not evidenced in relation to the results from the ecotoxicological tests. In general, the data suggest being the reservoir sediments with more toxic potential than the water. The data still suggest a toxicity gradient, beginning in the Billings and Guarapiranga reservoir, and for last Paiva Castro, being this one the less toxic. There was one possible correlation between the metal found in the more eutrophicated reservoir sediment and the toxicity obtained in the ecotoxicological tests. The data suggest that greater preventive and corrective efforts must be allocated to the more eutrophicated reservoirs with more toxicity occurrences, as the case of Guarapiranga and Billings, while in the third reservoir, Paiva Castro, the monitoring and preventive efforts will be satisfactory, keeping the water quality standard in relation to the parameters analyzed in this work.

O mexilhão marrom Perna perna (Linnaeus, 1758) auxilia no monitoramento de compostos químicos em ecossistemas marinhos. No entanto, os mecanismos moleculares de detoxificação e resposta ao estresse são desconhecidos. Elucidar esses mecanismos é crucial para entender os efeitos tóxicos dos poluentes químicos e desenvolver biomarcadores para avaliar a qualidade ambiental dos ecossistemas marinhos. No presente estudo, indivíduos da espécie P. perna foram expostos a antraceno (ANT) e os RNAs mensageiros (mRNA) das brânquias foram sequenciados com a plataforma Illumina. A análise química do tecido mole dos animais identificou concentrações de ANT 268 a 715 vezes mais alta no grupo exposto comparado ao grupo controle, demonstrando que a exposição foi realizada com sucesso. O sequenciamento do transcritoma do P. perna gerou 273.152.390 pares de reads, resultando na montagem de 231.728 contigs com tamanho médio de 720 pb e N50 de 1.083 pb, os quais 66.563 contigs (28,7%) pode ser anotado utilizando banco de dados como GenBank, Pfam, Gene Ontology e KEGG. Os resultados obtidos a partir da anotação funcional sugerem que as brânquias tenham papel na biotransformação de xenobióticos, resposta antioxidante, sinalização, resposta imunológica inata, e osmorregulação. Foi possível identificar genes de biotransformação de fase I, II e III, incluindo CYPs e GSTs. Transcritos similares a CYPs e GSTs estavam sendo expressos no grupo exposto, porém nenhum deles foram classificados como diferencialmente expressos. Contudo, muitos genes hipotéticos foram diferencialmente expressos, o que sugere que P. perna utilize mecanismos desconhecidos de biotransformação para lidar com a contaminação de ANT. Genes de sistema imune inato foram regulados tanto positivamente quanto negativamente, assim como observado para Perna viridis exposto a benzo(a)pireno, sugerindo que ANT promove alterações da capacidade de resposta do sistema imune inato do P. perna.
The brown mussel Perna perna (Linnaeus, 1758) helps the monitoring of chemical compounds in marine ecosystems. However its molecular mechanisms of detoxification and stress response remain unclear. Elucidating these mechanisms is crucial to understand the toxic effects of chemical pollutants and to develop biomarkers to assess marine ecosystems. In this study, P. perna individuals were exposed to anthracene (ANT) and its mRNA complement was sampled sequenced with Illumina technology. Chemical analysis of the soft tissue identified ANT concentrations 268 - 715 fold higher in the exposed group compared to controls, demonstrating that the exposure procedure was successfully accomplished. Transcriptome sequencing of P. perna generated 273.152.390 paired reads that were assembled in 231.728 contigs of average length 720 bp and N50 1.083 bp , which 66.563 contigs (28,7%) could be annotated using GenBank genes, Pfam domains, Gene Ontology (GO) terms and KEGG pathways. The results obtained from functional annotation suggest gills play a role in xenobiotics biotransformation, antioxidant response, signal transduction, innate immune response, and osmoregulation. It was possible to identify transcripts similar to genes related with biotransformation reactions of phases I, II and III, including CYPs and GSTs. Transcripts similar to CYPs and GSTs isoforms were highly expressed in the group exposed to ANT, however no CYP, GST, or even other genes related with biotransformation reactions were classified as differentially expressed. On the other hand, several hypothetical genes were differentially expressed, which suggests that P. perna uses unknown mechanisms of biotransformation to deal with ANT stress contamination. Immune related-genes were both up and down-regulated, as was also observed for Perna viridis exposed to benzo(a)pyrene, suggesting that ANT promotes alteration in the immune response of P. perna.

The major findings of the study were as follows: 1. Rats ate significantly more coumatetralyl than brodifacoum, and rats in an area of physiological resistance (central southern England) ate greater quantities of rodenticide than rats in the east midlands. Coumatetralyl failed to achieve rat control on farms in central southern England. 2. Physiological resistance was suspected on two farms in the east midlands (near Lincoln) where rats ate excessive quantities of coumatetralyl and the control programme was unexpectedly extended. 3. GCMS analyses performed on extracts of 169 whole rodents revealed that some rats had eaten levels of coumatetralyl that far exceeded a lethal dose for susceptible animals. Excessive bait consumption occurred mostly in the area of physiological resistance, but also on the two sites in the east midlands where resistance was suspected. Brodifacoum consumption by some rats was also high, but complete control was usually achieved with brodifacoum and there was no evidence of any resistance to brodifacoum. 4. HPLC analyses carried out on 10 rats from coumatetralyl sites (five from each region), revealed that trapped, physiologically-resistant animals are capable of carrying 50 times the LD50 of coumatetralyl without any obvious ill effect. Rats from the east midlands carried a significantly lower load of coumatetralyl. 5. Video observations gave no evidence to support a bait point monopoly theory. Interactions at feeding sites were common. 6. This study has revealed that the potential exposure of non-target predators and scavengers to rodenticides is considerably higher in areas where rats show physiological resistance. This fact is discussed with reference to the regulation and monitoring of pesticides in the UK.

Manufactured nanoparticles are increasingly being used in the production of consumer products and appliances. A release in the environment, either intended through remediation or unintended through a spill at production sites, through wastewater or product degradation, is most likely to occur. Due to their small size, nanoparticles have a far greater surface area to unit mass ratio than conventional substances, rendering them potentially more reactive. This project aims to obtain key data on the ecotoxicology of nanoparticles in the aquatic environment. Initially, data from acute and chronic toxicity tests were gathered by exposing the invertebrate Daphnia magna to nanoparticles of carbon black, cerium dioxide, silver and titanium dioxide. The endpoints were mortality, moulting frequency, growth and number of offspring. The results indicate that a gradient of toxicity can be drawn, with cerium dioxide being the least toxic, to silver being the most toxic. Also a size dependent increase of toxicity was observed, with exposures to nano sized particles being more toxic than micro sized particles. Uptake and fate of nano sized materials were studied by exposing D. magna to fluorescent polystyrene beads of 20 nm and 1000 nm sizes and the results were compared. Both particle sizes were readily taken up in the gut and relocated in storage droplets within the body of D. magna. A quantification of the results showed that the mass of 1000 nm sized particles taken up was higher at equal exposure concentrations than the 20 nm sized particle but the excretion rate was higher as well for the 1000 nm particles. However, when assessing uptake as surface area or particle number dose, uptake of 20 nm particles exceeds uptake of 1000 nm particles. To assess the effect of nanoparticles on oxidative stress, the total antioxidant capacity was measured as well as the glutathione concentration of exposed D. magna. A decrease in total glutathione in D. magna was detected due to exposure to nano sized carbon black, while measuring the total oxidant capacity proved to be impossible due to interferences with the method used. The results show that, when negative effects are observed, these are more severe in exposures to nanoparticles than their micro sized counterparts and furthermore a clear route of uptake of nanoparticles in the body of D. magna can be observed.

Landscape ecotoxicology is the study of dose-response relationships to toxicants and integrating environmental factors across a defined landscape. In this thesis, I contributed new knowledge to the field of landscape ecotoxicology by adapting analytical methods to assess spatial patterns of chemical exposure among different wildlife keystone species, quantify the relationships between contaminant sources and exposures, and quantify dose-response relationships across large landscapes. Currently, there are few landscape ecotoxicology tools available for quantifying geospatial patterns of environmental toxicology data. To address this gap, I adapted spatial and statistical methods and demonstrated how they can be used to 1) integrate data and assess spatial patterns of contaminant exposure; 2) assess spatial patterns of exposure to complex mixtures; and 3) examine dose-response patterns across landscapes. I developed fur Hg as a biomarker medium as a non-invasive biomonitoring tool in river otter (Lontra canadensis) and mink (Neovison vison) by developing conversion factors that can be used to estimate internal organ Hg from fur Hg, using a meta-regression approach. Based on these results, I suggest that the fur Hg screening guideline be reduced from 20 ug/g to 15 ug/g to be more conservative. I also quantified how the distribution of fur Hg changes across the pelt of river otters. Results from this study indicate that topcoat should be used for biomonitoring as it is less variable than the undercoat and samples should be taken from the forebody (head and legs) for the most accurate organ Hg estimation. Using biomarkers of exposure, I quantified the relationship between sources of Hg and factors that promote Hg bioaccumulation with dietary Hg from stomach contents and fur Hg to establish fur as a proxy for bioavailability of environmental Hg. I also assessed spatial dose-response patterns between fur Hg and fur cortisol using a geographically weighted regression (GWR). Based on these results I use my proposed fur screening guideline of 15 µg/g to categorize fur Hg exposures and demonstrate that at low exposures (<15 µg/g) in fur, Hg has a positive relationship with cortisol. Conversely, at high exposures (>15 µg/g) in fur, Hg has a negative relationship with cortisol. This research provides a field example of heterogeneous dose-response relationships. Finally, I assessed spatial patterns of complex metal exposures in a variety of biomonitoring datasets. I used normalization and transformation techniques to effectively combine datasets comprised of different species and life stages. I then used a spatial principal components analysis (sPCA) to exemplify clusters of complex exposures associated with oil and gas development in regions of Alberta, Canada. These advancements in the field of landscape ecotoxicology will help advance evidence-based long-term ecological monitoring programs.

All organisms studied so far respond to heat shock by inducing the synthesis of a number of proteins called heat shock proteins(LISPs). This universal response can also be induced by a variety of stressors, including heavy metal ions and organic and organo-metallic compounds. As a result, the stress response has recently attracted the attention of ecotoxicologists for use in environmental biomonitoring. In the present study, we have investigated the stress responses of two different organisms ; namely the free-living soil nematode Caenorhiabdities elegans(both wild-type and transgenic strains) and the freshwater crustacea Asellus aquazicus. We have also explored the possible use of these model systems in environmental monitoring using different techniques which include metabolic labelling with subsequent one-dimensional electrophoresis and autoradiography, and one- or two-dimensional western blotting using antibodies specific to stress protein 70. The study with A. aquaticus shows that this organism exhibits a classical stress response. The exposure of asellids to heat shock-treatment (26°C ; 13°C above the standard maintenance temperature) or to sublethal concentrations of metal ions (Cd++ and Cu++) resulted in the induction of at least 5 putative HSPs which belong to several major HSP families (HSP100, HSP90 and possibly HSP60). An increase in the synthesis of smaller sizes of polypeptides (25-35 kD) should be also noted. Moreover, the time-course of heat versus heavy metal stress-response in this organism suggests that the pattern of stress-protein synthesis changes considerably with increasing exposure time ; notably the response to heat is more transient than that to heavy metals. However, HSP70 does not appear to be the major stress protein induced in this organism. The presence of low molecular weight (LMW) proteins which react with anti-HSP70 antibodies and the apparent deficiency of classical 70 kD stress proteins in A. aquaticus, both suggest that HSP70s in this organism are for some reason prone to degradation. In the nematode C .elegans, shifting the culture temperature from 20°C to 34°C induces the synthesis of a set of HSPs corresponding to the HSP90, HSP70 and small HSP families. There are at least nine members of the hsp7O multigene family in C. elegans ; some members are expressed constitutively while others are stress inducible. W e have studied the effects of heat and heavy metal (cadmium) stress on the expression patterns of the HSP70 protein family in the nematodes by one- and two-dimensional Western blotting using a monoclonal anti-HSP70 antibody that recognises a conserved epitope shared by most HSP70 family members. Constitutive C. elegans HSP70s (expressed at 20°C) are almost undetectable on one-dimensional immunoblots, but chemiluminescent probing of two-dimensional blots reveals a complex pattern of several HSP70s pots .Mild heats hock at 31° C induces a doublet HSP70 band on one-dimensional blots, of which the heavier component (75 kD) is more prominent than the lighter (73 kD). On two-dimensional blots, this pattern is shown to be more complex with a prominent 75 kD spot newly induced and several other spots intensified. Severe heat shock at 34°C strongly induces both 75 and 73 kD bands on one-dimensional blots; two dimensional analysis reveals a series of novel and/or elevated 73 and 75 kD spots. Treatment with cadmium( 16 ppm) at 31° C gives a different pattern of spots as compared with 31 °C alone ; several spots show enhanced while some are newly expressed, and not all of these are present at 34°C. These results indicate that related members of the HSP70 protein family in C. elegans are independently regulated in response to different forms of stress. The possible significance of these findings is discussed in relation to the possible use of stress responses s environmental biomonitors. We have also utilised a stress-inducible C. elegans strain (CB4027) for monitoring environmental contamination. This transgenic strain carries integrated copies of the Drosophila hsp70 promoter fused to an E.coli lacZ reporter gene. When exposed to heat shock or to several environmentally relevant stressors, the transgenic strain expresses the reporter product, 3-galactosidase, which can easily be quantified or localised in situ in stained worms or on Western blots (apparently enzymatically active as a 170 kD form). We have exposed transgenic worms to a variety of toxicants at an elevated temperature (32°C) just below that required for heat shock (34°C), in order to obtain optimal transgene induction. Exposure of nematodes to several heavy metals (e. g. Cd+, Hg++, Zn+, Sn++, Mn++ and Ag+), organometallic toxicants (tributyltin) or organic pollutants (lindane) induces ß-galactosidase expression in a dose-dependent manner. Cadmium is found to be by far the strongest inducer of transgene activity amongst the agents tested, although tributyltin is an effective inducer at ppb levels. The effects of mixtures of divalent metal ions (Cd++/Ca++, Cd++/Zn++ and Cd++/Hg++) on ß-galactosidase expression have been also investigated. All three divalent ions tested in combination with cadmium significantly inhibit cadmium-induced transgene activity in comparison to cadmium alone. In the case of Cd++/C++ mixtures, a marked inhibition of Cd++ accumulation by worm tissues has also been demonstrated, directly related to the Ca++ concentration. These effects may represent competition for metal-ion uptake through calcium channels. Our results show that this transgenic system works well within strictly defined assay conditions, and can detect clear responses over a 7h exposure period to environmentally relevant toxicants at sublethal concentrations well below the 24 or 48h LCSO values. However, there is a need for careful characterisation and containment of any transgenic organism if it is to be used as environmental monitoring tool.

The use of nanoscale materials is growing exponentially, but there are also concerns about the environmental hazard to aquatic biota. Metal-containing engineered nanoparticles (NPs) are an important group of these new materials, and whilst there are undoubtedly a plethora of beneficial uses for these NPs, it is essential that an appropriate risk assessment is carried out in order to protect the environment and human health, with the consumption of contaminated fish a distinct possibility. The current study aimed to assess the bioavailability, uptake and toxicological effects of two metal-NPs (TiO2 NPs and Cu-NPs) to fish from both dietary and waterborne exposure routes and where appropriate compare them to their bulk counterpart. Whole body system effects were assessed along with the influences of the life stage of exposed fish and abiotic factors on toxicity. A technique to improve the quantification of Ti from TiO2 NPs in fish tissue was also developed. Effects from exposure to dietary TiO2 NPs manifested similarly to traditional dietary metal exposure, with no reduction in growth, but some sublethal affects. Exposure to waterborne Cu-NPs showed that rainbow trout were more acutely sensitive to CuSO4 than the NPs, but that despite limited uptake several body systems were affected (most notably ionoregulation). Larvae were more sensitive to CuSO4 than Cu-NPs, but no differences were seen with embryos, whilst larvae were more sensitive than embryos. Abiotic factors did have an effect on acute Cu-NP toxicity, though not always in a predictable manner, with some effects more pronounced than with CuSO4. Overall, it appears that metal-NPs are not as acutely toxic as their bulk counterparts, but sublethal effects, were routinely observed. As TiO2 NPs appear more toxic than its bulk counterpart, current legislation governing safe environmental limits may have to be adjusted, though the situation with Cu-NPs isn’t as clear and further investigation is required. However, the risk of human exposure via the consumption of NP contaminated fish fillets is extremely low.

The wood mouse (Apodemus sylvaticus) is a common and widespread species in Europe and therefore often used as a focal species in the risk assessments of pesticides. Recent years have seen a growing interest in the use of ecological models in ecological risk assessments. The purpose of this PhD project was to construct and apply population models of the wood mouse to support risk assessment of pesticides. In the thesis I first reviewed the current practice of risk assessment and the weaknesses and the advantages of ecological models, in particular individual-based models. Following a modelling cycle, the PhD project started with a literature review of the basic ecology of the wood mouse and relevant information for modelling purposes. A conceptual model was then constructed, based on which an individual-based model was developed. The model was fully described and its ecological credibility was evaluated by the "pattern-oriented" validation method. The model was then used to link the spatial choice of wood mice and their potential exposure to pesticide application in the field. Analyses of the model showed that when the population density reached a high level in the off-crop hedgerows, which act as the source habitat for wood mice, the surplus were driven to the crop fields and thus have a high risk of exposure if pesticides are applied. The model was further developed by incorporating a sub module of toxicokinetic processes which represented exposure as internal concentration instead of ingested dose. The interactions between the spatial patterns of foraging provided by the individual-based model and the temporal patterns of absorption and elimination provided by the toxicokinetic model were explored. The combined model showed little further reduction of risk than either of the two models respectively alone. It suggested that such a combined approach is most likely to bring added value when the spatio-temporal patterns of feeding are at the intermediate level, i.e. when mice divide their foraging between exposed crop and unexposed areas. I then used the model to explore the population-level sensitivity in terms of both population size and population growth rate, to detrimental effects on individual-level life-history traits. The results showed the two indices had different sensitivities to changes in life-history traits. Thus, endpoint from the models used in risk assessments should take into account whether the protection goal is to ensure that the population remains stable or that it grows. The overall high level of sensitivity to survival also indicated that protection should be more focused on acute effects than chronic effects. Finally I summarised the major findings in the thesis and discussed the need for future research, as well as the lessons learnt about conventional risk assessment approach and the opportunities ecological modelling brings for a more realistic and ecologically relevant risk assessment for pesticides.

Mestrado em Biologia Molecular e Celular
O organismo teste Enchytraeus albidus é frequentemente utilizado na avaliação de risco ambiental em solos. Existem testes biológicos padronizados para este organismo (ISO 16387, 2003; OECD 202, 2004) para a determinação de parâmetros tradicionais como a sobrevivência e reprodução (6 semanas). Mais recentemente, têm sido realizados testes de “evitamento” (48h), utilizando a capacidade destes organismos evitarem solos contaminados. Embora se conheçam os efeitos de vários tipos de stress nos níveis crónico e agudo, sabe-se muito pouco sobre os efeitos ao nível subcelular e molecular. O principal objectivo deste trabalho experimental consistiu na determinação de marcadores de stress oxidativo, neuro-musculares (cholinesterases) e de expressão génica em E. albidus expostos a diferentes factores abióticos e aos químicos cobre e fenmedifam durante diferentes períodos de exposição e correlacioná-los com efeitos populacionais e comportamentais. A escolha dos químicos cobre e fenmedifam deveu-se ao facto de representarem dois grupos de stressores químicos com diferentes propriedades, presentes frequentemente nos solos, metais e herbicidas, e devido ao conhecimento prévio dos seus efeitos a nível populacional. A escolha dos parâmetros moleculares e sub-celulares utilizados neste estudo deveu-se às seguintes razões: os biomarcadores de stress oxidativo determinados consistem em defesas antioxidantes que destoxificam espécies reactivas de oxigénio e produtos pró-oxidantes, assim como indicadores de dano oxidativo; as colinesterases são biomarcadores enzimáticos relacionados com funções neuronais e musculares e, em particular, as acetilcolinesterases desempenham um papel central no mecanismo de neurotransmissão; a análise de microarrays permite medir directamente as alterações na regulação da transcrição de centenas de genes simultaneamente. Os resultados obtidos mostram que os biomarcadores de stress oxidativo e neuro-musculares determinados são significativamente alterados em E. albidus exposto a diferentes factores abióticos e aos químicos, nos diferentes tempos de exposição. As hibridações dos microarrays revelaram genes diferentemente expressos nas condições testadas. Os resultados obtidos para todos os parâmetros determinados mostram claramente que os solos naturais são mais indicados para testes com E. albidus. Os resultados sugerem que as alterações bioquímicas e moleculares observadas em todas as condições podem estar associadas aos efeitos observados em termos de sobrevivência, reprodução e evitamento. Esta tese demonstra a importância da utilização de diferentes ferramentas em ecotoxicologia terrestre e aponta para o uso de novos parâmetros para uma determinação de stress em ecotoxicologia de solos mais rápida e eficaz. ABSTRACT: Traditionally, in soil Environmental Risk Assessment (ERA), a set of standardized biological tests are available and commonly performed with Enchytraeus albidus (ISO No 16387, 2003; OECD 202, 2004) for the determination of survival and reproduction endpoints (6 weeks test). More recently this organisms’ ability to avoid contaminated soil has been used to determine avoidance behaviour (48h test). Though stress effects are known at acute and chronic levels for a wide range of stress conditions, very little is known about the sub-cellular and molecular effects in these organisms. The main objective of this work was to assess oxidative stress, neuro-muscular (cholinesterases) and gene expression markers in E. albidus exposed to different soil properties and the chemicals copper and phenmedipham during different exposure periods and to link them with population and behaviour level effects. Copper and phenmedipham were chosen for this study because they are representative of two different groups of chemical stressors with different properties, present commonly in soils, metals and herbicides and due to previous knowledge of effects at population level. The endpoints assessed were chosen for the following reasons: the oxidative stress biomarkers determined consist of antioxidant defences that detoxify reactive oxygen species and prooxidant products, as well as indicators of oxidative stress damage; cholinesterases are biomarker enzymes related with neural and muscular functions and acetylcholinesterase, in particular, plays a central role in the mechanism of neurotransmission; the microarray analysis allows the direct measurement of changes in the transcriptional regulation of many thousands of genes simultaneously. The results showed that the oxidative stress and neuro-muscular biomarkers determined were significantly affected in E. albidus exposed to different soil properties and to the chemicals at the different exposure periods. The microarray hybridizations revealed differentially expressed genes for these stress conditions. The results obtained for all endpoints clearly show that natural soils are more appropriate for ecotoxicological testing with E. albidus. The results suggest that these biochemical and molecular alterations induced by different soil properties and chemicals might be associated with the effects observed on E. albidus survival, reproduction and avoidance behaviour. This thesis shows the importance of using different tools in soil ecotoxicology and points to the use of new endpoints for a quicker stress assessment in soil ecotoxicology.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Dentre os agrotóxicos comumente utilizados na piscicultura para controle de ectoparasitos e insetos aquáticos, destacam-se o diflubenzuron (Dimilin®) e o teflubenzuron (Nomolt®), inseticidas com ação reguladora do crescimento. Na aqüicultura, o tratamento com inseticidas pode ser realizado com a aplicação direta na água, ou administração na ração dos animais. Apesar de muito utilizados, poucos são os trabalhos sobre ecotoxicologia, comportamento e bioacumulação do diflubenzuron (DFB) e teflubenzuron (TFB) na água e seus efeitos em organismos aquáticos de clima tropical. Os objetivos deste trabalho foram: estimar a toxicidade (CL(50-96h)) do DFB e TFB para o pacu (Piaractus mesopotamicus), calcular o risco alimentar para o consumo humano, determinar resíduos de DFB e TFB na água, sedimento e músculo de pacus, após a exposição aos inseticidas na água ou na ração dos peixes e analisar a atividade hepática das enzimas glutationa-S-transefrase (GST) e catalase (CAT) em pacus alimentados com ração acrescida de DFB ou TFB. A CL50;96h do DFB para o pacu foi superior a 1200 mg.L-1 e a do TFB, superior a 1000 mg.L-1. Os resíduos de DFB foram detectáveis até 7 dias após a aplicação (com 18,3% de resíduo) e para o TFB, foram detectáveis ate o terceiro dia (8% de resíduo). No sedimento, as maiores concentrações de DFB e TFB foram observadas nas primeiras 24 h e em todos os períodos amostrados foram detectados resíduos dos inseticidas. Nos filés dos pacus foram detectados resíduos dos inseticidas após a alimentação com a ração tratada e após aplicação de DFB e TFB na água. A ingestão diária estimada (IDE) de DFB e TFB encontra-se acima dos valores de ingestão diária aceitável (IDA), mesmo para as amostras com menores valores de resíduos detectados nos filés do pacu. Em relação aos biomarcadores bioquímicos...
Diflubenzuron and teflubenzuron are growth regulator inhibitors insecticides, used to control ectoparasites in fish and aquatic insects. In aquaculture these pesticides can be injected directly in the water or in the fish food. Besides their large utilization, there are few works about the effects of these insecticides on the fish metabolism and biomagfication, especially in tropical aquatic organisms. This study aims to: evaluate the acute toxicities (LC(50-96h)) in pacu (Piaractus mesopotamicus), measure the risk food for human consumption, determinate residues of DFB and TFB in water, sediment and muscle of pacus after the management of the insect-powders on water or in the fish food and to analyze the hepatical activity of the enzymes catalase (CAT) and glutathione-S-transferase (GST) in fishes fed with ration plus DFB or TFB. The LC((50- 96h)) of DFB and TFB for the pacu was higher than 1.200 mg.L-1 and 1.000 mg.L-1, respectively. The residues TFB were detected until the thirth day (8% of residue) and DFB until seventh day after application in the water (18,3% of residue). In the sediment, the highest concentrations of DFB and TFB had been observed in the first 24 h and in all the samplings there were . In muscle of pacus, residues of the insecticides had been detected after the feeding with the ration treated and after application to DFB and TFB in the water. The daily ingestion estimate (DIE) of DFB and TFB meets above of the values of acceptable daily ingestion (DAI), exactly for the samples with lesser values of residues detected in filés of pacu. For the enzymes the CAT activity was not affected by both pesticides, but the GST activity was reduced five days after the feeding. Besides the importance of the evaluation of the toxicity of DFB and TFB for the fish, the determination of residues and the alimentary risk, it can subsidize the determination of measures of sanitary... (Complete abstract click electronic access below)

O presente estudo teve por objetivo avaliar a qualidade dos compartimentos água e sedimento do rio Monjolinho, localizado na sub-bacia hidrográfica do Alto Jacaré-Guaçu, na região centro-norte do estado de São Paulo. Para tanto, foram realizadas análises limnológicas e ecotoxicológicas em seis estações de amostragem ao longo do rio, durante cinco campanhas de coleta (julho, outubro de 2003 e janeiro, abril, julho de 2004). Os ensaios de toxicidade com amostras ambientais foram realizados com os cladóceros planctônicos Daphnia similis e Ceriodaphnia dubia como organismos-teste. Adicionalmente, foram realizados testes de sensibilidade aos metais cádmio e cobre utilizando Daphnia similis e Ceriodaphnia dubia, com o intuito de verificar se as concentrações permitidas pelo CONAMA 357/05 para estes metais são adequadas para a proteção da vida aquática em rios de classe 2. Os resultados obtidos quanto as análises limnológicas apontaram três trechos distintos do rio Monjolinho considerando-se a qualidade da água: a) trecho não poluído a ligeiramente poluído (estação 1); b) trecho poluído (estações 2, 3, e 4) e c) trecho fortemente poluído (estações 5 e 6). Os ensaios de toxicidade com amostras ambientais indicaram uma maior toxicidade do sedimento do que da água, particularmente nos períodos de outubro de 2003, janeiro e abril de 2004. Neste contexto, os testes com sedimento foram mais adequados para uma avaliação da real condição de toxicidade do sistema. Quanto aos testes de sensibilidade, os resultados para cádmio foram superiores ao limite estabelecido pela resolução 357 do CONAMA/05 (1 μg/L), demonstrando adequabilidade do valor estabelecido para a manutenção da vida aquática em rios de classe 2, no que se refere ao efeito isolado deste metal. Já os valores encontrados nos testes de sensibilidade ao cobre foram inferiores ao limite para cobre dissolvido estabelecido pelo CONAMA 357/05 (9 μg/L), mostrando que o valor definido para a manutenção da biota aquática ainda não está adequado, levando-se em consideração o efeito isolado deste contaminante.
The present study aimed to evaluate water and sediment quality of Monjolinho river, located at High Jacaré-Guaçu water basin, in the center of São Paulo state. Limnological and ecotoxicological analyses were carried out in six sampling stations along the river, during five sampling periods (July, October of 2003 and January, April, July of 2004). Bioassays with environmental samples were carried out with planktonic crustaceans, Daphnia similis and Ceriodaphnia dubia, as test organisms. Additionally, tests of sensibility to cadmium and copper were carried out with the same species to verify if their allowed concentrations by CONAMA 357/05 are appropriate for the protection of the aquatic life in class 2 rivers. The results of the limnological analyses indicated three distinct regions of water quality: a) not polluted to slightly polluted region (station 1); b) polluted region (stations 2, 3, and 4) and c) highly polluted region (stations 5 and 6). The bioassays with environmental samples indicated greater sediment toxicity than water toxicity, particularly in October of 2003, January and April of 2004. The tests of sensibility to cadmium, showed higher values than the limit established by resolution 357 of CONAMA/05 for this metal (1 μg/L), demonstrating adequacy of the established value for the maintenance of the aquatic life in class 2 waters, concerning the isolated effect of cadmium. However, the tests of sensibility to copper, presented lower values than its limit established by CONAMA 357/05 (9 μg/L), showing that the determined value of copper for the maintenance of the aquatic biota is still not adequate, considering the isolated effect of this pollutant.

Entre os diversos impactos nos ecossistemas aquáticos, a construção de reservatórios talvez represente um dos principais agentes modificadores, inserindo alterações físicas, químicas e biológicas nos sistemas antes e após a sua construção. No Brasil, tal impacto também é singular, verificando-se o aproveitamento dos mesmos para a construção de barragens, principalmente as construídas em cascatas (sistemas Paranapanema, Tietê e São Francisco por exemplo), sendo que, neste padrão de construção, espera-se, na maioria das vezes, um efeito crescente do controle da qualidade da água, com o primeiro reservatório mais eutrófico que os demais por sua capacidade de reter materiais provenientes dos principais tributários. No estado de São Paulo, na região Sudeste, destacam-se os reservatórios de Barra Bonita, Bariri, Ibitinga, Promissão, Nova Avanhandava, e Três Irmãos. Visando avaliar os impactos e contribuir com informações para monitorar as condições ambientais desses reservatórios, foram realizadas coletas de água e sedimento em quatro períodos distintos (outubro/99, fevereiro, maio e julho/00), em 15 estações de coleta, incluindo estações nos rios Tietê e Piracicaba e à montante e à jusante da barragem de cada um dos seis reservatórios, bem como no rio Bauru (um afluente bastante impactado de toxicidade com amostras de água e sedimento, utilizando-se Daphnia similis (em bioensaios de toxicidade aguda) e Ceriophnia dubia (em bioensaios de toxicidade crônica), análises de metais totais na água e biodisponíveis no sedimento, além do monitoramento de variáveis limnológicas (temperatura, pH, condutividade, oxigênio, dissolvido, material em suspensão, clorofila-a, nutrientes totais e dissolvidos). Os resultados obtidos para as variáveis limnológicas, de modo geral, revelaram diferenças na qualidade de água em relação às escalas espacial e temporal (efeitos de fatores climatológicos e hidrodinâmicos-vazão e tempo ) de retenção de água), com elevado aporte de nutrientes pelos rios Tietê e Piracicaba, além da incorporação, da sedimentação e da redução de materiais no reservatório de Barra Bonita, promovendo a melhoria da qualidade da água nos demais reservatórios, caracterizando assim, as primeiras estações como eutróficas e as demais como mesotróficas e oligotróficas. Concentrações de metais totais de água acima do limite estabelecido pela resolução CONAMA 20/1986 foram encontradas, bem como um aumento da fração biodisponível. Os ensaios revelaram toxicidade aguda para Dapnhia similis somente nos reservatórios localizados à justante da barragem de Barra Bonita, o que não seria inicialmente esperado. Tal fato pode estar relacionado às entradas difusas de poluição ou a presença de metais no sistema Tietê, os quais, no primeiro reservatório, estariam mais presentes na forma retida em função de sua maior incorporação (pela maior biomassa e pelo maior tempo de retenção), não causando toxicidade aguda aos organismos devido à parte dissolvida não ser tão elevada. Os resultados de toxicidade crônica, porém, configuraram um padrão diferente, verificando-se toxicidade em ordem decrescente (de Barra Bonita à Três Irmãos), na maioria dos períodos de amostragens, o que demonstra o estado de degradação ambiental nos reservatórios e a necessidade de estudos mais integrados para uma melhor avaliação ambiental.
Among the several impacts in the aquatic ecosystems, perhaps the construction of reservoirs represents one of the principal modifier agents, inserting physical, chemical and biological alterations in the systems before and after its construction. In Brazil, such impact is also singular, having verified this application for the construction of reservoirs, mainly the ones built in cascades (Paranapanema, Tietê and São Francisco system, for example) and, in that construction pattern, in most cases, a growing effect of the control of the quality of the water is anticipated, with the first reservoir being more eutrophic than the others for its capacity to retain materials that come from the principal tributary. In the southeast of São Paulo States, Barra Bonita, Bariri, Ibitinga, Promissão, Nova Avanhandava and Três Irmãos reservoirs stand out. Seeking to evaluate the impacts and contributing with information to evaluate the environmental conditions of those reservoirs, collections of water and sediment were accomplished in four different periods (October/99, February, May and July/00), in 15 sampling stations, including stations in the Tietê and Piracicaba Rivers and at the specified place after and before the line of the dam, from each one of the six reservoirs, as well as the Bauru River (a very impacted tributary of the Bariri Reservoir ). Toxicity bioassays were accomplished with samples of water and sediment using Daphnia similis (in acute toxicity bioassays) and Ceriodaphnia dubia (in chronic toxicity bioassays), analyses of total metals in the water and bioavailable in the sediment, besides the monitoring of limnological variables (temperature, pH, conductivity, dissolved oxygen, material in suspension, chlorophyll-a, total nutrients and dissolved). The results obtained for the limnological variables, in general, revealed differences in the quality of the water in relation to the spatial and temporary scales (effects of climatological factors and hydrodynamic- flowing out and residence time) with high contribution of nutrients from the Tietê and Piracicaba Rivers, besides the incorporation, the sedimentation and the reduction of materials in the Barra Bonita Reservoir, promoting the improvement of the water quality in the other reservoirs, characterizing, in this way, the first stations as eutrophic and the others as mesotrophic and oligotrophic. Concentrations of total metals in the water above the established limit for the resolution CONAMA 20/1986 were found out, and an increase of the fraction bioavailable of metals was verified as well. The bioassays only revealed acute toxicity for Daphnia similis to the points after the Barra Bonita Reservoir, something not initially awaited for. Such fact can be related to the diffuse entrances of pollution or to the presence of metals in the Tietê System, which, in the first reservoir, would be more present in the form retained in function of their largest incorporation (for the largest biomass and for the largest residence time), not causing acute toxicity to the organisms due to the dissolved part not to being so high. However, the results of chronic toxicity, configured a different partem, being verified toxicity in decreasing order (from Barra Bonita to Três Irmãos), during most of the samplings periods, which demonstrates the state of environmental degradation in the reservoirs and the need for more integrated studies for a better environmental evaluation.

A intervenção das atividades antrópicas ao meio ambiente tem repercutido na estrutura e funções dos ecossistemas, sendo que o uso de aditivos químicos, como agrotóxicos e fertilizantes, destacam-se pela parcela de responsabilidade que assumem diante desses desequilíbrios. O agrotóxico Vertimec® 18CE, cuja toxicidade para diversas taxóns já é conhecida, é amplamente utilizado em lavouras agrícolas, e têm-se conhecimento a respeito das intensas práticas de manuseio, muitas vezes irregulares, o que pode fazer com que concentrações muito maiores do que as previstas atinjam o ambiente aquático. Um dos grupos que nos últimos anos vem se destacando pelo declínio crescente associado ao uso destes produtos são os anfíbios. Os anfíbios possuem papel central na cadeia alimentar, e muitos estudos relatam o desequilíbrio ecológico causado pelo declínio desse grupo em relação a adição de contaminantes, incluindo os agrotóxicos. No presente estudo foram desenvolvidos testes laboratoriais a fim de determinar o estágio dos girinos de Lithobates catesbeianus mais suscetível à intervenção do agrotóxico e à concentração capaz de causar mortalidade em 50% dos indivíduos (CL50, 96h). A partir desses resultados, experimentos in situ (mesocosmos) com girinos no estágio mais avançado (25 da tabela de Gosner) foram desenvolvidos com o intuito de comparar as diferenças de toxicidade do Vertimec® 18CE quando manipulado em campo e laboratório, e quais os efeitos causados por uma única aplicação do produto e por aplicações múltiplas. Os testes laboratoriais demonstraram que os girinos em estágios mais precoce (21G) foram mais sensíveis ao agrotóxico Vertimec® 18CE do que àqueles mais avançados (25G). O experimento em campo indicou que a CL50, 96h para L. catesbeianus (25G) foi muito mais agressiva quando próxima às condições naturais, causando a mortalidade de todos os indivíduos no mesmo estágio de desenvolvimento em um período inferior a 24 horas. Entretanto, a concentração inferior à CL50, 96h (0,002 ml de Vertimec.L-1) quando ministrada em campo, assim como no laboratório, não causou mortalidade, mas implicou em atraso no desenvolvimento e maior período larval do que àqueles organismos que não estiveram em contato com o agrotóxico, já que atingiu os recursos disponíveis, promovendo um efeito indireto no sucesso dessa espécie. Não foi possível observar diferença entre as aplicações única e múltipla do Vertimec® 18CE, em decorrência da morte dos girinos do tratamento em dosagem múltipla, porém observou-se desequilíbrio das condições limnológicas do sistema ao sofrer nova intervenção do Vertimec® 18CE. Diante dos resultados obtidos é possível concluir que o Vertimec® 18CE é capaz de afetar diretamente (mortalidade, estágios de desenvolvimento e comportamento) e indiretamente (redução de recursos alimentares) a população de Lithobates catesbeianus, contribuindo para o declínio de anfíbios nos sistemas naturais.
The intervention of anthropogenic activities to the environment has reflected in the structure and functions of ecosystems and the use of chemical additives, such as pesticides and fertilizers, has contributed for the disequilibrium. The Vertimec® 18EC, whose toxicity is described to different taxa, is widely used in agricultural crops, and it has been known about the intense handling practices, often irregular, which can reach higher concentrations than those expected in the aquatic environment. One group that has been highlighted in recent years by the increasing decline associated with the use of these products is the amphibians. Amphibians plays a central role in the food chain and several studies have related the ecological disequilibrium caused by the decline of this group due to the addition of contaminants, including pesticides. In this study, laboratory bioassays were developed to determine the more susceptible stage and the LC50,96h of Lithobates catesbeianus to Vertimec® 18EC. From these results, in situ experiments (mesocosms) were conducted with tadpoles in more advanced stage (25 of Gosner table) in order to compare the differences in toxicity of Vertimec® 18EC when manipulated both in field and in laboratory. Also, the effects caused by a single and multiple application of the pesticides were evaluated. The laboratorial results showed that earlier stages (21G) were more sensitive than more developing tadpoles (25G), and the effects of the pesticide were more aggressive under natural conditions (mesocoms), with 100% of mortality before 24 hours, at the same developmental stage. The results of laboratorial and in situ bioassays indicated that the lower concentration (0.002 ml Vertimec.L-1) didn\'t cause mortality, but alterations on development stage and larval period time were observed, indicating indirect effects (available resources) on the success of this species. On the other hand, no difference was found between the single and multiple applications of Vertimec® 18EC because all organisms died in the unique dosage treatment, but it was possible to verify limnological alterations after the second application of the pesticide. Considering the results obtained we conclude that Vertimec® 18EC can affect directly (mortality, development stages and behavior) and indirectly (food reduction) the population of Lithobates catesbeianus, contributing to the decline of amphibians in the natural systems.

O presente estudo avaliou os efeitos letais e subletais de dipirona sódica e paracetamol para organismos de água doce. O efeito letal foi determinado pela realização de ensaios agudos com D. similis, C. dubia, C. silvestrii e D. rerio. A influência da temperatura, tipo de água de diluição e fotoperíodo na ecotoxicidade aguda foram avaliadas. Os efeitos subletais foram determinados por meio de ensaios de embrioxicidade com D. similis (20°C), crônicos individuais e populacionais com D. similis, C. dubia e C. silvestrii. A influência da temperatura na ecotoxicidade crônica individual e populacional foi determinada. Os critérios para aceitabilidade para o controle (número de neonatas) nos ensaios populacionais com D. simlis (20 e 25°C) e C. dubia, foram estabelecidos. Nos ensaios de ecotoxicidade aguda, D. similis (20°C) foi mais sensível a dipirona sódica que a 25°C e, para paracetamol, D. similis (25°C) foi mais sensível. A água de diluição influenciou na ecotoxicidade aguda apenas do paracetamol e o fotoperíodo não influenciou na ecotoxicidade aguda de ambos os fármacos. Os valores de CL(I);96H obtidos para D. rerio foram 3670 e 590mg.L-1 para dipirona sódica e paracetamol, respectivamente. Dipirona sódica e paracetamol induziram malformações nas neonatas e embriões de D. similis e os valores de CI50 obtidos foram 21,1 e 94,00mg.L-1, respectivamente. Os valores de CI50 nos ensaios crônicos individuais com dipirona sódica para D. similis (20°C e 25°C) foram 7,53mg.L e 8,08mg.L-1, respectivamente. Para C. dubia e C. silvestrii a CI50 para ensaios crônicos individuais com dipirona sódica foram 5,38 e 3,57mg.L-1, respectivamente. Nos ensaios crônicos individuais com paracetamol, a CI50 para D. similis (20°C) foi 21,84mg.L-1 e 10,72mg.L-1 para D. similis (25°C). Para C. dubia e C. silvestrii a CI50 nos ensaios crônicos individuais com paracetamol foram 7,24 e 4,15mg.L-1, respectivamente. Como critérios de aceitabilidade para os ensaios crônicos populacionais estabeleceu-se para o controle de D. similis (20 e 25°C) e C. dubia 137, 143 e 80 neonatas, respectivamente. Os valores de CI50 nos ensaios populacionais com D. similis (20 e 25°C), C. dubia e C. silvestrii para dipirona sódica foram 8,84, 10,82, 4,68 e 2,81mg.L-1, respectivamente. Para os ensaios populacionais com paracetamol os valores de CI50 para D. similis (20 e 25°C), C. dubia e C. silvestrii foram 9,57, 10,1, 6,48 e 4,26mg.L-1, respectivamente. Os valores das concentrações que causaram ecotoxicidade aguda e crônica não são superiores as concentrações destes compostos no ambiente porém, de acordo com a classificação baseada na Diretiva Européia 93/67/EEC, estes compostos são classificados como nocivos para o ambiente.
The present study evaluated the lethal and sublethal effects of dipyrone and paracetamol to different freshwater organisms. The lethal effect was determined by acute toxicity assays with D. similis, C. dubia, C. silvestrii and Danio rerio. The influence of temperature, type of dilution water and photoperiod were evaluated in acute toxicity assays. The sublethal effects were determined by Embryotoxicity with D. similis (20°C) and chronic toxicity assays both individual and population methods with D. similis, C. dubia and C. silvestrii. The influence of temperature upon individual and population chronic ecotoxicity was also determined. The criteria for acceptability for the population control assays with D. simlis (20 and 25°C) and C. dubia were established. D.similis (20°C) was more sensitive for dipyrone that 25 °C and for paracetamol, D.similis (25°C) was more sensitive. Dilution water interfere on acute ecotoxicity paracetamol, only and the photoperiod did not influence the acute ecotoxicity of both drugs. The LC(I);96H to Danio rerio was 3670mg.L-1 and 590mg.L-1 for dipyrone and paracetamol, respectively. Dipyrone and paracetamol induced malformations in D. similis neonates and embryos. The IC50 for these assays with dipyrone were 21,1mg.L-1 and 94mg.L-1 for tests with paracetamol. The IC50 of dipyrone to D. similis (20°C) was 7,53 and 8,08 at 25°C. Values of IC50 to C. dubia and C. silvestrii on individual chronic toxicity tests with dipyrona was 5,98 and 3,57mg.L-1respectively. The IC50 of paracetamol for D. similis (20°C) was 21,84 and 10,72mg.L-1 for at 25°C in individual chronic tests. The IC50 to C. dubia and C. silvestrii in individual chronic tests with paracetamol was 7,24 and 4,15mg.L-1 respectively. As criteria of acceptability for the population chronic tests were established to control the D. similis (20 and 25°C) and C. dubia, 137, 143 and 80 respectively. The IC50 obtained to D. similis (20 and 25°C), C.dubia and C.silvestrii for Dipyrone 8,84; 10,82; 4,68 and 2,81mg.L-1 respectively. For chronic population tests with paracetamol the IC50 to D. similis with 20 and 25°C, C.dubia and C.silvestrii was 9,57; 10,1; 6,48 and 4,26mg.L-1 respectively. The concentrations that cause acute and chronic ecotoxicity on organisms in this study are higher than environmental concentrations of these compounds. However, according to the classification based on the European Directive 93/67/EEC, these compounds are hazardous to the environment.

Mestrado em Biologia Aplicada - Toxicologia e Ecotoxicologia
The growing interest in large-scale biochar application to soils, either for improving crop yield, as a tool for carbon sequestration or for replacing inorganic fertilizers, highlights the urgency for an effective evaluation of potential negative effects on soil biota, and the soil processes and functions that they mediate. While the field of biochar ecotoxicology is becoming more important, studies that focus on biochar effects on selected terrestrial species remain scattered and lacking environmental, ecological and practical relevance. This study focused on adapting and optimising a range of ecotoxicological and ecological tools (that have been established or standardized for contaminated soils), in order to test their suitability for evaluating the toxic potential of biochar-amended soils. Firstly, it was tested the suitability of invertebrate avoidance behaviour assays (using earthworms, collembolans and isopods) to assess the potential toxicity of soils enriched with wood-biochar, alone and in combination with traditional compost, over a 5 month period, in a real field trial at the Estação Vitivinícola da Bairrada. Nevertheless, there is increasing need for more representative conditions in testing, that account for longer study durations and greater environmental (e.g. soil moisture, temperature) and ecological variation (e.g. interactions among co-existing test organisms and their vertical distribution in soil). The second part of this work therefore, focused on optimizing a methodology using Small-scale Terrestrial Ecosystem Models (STEMs) containing earthworms and plants, for higher-tier studying of the potential ecological impact of manure-biochar on terrestrial ecosystems, at reported application rates. Results suggest that avoidance behaviour responses using representative invertebrates can be used for evaluating the impact of wood-biochar on soil biota, in a real case application, over 5 months. This can have implications for complementing other strategies for characterizing or managing biochar field applications, such as help with the choice of biochar type and safe application rates. Further, the use of STEMs containing earthworms and turnip seeds exposed to manure biochar under more representative conditions, has shown to be more conservative when compared to the standardised single species test and therefore, may be adequate as higher-tier evaluation of the toxic potential of soils with manure biochar. Studies like the present can be an important contribution for establishing suitable biochar risk assessment methodologies and support both, on-going development of biochar standardization schemes and development of adequate biochar regulations.
O crescente interesse da aplicação de biochar em grande escala em solos, seja para melhorar o rendimento das culturas, como uma ferramenta para o sequestro de carbono ou para a substituição de fertilizantes inorgânicos, realça a urgência de uma avaliação eficaz dos potenciais efeitos negativos sobre a biota do solo, assim como nos processos e funções do solo que medeiam. Enquanto a ecotoxicologia do biochar está a tornar-se progressivamente mais importante, os estudos que se concentram nos efeitos do biochar em espécies terrestres representativas são poucos e ainda sem relevância prática a nível ambiental e ecológico. Este estudo centrou-se na adaptação e otimização de uma série de ferramentas ecotoxicológicos e ecológicos (que foram criados ou padronizado para solos contaminados), a fim de testar a sua adequabilidade na avaliação do potencial tóxico de solo alterados com biochar. Em primeiro lugar, foi testada a adequabilidade de testes de evitamento em invertebrados (utilizando minhocas, colêmbolos e isópodes) na avaliação do potencial toxico de solos enriquecidos com biochar de origem vegetal, quer isoladamente quer em combinação com adubo tradicional, ao longo de um período de cinco meses, num teste de campo realizado na Estação Vitivinícola da Bairrada. No entanto, existe uma crescente necessidade de mais condições de representatividade em testes, que tenham em conta períodos de tempo mais longos e maior variabilidade ambiental (por exemplo, a umidade do solo, temperatura) e ecológica (ex: interacções entre organismos teste coexistentes e sua distribuição vertical no solo). Desta forma, a segunda parte deste trabalho foca-se na optimização de uma metodologia, utilizando um modelo de ecossistema terrestre de pequena escala contendo minhocas e plantas, para se obter um estudo superior e mais representativo dos impactos potenciais de biochar produzido através de estrume em ecossistemas terrestres a taxas de aplicação propostas. Os resultados sugerem que as respostas de comportamento de evitamento usando organismos invertebrados representativos podem ser usadas para avaliar o impacto do biochar de origem vegetal na biota do solo, num cenário real, ao longo de 5 meses. Isto pode ter implicações para complementar outras estratégias que tenham em vista a caracterização ou gestão das concentrações de referência de biochar a aplicar em solos, assim como na ajuda a escolher o tipo de biochar e taxas de aplicação de segurança. Além disso, a utilização de STEMs contendo minhocas e sementes de nabo expostos ao biochar produzido através de estrume sob condições mais representativas mostrou ser mais conservador quando comparada com os testes normalizados de apenas um teste e, portanto, pode ser adequado numa avaliação de nível superior do potencial tóxico de solos com biochar feito a partir de estrume. Estudos como o presente poderão ser uma contribuição importante para o estabelecimento de metodologias adequadas de avaliação de risco de biochar e assim apoiar tanto no desenvolvimento contínuo dos sistemas de normalização de biochar e também no desenvolvimento de regulamentos biochar adequados.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
In aquatic environments, phytoplankton consists mostly of photosynthetic microorganisms that serve as the basis of food chains. However, besides photoautotrophy, it is widely reported in the literature that many microalgae can take up dissolved organic matter present in the environment concomitantly with the photosynthesis, a metabolic pathway known as mixotrophy. Little is known about the ecophysiology of mixotrophy in microalgae, and almost all studies are focused on the use of this metabolic pathway to increase the production of algal biomass and stimulate the production of specific biomolecules. Another important issue, considering the current anthropic activity, is that most of the contaminants eliminated in aquatic environments, such as metals and nanoparticles, affect the phytoplankton. However, so far, no ecotoxicological study involving mixotrophic metabolism was found in the literature. To better understand mixotrophy in microalgae, this work chose the chlorophycean freshwater Chlorella sorokiniana as test organism. We divided the study into two parts: the first focused on the physiological/biotechnological interest through the study of growth, photosynthetic parameters, changes in cellular volume, and production of biomolecules (proteins, carbohydrates and lipids); the second part focused on the ecotoxicological effects of cadmium (Cd) and titanium dioxide nanoparticles (NPs-TiO2). To stimulate mixotrophy, glucose (1.0 g.L-1 or 5 x 10-3 mol.L-1) was used as the organic carbon source. The results showed that during mixotrophy, the microalga C. sorokiniana presented higher population growth and production of biomolecules, such as chlorophyll a and lipids, when compared to photoautotrophic cultures. It was also observed that the photosynthetic parameters were affected by mixotrophy, although they did not interfere in the growth of the microalga, and that the presence of bacteria in the cultures acted as a stimulant factor in the production of algal biomass. Regarding the ecotoxicological effects of contaminants, microalgae in mixotrophy were more resistant to both Cd and NPs-TiO2 than those in photoautotrophy, but with changes in the biochemical composition what can affected the energy transfer in the environment. In general, we can conclude that mixotrophy should be considered in studies with phytoplankton, since aquatic environments present a myriad of organic carbon that can be used by these microorganisms. As general conclusions, we can mention that organic carbon acted as an extra source of structural carbon and energy for microalgae, not necessarily relying solely on photosynthesis to survive, so stimulating the growth and production of biomolecules of biotechnological interest, and increased cellular viability in environments contaminated with metals and nanoparticles. This study is a contribution to the understanding of mixotrophy and photoautotrophy metabolisms in a freshwater Chlorophyta with biotechnological potential.
Nos ambientes aquáticos, o fitoplâncton é formado basicamente de microrganismos fotossintetizantes que servem como base das cadeias alimentares. Entretanto, além da fotoautotrofia, é vastamente citado na literatura que muitas microalgas alimentam-se de matéria orgânica dissolvida presente no ambiente concomitantemente à realização da fotossíntese, uma via metabólica conhecida como mixotrofia. Sabe-se pouco sobre a ecofisiologia em metabolismo mixotrófico nas microalgas, sendo os estudos, em sua quase totalidade, voltados ao uso dessa via metabólica para aumentar a produção de biomassa algal e estimular a produção de biomoléculas específicas. Outra questão importante, considerando a atividade antrópica atual, é que a maioria dos contaminantes eliminados nos ambientes aquáticos, como metais e nanopartículas, são estudados em fitoplâncton sob metabolismo fotoautotrófico, não sendo encontrados trabalhos ecotoxicológicos envolvendo o metabolismo mixotrófico na literatura. Para entender melhor o metabolismo algal em mixotrofia, este trabalho escolheu a microalga Chlorophyta de água doce Chlorella sorokiniana como organismo-teste. Para melhor organizá-lo, foi dividido em duas partes: a primeira focou no interesse fisiológico/biotecnológico através do estudo do crescimento, parâmetros fotossintéticos, volume celular, e produção de biomoléculas (proteínas, carboidratos e lipídeos); a segunda parte focou nos efeitos ecotoxicológicos de cádmio (Cd) e de nanopartículas de dióxido de titânio (NPs-TiO2). Para estimular a mixotrofia, glicose (1.0 g.L-1 ou 5 x 10-3 mol.L-1) foi utilizada como fonte de carbono orgânico. Os resultados mostraram que durante a mixotrofia, a microalga C. sorokiniana apresentou maiores crescimento populacional e produção de biomoléculas, como clorofila a e lipídeos, quando comparada com as culturas em fotoautotrofia. Também foi observado que os parâmetros fotossintéticos foram afetados em mixotrofia, porém não interferindo no crescimento da microalga, e que a presença de bactérias pode ter atuado como fator estimulante na produção de biomassa algal. Em relação aos efeitos ecotoxicológicos dos contaminantes, as microalgas em mixotrofia foram mais resistentes tanto ao Cd quanto às NPs-TiO2 do que em fotoautotrofia, porém com mudanças na composição bioquímica, podendo afetar a transferência de energia nos ecossistemas aquáticos. De modo geral, podemos concluir que a mixotrofia deve ser considerada em estudos com fitoplâncton, visto que os ambientes aquáticos apresentam uma miríade de fontes de carbono orgânico para esses microrganismos. Na mixotrofia, o carbono orgânico funciona como uma fonte extra de carbono estrutural e de energia para as microalgas, não dependendo obrigatoriamente somente da fotossíntese para sobreviver, estimulando o crescimento e produção de biomoléculas de interesse biotecnológico, além de aumentar a viabilidade celular em ambientes contaminados tanto com Cd quanto com NPs-TiO2. Este estudo é uma contribuição ao entendimento dos metabolismos mixotróficos e fotoautotróficos em uma Chlorophyta de água doce com potencial biotecnológico.
CNPq: 302175/2015-6
FAPESP: 2014/15894-0

In this thesis the effects of individual and multiple environmental stressors (physical and chemical) are examined in inbred and outbred zebrafish (Danio rerio, Hamilton), a model species used in ecotoxicology and environmental risk assessment (ERA). The central question addressed is, are inbred laboratory animals representative and protective of wild populations? That is, are inbred fish equally or more sensitive to chemicals and other stressors compared with more outbred (wild) fish? A combination of tools and approaches incorporating traditional (eco)toxicology and population genetics have been employed, together with more contemporary molecular genetics and population modelling, to compare and contrast a range of responses in inbred and outbred zebrafish exposed to the endocrine disrupting chemical clotrimazole and/or temperature elevation in the laboratory. The choice of test species was based on our broad understanding of its basic biology, extending from the molecular level to the population level, and its wide use as a model organism in (eco)toxicology. Selection of the test chemical clotrimazole and temperature was based on a shared mode of action, aromatase inhibition, and therefore their ability to block oestrogen production, impair reproduction, promote male development and skew population sex ratios in zebrafish. A cascade of responses were compared in inbred and outbred zebrafish, including changes in the levels of expression of genes for gonadal aromatase and other steroidogenic enzymes, circulating sex steroid hormones, gonadal sex differentiation and development (via gonadal histopathology) and reproductive fitness (female fecundity, paternity and viability of embryos). Amongst the most striking results were directional skews in sex ratio towards males in response to clotrimazole (Chapter 5) and elevated temperature exposure (Chapter 7). Inbred fish were generally more responsive compared to outbreds, which showed evidence of physiological and developmental compensation, resulting in lower male-sex skews and superior fitness in terms of male reproductive success (paternity and viability of embryos). The greater effects observed in inbred fish were attributed to inbreeding×environment interactions and the amplification of inbreeding depression. Although no empirical genetic evidence of this mechanism is presented (loss of heterozygosity at quantitative trait loci and concomitant loss of heterosis and/or the expression of recessive, deleterious alleles in homozygotes), supporting evidence was provided by increased phenotypic variance in some apical endpoints in inbred fish, including specific growth rate and fecundity. This increased variance also has the potential to counteract the higher levels of response observed in inbreds, because the power to detect statistically significant changes in responses is reduced. This trade-off was demonstrated for specific growth rate. Crucially, significant male-sex ratio skews (>80%) were induced at substantially lower clotrimazole exposure concentrations (1.7 µg l-1) in combination with elevated temperature (33°C), compared with exposure concentrations (43.7 µg l-1) generating similar sex ratio skews at the standard test temperature of 28°C. These temperatures represent current and predicted 2100 (elevated) mean temperatures in the zebrafish’s native India and Bangladesh. Although the lowest observed effect concentration was an order of magnitude above the predicted environmental concentration for clotrimazole, it is conceivable that combined environmental exposures to similarly acting chemicals (e.g. other azole compounds used in crop protection, veterinary and human medicine) could produce similar effects to those we observed. The consequent effects of sex ratios skews and reduced fitness (fecundity and embryo viability) on per capita population growth rate (r) and extinction probability were predicted in inbred versus outbred zebrafish populations using stochastic population viability analysis. The results showed that the observed male-skews >80% threaten small zebrafish populations with fewer than 100 breeding adults (<20 adult females). However, small reductions of 2-3% in embryo-juvenile (age 0+) survivorship (including simulated inbreeding depression) were more influential on r and extinction probability than large sex ratio skews and/or reduced female fecundity. The results presented in this thesis support the contention that chemical effects may be exacerbated by other environmental stressors, but also illustrate the importance of considering biological (genetic), as well as physical and chemical interactions in cumulative ERA. Greater sensitivity of inbred versus outbred organisms to the effects of environmental stressors on sexual differentiation and reproductive fitness offers a margin of safety to ERA and the protection of wildlife populations (excluding those that are severely inbred and critically endangered). This is because, as originally stated, laboratory organisms used in ERA are generally more inbred than their wild counterparts. Nevertheless, more attention should be paid to the origin, breeding history and genetics of laboratory strains. This will help to ensure consistency between studies and testing laboratories and provide more confidence in extrapolating the results to wild populations.

The Silk Stream in north London is one of two principal surface feeders into the Welsh Harp reservoir. The reservoir represents an important amenity and recreational site and is internationally recognized for its wintering wildfowl. However, the larger part of the catchment area lies within a highly urbanized zone and is therefore subjected to a variety of point and non-point pollution sources rendering the ecological balance considerably more delicate than corresponding rural areas. The construction of a litter screen and oil boom on the lowest reaches of the Silk Stream has highlighted the particular problem of oil inputs from urban runoff. In order to assess the ecotoxicological damage resulting from hydrocarbon pollution in the Silk Stream and at the receiving basin, two common, physiologically contrasting macroinvertebrates, Asellus aquaticus and Lymnaea peregra were used as caged biomonitors. Both organisms were found to be bioaccumulators of hydrocarbons when transferred from rural sites to the caged urban sites although tissue levels were substantially lower at the receiving basin site. Elevated mortality rates of both organisms were also observed at the three stream sites indicating the considerable recovery that occurs at the receiving basin. Hydrocarbon tissue concentrations were found to mirror those of the surrounding sediments in which the combustion derived compounds, fluoranthene and pyrene, were consistently the most abundant polycyclic aromatic hydrocarbons (PAHs). The alkane profiles were generally unimodal, peaking in the C20-C23 range. Inputs of biogenically derived hydrocarbons and of lubricating oils were also identified in the abiotic environment as well as in the organism tissues. The application of Principal Component Analysis revealed an association between a lubricating hydrocarbon source and mortality in L. peregra at two sites and with A. aquaticus at one site. Links between mortality and rainfall were also established at the Silk Stream for L. peregra and to a lesser extent for A. aquaticus. Neither hydrocarbon tissue burdens nor rainfall were linked to the low mortalities at the receiving basin. Laboratory toxicity tests showed that both organisms accumulate and depurate hydrocarbons rapidly and that lower mortalities were attained for similar tissue burdens compared with the field results. The depuration patterns indicated that a substantial proportion of the measured hydrocarbons were in non-assimilated forms.

With their high aspect ratio, strength, light weight and electrical conductivity single-walled carbon nanotubes (SWCNTs) provide properties of great interest to industry, and, consequently, are finding use in an ever increasing number of products and applications, which will eventually lead to their appearance in the environment. The toxicity of SWCNT, however, is controversial, mainly because of the inconsistency of results, owing to a lack of understanding of the behaviour of SWCNTs in environmental media. The present study explored the ecotoxicology of engineered SWCNTs and their interacton with other contaminants as well as their potential for trophic transfer. A series of co-contaminant experiments was performed to understand the interaction of SWCNTs with dissolved metals at low-level and non-toxic concentrations of both. The results showed that any nano-scale effects were negated by the tendency of uncoated SWCNTs to agglomerate in seawater. However, SWCNTs, in combination with natural organic matter (NOM), remained suspended for long enough to become available to filter-feeding mussels. A potentiating toxicological effect was observed, expressed as DNA strand breaks obtained using the comet assay and oxidative stress, on divalent metals afforded by negatively charged SWCNT agglomerates in seawater at concentrations as low as 5μg L-1. This is supported by the observation that SWCNTs alone were only toxic at concentrations ≥100μg L-1 and that the SWCNT-induced DNA damage was correlated with oxidative stress only in the absence of metals The potential for trophic transfer was assessd using the green algae (Tetraselmis suecica). Light microscopical observations, confirmed by SEM and Raman spectroscopy, showed that SWCNTs adhered to the external algal cell walls and TEM results suggested internaization. A direct effect of SWCNT exposure on the algae was a significant decrease in chlorophyll a concentrations and cell viability. Algea fed to mussels in the presence of SWCNTs led to a signifiacntly increased pseudofaeces production, suggesting selective feeding. However, histological sections of the mussel digestive gland following exposure showed evidence of SWCNTs containing algal and toxicological tests signs of DNA dmage and oxidative stress. In conclusion, the observed SWCNT-algal interaction may facilitate trophic transfer of SWCNTs up the food chain with potential consequences for human health. If these laboratory experiments are confirmed in the natural environment, the present results will have implications for the understanding of the role of carbon nanotubes in environmental metal dynamics, toxicology, and consequently, regulatory requirements.

In meeting the challenges associated with the study of nano-sized objects, a major area of research is represented by the interaction of nanoparticles (NPs) with biological systems. Owing to their characteristics, NPs interact in a fundamentally different fashion with biological entities than molecular species. When in contact with biological fluids, the particle surface is covered by a layer of adsorbed proteins and other biomolecules, in dynamic equilibrium with the surrounding environment, termed the “biomolecular corona”, which provide the as-synthesized particle with a unique biological identity. While within the field of nanotoxicology the importance of the study of the biomolecular corona has been largely recognized as key to untangle the complicate interactions of NPs with cells, environmental toxicology has just begun to consider the study of coronas in facing the upcoming issue of NP pollution of the marine ecosystems. The main aim of this PhD was to investigate the interactions of NPs with biomolecules in the marine environment in order to understand their ecotoxicological implications. As a proxy of the emerging issue of plastic contamination of marine ecosystems, polystyrene nanoparticles, with positive and negative surface charges, were employed throughout the study. Firstly, the nanoparticles interactions with the sea urchin Paracentrotus lividus, as a model invertebrate species, were considered. The biological identity of positive and negative polystyrene nanoparticles entails the potential to promote particlecell association, by assisting their interaction with the highly specialized immune systems of P. lividus. Both positive and negative particles maintained hydrodynamic sizes indicative of a narrow size distributions, mostly encompassing protein coated monomers and dimers, acquiring a net negative surface charge. The observed corona-derived colloidal stability over time suggests that, irrespectively of initial surface charge, in the biological milieu polystyrene nanoparticles can acquire the same physical-chemical characteristics, possibly being presented for bio-nano interactions in a comparable manner. The role of an adsorbed protein in promoting the association of positivelycharged polystyrene nanoparticles was proven to lead to toxic effects sustained by the phagocytic immune cells of the sea urchin, confirming that biomolecular coronas dictate the biological fate of nanoparticles in marine invertebrate species, with a mode of action comparable to what established in “classical” nanotoxicology. The subsequent studies focused on polystyrene NPs in marine waters, as a primary target of their release in the environment. In such abiotic, yet biomolecule rich, environment the interaction with extracellular polymeric substances (EPS) extruded by ubiquitous photoautotrophic planktonic species (e.g. diatoms and cyanobacteria) determine the particle fate. The formation of an “eco-corona” reverses the typical colloidal behavior of positive and negative polystyrene NPs, as solely determined by surface charge, when in high-salinity media. Here, positively-charged particles underwent remarkable aggregation while negative ones were stabilized and aggregates formation was limited upon interaction with EPS, prospecting an opposite environmental destiny in marine ecosystems. Surface charge influenced ecotoxicity, with negative PS NPs being generally non-toxic to microalgae and cyanobacteria. Positively-charge counterparts, however, caused loss of cell viability and oxidative stress, but such effects were significantly reduced when an eco-corona was present, suggesting a buffering action carried out by the adsorbed EPS layer, capable of limiting direct NP-cell interactions and their resulting noxious effects. Overall, a multifaceted scenario for bio-nano interactions in the marine environment emerges from this study, with important ecotoxicological implications. If biological recognition of the biomolecular corona seems the underlying mechanism to ecotoxic effects in complex biological milieus, mere physical effects entailed by the eco-corona may be responsible for modulating the toxicity of NPs dispersed in seawater. This fundamentally different characters of bio-nano interactions forecast an additional dimension of complexity of nano-ecotoxicological research in the marine environment, worthy of being considered in future studies.

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul
Essential to life, water is present in almost all human activities, mainly in the consumption of drinking water and agricultural activities. Considering that the world population has increased enormously, food production must be increased and this need has encouraged cooperatives and producers to seek alternative techniques to improve productivity and reduce costs. In this context, the addition of hydrogels on the ground, optimizes the availability of water due to its large storage capacity, accelerating the development of the root system and aerial part of the plant. Most hydrogels is done based on polyacrylamide polymer of acrylamide (AM) monomer which is in the form odorless and white colored water soluble and highly toxic. The damaging effects of AM, which has carcinogenic risk and endocrine disrupter in living beings are known for over 30 years. In soil, acrylamide has a high time to be degraded and due to leaching, the monomer can be found in aquatic environments. Due to its toxicity, acrylamide in a concentration of 5.88 mg / L-1 restates the EC50 microalgae Chlorophyceae Desmodesmus quadricauda (Turpin) Brébisson. In addition to the influence of pollution, various abiotic factors such as temperature and pH, interfere with aquatic communities. The aim of this study was to evaluate the toxic effects of acrylamide in predetermined concentration with changes in temperature (24 ° C and 17 ° C) and pH variation on physiological parameters of microalgae D. quadricauda. The green algae were grown in Water Culture (WC) with its exposed cells in exponential growth phase for a period of 72 hours at concentrations of 5.88 mg l-1 of acrylamide, pH values ranging from 5, 6, 7 and 8. Periodic samples counts were performed under the microscope, in which it took into account the phenotypic plasticity and cellular measures, the biovolume and cell density. When comparing this study with acrylamide EC50 obtained in a previous study, the results indicated at low temperatures (17 ° C) the toxicity of acrylamide and decreased at high temperatures (24 ° C) increased, not having the pH interfered in this parameter. Analyzing cellular measures of width and length, results were significant with the control of pH 8 and temperature 15 ° C, as in biovolume values where only the variation of pH shows variation among the tested controls and treatments. Phenotypic gender plasticity shows that when analyzing the groups separately for periods of 24, 48 and 72 hours, although no significant differences, most phenotypic change was found in the monasteries of a cell that prevailed most in the group treated with acrylamide and pH change in almost all periods. Therefore, the concentration of acrylamide in this test along with changes in temperature and pH tested, indicating that the toxicity of the substance changes the parameters tested in microalgae, becoming a risk to aquatic communities exposed to acrylamide.
Essencial à vida, a água está presente em quase todas as atividades humanas, principalmente no consumo de água potável e em atividades agrícolas. Tendo em vista que a população mundial vem aumentando enormemente, a produção de alimentos tem de ser aumentada sendo que esta necessidade tem-se estimulado cooperativas e produtores a buscarem técnicas alternativas para a melhoria da produtividade e redução de custos. Neste contexto, a adição de hidrogéis no solo, otimiza a disponibilidade de água , devido à sua grande capacidade de armazenamento, acelerando o desenvolvimento do sistema radicular e da parte aérea das plantas. A maioria dos hidrogeis é feito à base de poliacrilamida, polímero da acrilamida (AM), monômero que se apresenta na forma inodora e de coloração branca, solúvel em água e altamente tóxica. Os efeitos prejudiciais da AM, que apresenta risco carcinogênico e desregulador endócrino nos seres vivos, são conhecidos há mais de 30 anos. No solo, a acrilamida apresenta um elevado período para ser degradada e devido a lixiviação, o monômero pode ser encontrada em ambientes aquáticos. Devido a sua toxicidade, a acrilamida em concentração de 5,88 mg/L-1 reapresenta a EC50 de microalgas clorofíceas Desmodesmus quadricauda (Turpin) Brébisson. Além da influencia da poluição, vários fatores abióticos como temperatura e pH, interferem nas comunidades aquáticas. O objetivo deste estudo foi avaliar os efeitos tóxicos da acrilamida em concentração pré-estabelecida juntamente com alterações de temperatura (24 ºC e 17 ºC) e variação de pH sobre parâmetros fisiológicos da microalga D. quadricauda . As clorofíceas foram cultivadas em meio Water Culture (WC), com suas células em fase exponencial exposta por um período de 72 horas a concentração de 5,88 mg.L-1 de acrilamida, valores de pH que variaram de 5, 6, 7 e 8. Amostras periódicas foram realizadas contagens ao microscópio, em que se levou em consideração a plasticidade fenotípica e medidas celulares, o biovolume e densidade celular. Ao comparar este estudo com a EC50 da acrilamida obtido em estudo prévio, os resultados indicaram que a 17 ºC a toxicidade da acrilamida diminuiu e em temperaturas a 24 ºC aumentou, não tendo o pH interferido neste parâmetro. Analisando as medidas celulares de largura e comprimento, resultados foram significativos com o controle de pH8 e com temperatura a 15ºC, assim como nos valores do biovolume, onde a variação apenas do pH apresenta variação entre os controles e tratamentos testados. A plasticidade fenotípica do gênero aponta que ao analisar os grupos separadamente nos períodos de 24, 48 e 72 horas, que apesar de não haver diferenças significativas, maior alteração fenotípica foi encontrada nos cenóbios de uma célula, que prevaleceu maior no grupo tratado com acrilamida e alteração de pH em praticamente todos os períodos. Portanto, a concentração de acrilamida presente nos ensaios juntamente com alterações de temperatura e pH testadas, indica que a toxicidade da substância altera os parâmetros testados na microalga, se tornando um risco às comunidades aquáticas se expostas a acrilamida.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
With the advance of the technology in agriculture, diverse substances have been used for the control of pests. As the fungicides to the copper base (metal heavy), these if accumulate in the environment compromising the activity of the organisms, over all the earthworms, essentials to the maintenance of the chemical and biological processes. The objective of this study was to evaluate the effect of the fungicide cooper oxychloride on earthworms considering parameters mortality and rejection to the contaminate the soil. Two species, Eisenia fetida (standard test species) and Pontoscolex corethrurus (Amazonian native species) have been selected as indicators of soil contamination of. E. fetida created in laboratory, from samples acquired in Manaus and, P. corethrurus collected in ground natural. For exposition to the contaminante two types of soil had been used, Gleissolo and Argissolo. The tested substance contained 588 g/kg of the active ingredient cooper oxychloride -35% of metallic copper. The methodology for determination of the acute toxicity and the effect on the behavior of rejection was based on OECD and ISO international protocols. The toxicity tests consisted on a contaminated soil with increased concentrations of cooper oxychloride. The acute toxicity was evaluated by the medium lethal concentration (CL50) using a Probit analysis, followed by the generation of the dose-response curve. Differences between the control and treatments had been evaluated through the analysis of variance (ANOVA) and for comparison of averages used the Dunnett test was used with a 5% of significance. For the data of rejection to the substratum the t-Student test for comparison of the averages of the number of individuals between the contaminated sections and control. The esteem value of the CL50 after 14 days of exposition in the contaminated Gleissolo for E. fetida, was of 1162.3 mg Cu/kg (IC 95% of 975-1385.2), showing low lethality for this species. However, the native showed to greater sensitivity to copper, being the estimated value of CL50: 154.6 mg Cu/kg (58.9-405.7). In contrast to E. fetida the reduction of biomass in P. corethrurus was not significant in the Gleissolo acute test. However, for the Argissolo test it was observed a significant reduction for the concentration of 56. Due to the great variability of response for P. corethrurus in Argissolo, the data had not been adjusted in a typical dose-response curve, however the estimated CL50 in 84.3 mg Cu/kg (35-202.8). The rejection for the contaminated Gleissolo was observed for both the species, being that P. corethrurus showed to greater sensitivity to the copper that E. fetida with significant results at 14 and 28mg Cu/kg, respectively, and in Argissolo the responses of P. corethurus did not show a significant correlation for the increase of concentrations. The rejection tests had shown that, for low concentrations of the fungicide, the earthworms had prevented contaminated ground, confirming the high sensitivity of these organisms in detecting a decrease of concentrations in the ground. In Argissolo the responses of rejection of P. corethurus had not been significant according to the increase of concentrations of cooper. For a further evaluation of the risks of pesticides for tropical soils toxicity assays must be also carried out with a native species in order to verify its sensitivity in relation to the standard test species.
Com o avanço da tecnologia na agricultura, diversas substâncias têm sido utilizadas para o controle de pragas. Como os fungicidas à base de cobre (metal pesado), estes se acumulam no ambiente comprometendo a atividade dos organismos, sobretudo as minhocas, essenciais à manutenção dos processos químicos e biológicos. O objetivo deste estudo foi avaliar os efeitos do fungicida oxicloreto de cobre em minhocas considerando características de mortalidade e rejeição ao solo contaminado. Foram selecionadas duas espécies, Eisenia fetida (espécie-teste padrão) e Pontoscolex corethrurus (espécie nativa) como indicadores de contaminação do solo. E. fetida criadas em laboratório, a partir de amostras adquiridas em Manaus e, P.corethrurus coletadas em solo natural. Para exposição ao contaminante foram usados dois tipos de solo, Gleissolo e Argissolo. A substância teste usada foi um fungicida cúprico, que contém 588 g/kg do ingrediente ativo oxicloreto de cobre (35% de cobre metálico). A metodologia para determinação da toxicidade aguda e do efeito comportamental de rejeição ao substrato contaminado baseou-se em protocolos internacionais OECD e ISO. Os testes de toxicidade consistiram na exposição das minhocas em solo contaminado em concentrações crescentes com oxicloreto de cobre. A toxicidade aguda foi avaliada pela estimativa da concentração letal mediana (CL50) utilizando análise de Probit, seguido da geração da curva dose resposta. Diferenças entre o controle e tratamentos avaliadas através da análise de variância (ANOVA) e para comparação de médias utilizado o teste de Dunnett, a 5 % de significância. Para os dados de rejeição utilizou-se o Teste t-Student para comparação das médias do número de indivíduos entre as seções contaminadas e de controle. O valor estimado da CL50 após 14 dias de exposição em Gleissolo para E. fetida, foi de 1162,3 mg Cu/kg (IC 95% de 975-1385,2), mostrando baixa letalidade para esta espécie. Entretanto, a nativa mostrou maior sensibilidade ao cobre, estimando a CL50 em 154,6 mg Cu/kg (58,9-405,7). Ao contrário de E. fetida a redução de biomassa em P. corethrurus, não foi significativa em teste agudo em Gleissolo. Entretanto, em Argissolo foi observada redução significativa apenas na concentração de 56 mg Cu/kg. Devido a grande variabilidade da resposta de cobre para P. corethrurus em Argissolo, os dados não foram ajustados em uma típica curva dose- resposta, estimando a CL50 em 84,3 mg Cu/kg (35-202,8). A rejeição ao Gleissolo contaminado foi observada para ambas as espécies, sendo que P. corethrurus mostrou maior sensibilidade ao cobre que E. fetida (respostas significativas a 14 e 28mg Cu/kg, respectivamente), e em Argissolo as respostas de rejeição de P. corethurus não foram significantes conforme o aumento de concentração. Os testes de rejeição mostraram que, mesmo em baixas concentrações do fungicida, as minhocas evitaram solos contaminados, confirmando a sensibilidade destes organismos em detectar baixas concentrações. Em Argissolo as respostas de rejeição de P. corethurus não foram significantes conforme o aumento de concentração. Para avaliação do risco de pesticidas para solos tropicais, os ensaios de toxicidade devem ser realizados também com uma espécie nativa, para verificar a sensibilidade desta em relação à espécie teste padrão.

Os girinos de rã-touro, Lithobates catesbeianus, podem ser bons bioindicadores de condições ambientais. O objetivo desse trabalho foi determinar o potencial de toxicidade para L. catesbeianus de alguns dos principais agrotóxicos utilizados no cultivo de arroz irrigado. Foram realizados testes de toxicidade aguda para a determinação da CL50-96h do bentazon, penoxsulam, óleo vegetal, permetrina e carbofuran, separadamente, e da mistura desses agrotóxicos. Com esses resultados foram estimados os índices de segurança dos produtos. Girinos em fase pré-metamorfose foram expostos aos agrotóxicos na própria lavoura de arroz e em laboratório por 21 dias, para avaliar os possíveis efeitos crônicos destas substâncias, separadamente e da mistura, sobre o quadro hematológico, metamorfose (regulada pelo eixo tiroideano), e também o possível potencial mutagênico através do teste do micronúcleo. A CL50-96h para girinos foi de 4530 mg/L para o bentazon; 7,52 mg/L para o penoxsulam + 145,66 mg/L do óleo vegetal; 81,57 mg/L para o óleo vegetal, 0,10 mg/L para a permetrina, 29,90 mg/L para o carbofuran (ingredientes ativos) e, 38,79 vezes a dose utilizada no campo para a mistura desses produtos. Foi determinado risco ambiental apenas para o inseticida permetrina. Nos testes in situ, as águas de irrigação não apresentaram toxicidade aguda para os girinos. A taxa de metamorfose não diferiu entre os tratamentos, demonstrando que os agrotóxicos utilizados nas doses indicadas não tem ação desreguladora do eixo tiroideano. As análises do micronúcleo mostraram aumento significativo de eritrócitos micronúcleoados para os testes in situ e, no laboratório, para o herbicida bentazon e para a mistura dos agrotóxicos. As análises hematológias mostraram diminuição da hemoglobina e número de eritrócitos no teste de campo, retornado aos padrões normais na semana seguinte. No laboratório houve queda na contagem de eritrócitos para o bentazon, aumento do VCM e HCM para o bentazon e penoxsulam; aumento do CHCM para o penoxsulam e para a mistura dos agrotóxicos. Para a série branca não houve diferenças no teste in situ, mas obtivemos aumento dos números de neutrófilos dos girinos tratados com o bentazon.
American bullfrogs, Lithobates catesbeianus could be good environmental indicators. The aim of this study was evaluate the potential toxicity of some principal pesticides used in irrigated rice crops to L. catesbeianus tadpoles. The pesticides Bentazon, Penoxsulam, Vegetable oil, Permetrina, Carbofuran and the mixture of them were assessed. Pre-metamorphose tadpoles were exposed to all of these agrochemicals in the laboratory to determinate de LC50-96h and so estimate the index of security by each product. Animals in the same phase were exposed to these pesticides on the rice crops, in situ and in laboratory per 21 days to evaluate the possible chronic effects of the substances, separated and in the mixture of them. The hematological results, red and white series, the mutagenic potential (micronucleous test), and the metamorphose rate (regulated by thyroid axis) were evaluated. The LC50-96h to tadpoles was 4530 mg/L to Bentazon; 7.52 + 145.66 mg/L to Penoxsulam + vegetable oil; 81.57 mg/L to vegetable oil; 0.10 mg/L to Permetrina; 29.90 mg/L to Carbofuran (active ingredients) and 38.79 times to the dose used in the field to the mixture of the products. Only to Permetrina insecticide was observed environmental risk. The metamorphose rate showed no difference between the treatments suggesting that these pesticides, used on indicated doses did not promote deregulated action on the thyroid axis. In situ tests the irrigated waters showed low mortality to the animals. The red series showed in situ, a decrease in the haemoglobin tax and in the counting of erytrocyte\'s number however return to the normal values in the follow week. In laboratory tests showed a decrease in the counting of erytrocyte\'s number to the animals exposed to Bentazon, an increase in the MCV and MCH to the animals exposed to Bentazon and Penoxsulam, an increase in the MCHC to those exposed to Penoxsulam and to the \"mixture\". The white series showed no difference in situ test however an increase in the neutrophils number was observed to the animals exposed to Bentazon in laboratory. The micronucleous analyze showed significant increase in the erytrocyte\'s micronucleated number in situ and in laboratorial tests to animals exposed to Bentazon and to the \"mixture\".

Els encoixinats plàstics agrícoles són una peça fonamental del sistema agrícola, contribuint a fer front a la demanda d'alimentació de la creixent població mundial. El seu ús incrementa la producció, precocitat i qualitat de les collites, redueix el consum d'aigua i l'aplicació de pesticides i prevé el desenvolupament de males herbes. Els encoixinats són majoritàriament de polietilè (PE), no biodegradables, i encara que s'han de retirar després de la collita, molts fragments romanen en el camp i es van acumulant, disminuint la qualitat del sòl i de les collites. Els encoixinats de plàstic biodegradable (BDM) s'han presentat com una alternativa sostenible que evita aquesta acumulació; després de la collita seran biodegradats pels microorganismes del sòl en el qual s'integren. Tanmateix, això implica l'aportació al sòl dels diversos compostos (polímers i additius) presents en els fragments, dels que a penes s'han estudiat els seus efectes en les plantes conreades i en els organismes del sòl. L'objectiu d'aquesta tesi és avaluar l'efecte que tenen vuit BDM de diferent formulació i els seus components en el microbioma del sòl agrícola i en plantes conreades. Per a això es van triar dues espècies comunament conreades amb encoixinats que estan entre els principals productes hortícoles a nivell mundial, l’enciam (Lactuca sativa L.) i el tomàquet (Lycopersicon esculetum Mill.). Com a control es va incloure un encoixinat de PE. En primer lloc, es va avaluar si els BDM poden alliberar compostos per contacte amb un mitjà aquós abans d'iniciar la seva biodegradació, i si els compostos alliberats poden afectar el desenvolupament de les plantes. Es va trobar que tots els BDM assajats van alliberar una diversitat de compostos, que en diversos casos (Bioplast SP4 i SP6, Mirel i Biofilm) van afectar negativament la germinació, la morfologia de les arrels o el desenvolupament i fisiologia de totes dues espècies, mentre que els altres BDM van causar efectes menors (Ecovio, Mater-Bi) o no significatius (Bioflex). A continuació, es van identificar els compostos alliberats, que van resultar ser diversos, tant components de la seva estructura polimèrica (1,4-butanediol, àcid làctic, àcid tereftàlic, etc.) com a additius (àcids grassos, glicerol, etc.). D'entre els identificats es va quantificar principalment els que anteriorment havien mostrat afectar el desenvolupament de plantes de tomàquet i d'enciam (1,4-butanediol, àcid làctic i àcid adípic). Les concentracions en què es van trobar van resultar ser substancialment menors que les responsables de causar efectes en les plantes, la qual cosa no permet establir una relació directa entre el seu alliberament dels BDM i els efectes que puguin tenir en les plantes. En tercer lloc, es va estudiar l'efecte de l’acumulació de fragments de BDM en el sòl sobre la germinació i desenvolupament de plantes de tomàquet i d'enciam. La presència de fragments de la majoria dels BDM no va afectar la germinació però si va reduir el creixement i el nivell de clorofil•la en tomàquet i especialment en enciam. En general, els efectes identificats van ser consistents amb els dels compostos alliberats dels BDM trobats anteriorment, i els fragments de PE no van causar efectes. En conjunt, els resultats suggereixen que la composició química del BDM té un paper rellevant en la seva interacció amb el sistema radical de les plantes, i que les conseqüències de la presència de fragments de BDM en el sòl es relaciona amb aquesta composició, probablement pel fet que alliberen components, més que a la seva presència física. Finalment, es va estudiar l'impacte de l'acumuació en el sòl de fragments de BDM en l'estructura i funcions de les comunitats microbianes del sòl agrícola. Després de tres mesos d'incubació, aquesta acumulació va tenir un baix impacte en la diversitat i estructura de les comunitats microbianes del sòl. No obstant això, alguns materials van provocar canvis significatius en l'abundància i diversitat de determinats grups bacterians (Mater-Bi), fúngics (paper MIMGreen) i protistes (Ecovio). Encara que l'activitat microbiana total no es va veure alterada, l'activitat quitinasa, implicada en el cicle del nitrogen, va disminuir significativament per la presència tant de BDM com de PE. Els resultats obtinguts en aquesta tesi doctoral aporten nous coneixements sobre els BDM i els seus potencials efectes. Principalment evidencien que els BDM (1) poden alliberar amb facilitat diversos compostos molt abans que s'iniciï la seva biodegradació, (2) que la solució que conté els compostos alliberats, en funció de la seva composició, pot tenir efectes sobre les plantes i (3) que l’acumulació de fragments de BDM en el sòl presenta capacitat d'afectar el desenvolupament de les plantes i de modificar l'abundància i diversitat del microbioma del sòl en funció de la composició del BDM. Tot això, resulta rellevant per al disseny i desenvolupament d'encoixinats plàstics biodegradables que tinguin sota impacte sobre plantes conreades i sobre el medi ambient.
Los acolchados plásticos agrícolas son una pieza fundamental del sistema agrícola, contribuyendo a hacer frente a la demanda de alimentación de la creciente población mundial. Su uso incrementa la producción, precocidad y calidad de las cosechas, reduce el consumo de agua y la aplicación de pesticidas y previene el desarrollo de malas hierbas. Los acolchados son mayoritariamente de polietileno (PE), no biodegradables, y aunque se deben retirar tras la cosecha, muchos fragmentos permanecen en el campo y se van acumulando, disminuyendo la calidad del suelo y de las cosechas. Los acolchados de plástico biodegradable (BDM) se han presentado como una alternativa sostenible que evita este acúmulo; tras la cosecha serán biodegradados por los microorganismos del suelo en el que se integran. Sin embargo, ello implica el aporte al suelo de los diversos compuestos (polímeros y aditivos) presentes en los fragmentos, pero apenas se han estudiado sus efectos en las plantas cultivadas y en los organismos del suelo. El objetivo de esta tesis es evaluar el efecto que tienen ocho BDM de diferente formulación y sus componentes en el microbioma del suelo agrícola y en plantas cultivadas. Para ello se eligieron dos especies comúnmente cultivadas con acolchados que están entre los principales productos hortícolas a nivel mundial, lechuga (Lactuca sativa L.) y tomate (Lycopersicon esculetum Mill.). Como control se incluyó un acolchado de PE. En primer lugar, se evaluó si los BDM pueden liberar compuestos por contacto con un medio acuoso antes de iniciar su biodegradación, y si los compuestos liberados pueden afectar al desarrollo de las plantas. Se encontró que todos los BDM ensayados liberaron una diversidad de compuestos, que en varios casos (Bioplast SP4 y SP6, Mirel y Biofilm) afectaron negativamente a la germinación, la morfología de las raíces o el desarrollo y fisiología de ambas especies, mientras que los de otros BDM causaron efectos menores (Ecovio, Mater-Bi) o no significativos (Bioflex). A continuación, se identificaron los compuestos liberados, que resultaron ser diversos, tanto componentes de su estructura polimérica (1,4-butanediol, ácido láctico, ácido tereftálico, etc.) como aditivos (ácidos grasos, glicerol, etc.). De entre los identificados se cuantificó principalmente los que anteriormente habían mostrado afectar al desarrollo de plantas de tomate y de lechuga (1,4-butanediol, ácido láctico y ácido adípico). Las concentraciones en que se encontraron resultaron ser sustancialmente menores que las responsables de causar efectos en las plantas, lo que no permite establecer una relación directa entre su liberación de los BDM y los efectos que puedan tener en las plantas. En tercer lugar, se estudió el efecto del acúmulo de fragmentos de BDM en el suelo sobre la germinación y desarrollo de plantas de tomate y de lechuga. La presencia de fragmentos de la mayoría de los BDM no afectó a la germinación pero si redujo el crecimiento y el nivel de clorofila en tomate y especialmente en lechuga. En general, los efectos identificados fueron consistentes con los de los compuestos liberados de los BDM encontrados anteriormente, y los fragmentos de PE no causaron efectos. En conjunto, los resultados sugieren que la composición química del BDM tiene un papel relevante en su interacción con el sistema radical de las plantas, y que las consecuencias de la presencia de fragmentos de BDM en el suelo se relaciona con esta composición, probablemente debido a que liberan componentes, más que a su presencia física. Finalmente, se estudió el impacto del acumulo en el suelo de fragmentos de BDM en la estructura y funciones de las comunidades microbianas del suelo agrícola. Tras tres meses de incubación, este acúmulo tuvo un bajo impacto en la diversidad y estructura de las comunidades microbianas del suelo. Sin embargo, algunos materiales provocaron cambios significativos en la abundancia y diversidad de determinados grupos bacterianos (Mater-Bi), fúngicos (papel MIMGreen) y protistas (Ecovio). Aunque la actividad microbiana total no se vio alterada, la actividad quitinasa, implicada en el ciclo del nitrógeno, disminuyó significativamente por la presencia tanto de BDM como de PE. Los resultados obtenidos en esta tesis doctoral aportan nuevo conocimiento sobre los potenciales efectos de los BDM en las plantas cultivadas y los microorganismos del suelo. Principalmente evidencian que los BDM (1) liberan con facilidad diversos compuestos mucho antes de que se inicie su biodegradación, tras el contacto con el agua, (2) la solución que contiene los compuestos liberados, en función de su composición, puede tener efectos sobre las plantas, (3) que el acúmulo de fragmentos de BDM en el suelo presenta capacidad de afectar al desarrollo de las plantas y de modificar la abundancia y diversidad de los microorganismos del suelo en función de la composición del BDM. Todo ello resulta relevante para el diseño y desarrollo de acolchados plásticos biodegradables que tengan un bajo impacto sobre las plantas cultivadas y sobre el medio ambiente.
Agricultural plastic mulches are an essential part of the agricultural system, contributing to face the food demand for the growing world population. Its use increases crop production, earliness and quality, reduces water consumption and pesticide delivery and prevents weed development. Mulches are mostly made of polyethylene (PE), non-biodegradable. Although they must be removed after harvesting, many fragments remain and accumulate in the field, reducing soil and crop quality. Biodegradable plastic mulches (BDM) have been fostered as a sustainable alternative preventing this accumulation. After harvest they will be biodegraded by the soil microorganisms in which they are integrated. However, this entails the various compounds (polymers and additives) present in the fragments are supplied to the soil, but their effects on cultivated plants and on soil organisms have hardly been studied. The objective of this PhD thesis is to evaluate the effect of eight BDM of different formulation, and their components, on the agricultural soil microbiome and on plants. For this purpose two plant species commonly cultivated with mulches which are among the main horticultural products were targeted, lettuce (Lactuca sativa L.) and tomato (Lycopersicon esculetum Mill.). One PE mulch was included as control mulch. Firstly, it was evaluated whether BDM can release compounds by contact with an aqueous environment before the onset of their biodegradation, and whether the released compounds can affect plant development. It was found that all the BDM tested released a diversity of compounds, which in several cases (Bioplast SP4 and SP6, Mirel and Biofilm) inhibited germination, root morphology or the development and physiology of both plant species, while those from other BDM caused minor (Ecovio, Mater-Bi) or non-significant (Bioflex) effects. Next, the released compounds were identified, which were eventually diverse, both components of its polymeric structure (1,4-butanediol, lactic acid, terephthalic acid, etc.) and additives (fatty acids, glycerol, etc.). Among those identified, the ones having previously shown to affect tomato and lettuce plant development (1,4-butanediol, lactic acid and adipic acid) were quantified. They were found to be in substantially lower concentrations than the ones responsible for causing effects on plants, which does not allow establishing a direct relationship between their release from BDM and the effects they may have on plants. Thirdly, the effect of the accumulation of BDM fragments in the soil on tomato and lettuce germination and plant development was studied. For most BDM, the presence of their fragments did not affect germination but it reduced plant growth and chlorophyll content in tomato and especially in lettuce. In general, the identified effects were consistent with those of compounds released from BDM previously found, and PE fragments caused no effects. Altogether, results suggest that the BDM chemical composition plays a relevant role in its interaction with the plant root system, and that the consequences of the presence of BDM fragments in the soil is related to this composition, likely due to the release of components, rather than to their physical presence. Finally, the impact of the BDM fragments’ accumulation in the soil on the structure and functions of the agricultural soil microbial communities was studied. After incubation for three months, this accumulation had a low impact on the soil microbial communities’ diversity and structure. However, some materials caused significant changes in the abundance and diversity of selected bacterial (Mater-Bi), fungi (MIMGreen paper) and protists (Ecovio) groups. Although the total microbial activity was not altered, the chitinase activity, involved in the nitrogen cycle, was significantly decreased by both BDM and PE presence. The results obtained in this doctoral thesis provide new knowledge on the potential effects of BDMs on cultivated plants and soil microorganisms. They mainly show that BDM (1) easily release several compounds soon before their biodegradation starts, after contact with water, (2) the solution containing the released compounds, depending on its composition, may have effects on plants and (3) the accumulation of BDM fragments in the soil has the capacity to affect plant development and to modify the abundance and diversity of soil microorganisms depending on the composition of the BDM. The results will contribute to the design and development of biodegradable plastic mulches that have a low impact on cultivated plants and the environment.

The uptake and impact of CdS and ZnO NPs in/on maize roots and shoots was investigated and compared with their soluble bulk materials (ZnClz and CdClz). The plants were grown in Eutric Cambisol soil for 21 days. The soil was treated with seven concentrations (0.1-1.25 mg kg-I) for each metal. The Tolerance Index (T!), the Agronomical Efficiency (AE), the Bio-Concentration Ratio (BCR), the Relative Increase Percentage (RI), uptake and uptake %. were calculated for maize roots and shoots. The concentration of metals in maize roots and shoots following treatments of soil with either NPs or bulk materials increased relative to control samples. In addition, the concentration of all metals was higher in maize roots than in shoots across all metals concentrations studied. The uptake of Cd and Zn by maize roots and shoots grown in soils treated with bulk compounds was higher than for those grown in equivalent treated soil with NPs. The majority of NPs and their bulk materials had no significant negative effects on maize growth parameters. However, CdS NPs, CdClz and ZnO NPs had negative effects on the length of maize roots and shoots at the highest metal soil ratio (1.0 and 1.25 mg kg-I). The calculated maize growth parameters (TI, AE, BCR, RI, uptake and uptake %) were varied in maize roots and shoots depending on the plant part, growth period and metal treatments. The toxic effects of CdS NPs (0-100 mg L-1) and ZnO NPs (0-1000 mg L-1) on the germination and the development of maize root were studied for 8 days. The results indicated that the concentration of Zn in maize seeds and roots was higher than Cd for equivalent initial NPs concentrations. Most of the NP concentrations studied had a negative effect on the length and dry weight of maize roots. Germination of maize seed was reduced by the ZnO NPS (68.6%) more than that of CdS (58.1 %). The uptake of CdS, ZnO, and CuO NPs was also investigated for maize plants grown in Eutric Cambisol soil and hydroponic culture over 21 days. High NPs concentrations were used across both growth mediums (0.01-1.0 g kg-1 /g L-1). The TI, AE, BCR, RI, uptake and uptake % were also calculated for maize roots and shoots. The concentration of all NPs showed a similar trend of accumulation behaviour in maize roots and shoots to those found in low concentrations of NPs « 1.25 mg kg-1). The concentration of all metals in maize roots and shoots grown in nutrient solution containing NPs was higher than those grown in the NP treated soil. In addition, the impact of all NPs indicate that CuO and CdS NPs has negative effect on the length of maize roots and shoots at the highest concentrations in both cultures. Moreover, the dry weight of maize shoots was decreased by CdS NPs at the highest concentration in hydroponic culture. The calculated maize growth parameters were also varied in maize roots and shoots depending on the plant part, growth period and metal concentrations. The adsorption kinetics and desorption % of CdS, ZnO, and CuO NPs was studied on the surface of four soils using the batch method. Adsorption isotherms were evaluated by Freundlich and Langmuir model. The results of study suggest that the adsorption of all NPs increased as a function of increasing NPs concentrations until the adsorption equilibrium was reached across all soils. The relative mean adsorption of NPs in four soils was found to follow the following order: Cu > Cd >' Zn. Results also indicated that the highest adsorption of NP on soils was as follows: Libyan sandy soil> Eutric Cambisol soil > Sandy soil > Haplic podzol soil. The adsorption results for all NPs were best modelled using Freundlich equation across all soils. The kinetic behaviour of all studied NPs toward four soils showed the pseudo-second order rather than pseudo-first order kinetics. The mean desorption % of NPs in four soils was found to follow the following order: Zn > Cd > Cu. The effect of CdS, CuO, and ZnO NPs on the rate of nitrogen mineralization was investigated in Eutric Cambisol, Haplic podzol, and Sandy soil over 28 days, three concentrations of each metal NP were used (0.01-l.0 g kg-1). The influence of all test NPs on soils respiration rate was also examined for 48 hours using the same NPs concentrations above. The results of nitrogen mineralization revealed that, the concentration of nitrate (N03-) accumulated readily in three soils; however, the concentrations of ammonium (NH/), dissolved organic nitrogen (DON) and free amino acids had low levels of accumulation across all of test soil and NP types. The comparison results of NPs impact indicated that the large majority of NPs failed to reveal any significant effect upon nitrogen mineralization under any of the NP concentrations save that for amino acid concentrations. Results of soil respiration revealed that no negative significant impacts for all NPs on soil respiration across all NPs and soil types.

Mestrado em Estudos Ambientais (JEMS)
While the appeal of biochar application to soils continues growing, so does the concern about the possibility for surface and groundwater contamination, due to biochar-bound contaminants, such as polycyclic aromatic hydrocarbons (PAHs). Up to now, insufficient information exists on to what extent that PAH fraction can become bioavailable in the soil solution over time and which are the associated potential ecotoxicological implications, as a result of processes such as runoff. This pilot study aimed to evaluate the ecotoxicological effects of biochar-bound PAHs in runoff from soils amended with biochar, having been identified as a gap in current knowledge. Combining soil wetting-drying cycles with PAH water-extraction, a good first approach was obtained for evaluating their potential occurrence in soil solution, while taking into consideration natural soil processes and soil-biochar interactions. LUFA 2.2 soil alone and containing biochar (at usual field rates, 4% ww-1) was subjected to 0, 6 or 12 (sampling times ST-0, ST-1 or ST-2 respectively) consecutive wetting-drying cycles, after which the corresponding test elutriates were extracted. Alongside PAH quantification, a battery of standard aquatic bioassays were used with representative test organisms (Vibrio fischeri, Pseudokirchneriella subcapitata and Daphnia magna), for a robust ecotoxicological evaluation of the biochar-soil (BS) aqueous extracts, while LUFA soil elutriates were used as control (SS). Compared to the control (SS) and to elutriates of biochar alone (B), BS extracts showed the highest total PAH contents, suggesting that a relevant PAHs fraction in biochar-amended soil may be easily water-extractable, perhaps due to interactions between biochar and soil components. Yet, the number of soil wet-dry cycles on aqueous total PAH concentrations was often not significant, suggesting that natural soil wetting-drying events might have little influence on increased PAH bioavailability in pore water, on the short term. BS extracts induced toxicity in all tested species, although its extent was species-specific and varied with the number of wet-dry cycles. For example, the highest sensitivity was observed in the acute assay with D. magna exposed to BS extract for ST-0, while P. subcapitata and V. fischeri were most sensitive when exposed to BS, ST-1. Nevertheless, sub-lethal effects were also observed for P. subcapitata and V. fischeri, when exposed to the control (SS) extracts. Although the levels of individual PAHs in all samples (BS, SS and B) were below the acutely toxic concentrations reported in the literature, it cannot be excluded the combined effects of the multiple PAHs in the test elutriates when explaining these results. Furthermore, although individual PAH concentrations were below that to produce acute effects, chronic effects can occur, and therefore, long-term exposure to these elutriates and using additional non-target species, various biochars and soil properties are necessary for a full evaluation of the bioavailability and ecotoxicity of biochar-bound PAH contaminants in runoff from treated soils.

Orientadores: Marta Sivieiro Guilherme Pires, Gisela de Aragão Umbuzeiro
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Tecnologia
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Resumo: O lodo gerado em estações de tratamento de esgoto (ETE) é um resíduo que possui grande quantidade de nutrientes (nitrogênio e fósforo) e matéria orgânica, podendo ser reaproveitado na agricultura. No entanto, pode conter substâncias tóxicas que inviabilizem esse uso, sendo assim, é necessário caracterizá-lo quanto a diversos parâmetros antes da sua utilização. Este trabalho tem como objetivo caracterizar a toxicidade do lodo de esgoto, proveniente de diferentes tipos de estações de tratamento, para avaliar a viabilidade do seu uso na agricultura. Foram coletadas amostras de 6 ETEs diferentes, que tratam efluentes predominantemente domésticos, e realizadas análises de Nitrogênio total, amoniacal e nitrato/nitrito, Fósforo, Carbono Orgânico, Enxofre, Potássio, Magnésio e Cálcio para caracterizar seu potencial agronômico, análises físico-químicas como pH, condutividade, sólidos e umidade, e metais previstos na Resolução CONAMA 375/2006. Os ensaios ecotoxicológicos foram feitos em amostras de lodo in natura utilizando organismos representativos do solo (Lactuca sativa) e aquáticos (Daphnia similis e Vibrio fischeri). Os resultados dos testes foram aplicados ao Índice de Perigo de Lodo de Esgoto (IPLE) proposto por Da Matta (2011). Nesse trabalho pode-se concluir que os lodos apresentam potencial agronômico relevante. O lodo mais estável apresentou menor toxicidade e, consequentemente, menor valor de IPLE. Os resultados de metais de 5 estações foram menores que os valores preconizados na Resolução CONAMA nº 375/2006, mas, mesmo assim, as amostras apresentaram alta toxicidade. O índice, baseado em testes ecotoxicológicos, demonstrou ser uma ferramenta que pode auxiliar na tomada de decisão mais segura quanto à disposição do lodo ao solo
Abstract: The sludge generated at wastewater treatment plants (WWTP) is a residue that has lots of nutrients (nitrogen and phosphorus) and organic matter, which can be reused in agriculture. However, it may contain toxic substances that can not use it, so it is necessary to characterize the various parameters as before use. This work aims to characterize the toxicity of sewage sludge from different treatment plants, to assess the viability of their use in agriculture. Samples of 6 different WWTP, dealing predominantly domestic sewage, and performed analyzes of the total nitrogen, ammonia and nitrate / nitrite, Phosphorus, Organic Carbon, Sulfur, Potassium, Magnesium and Calcium to characterize the agronomic potential, physical and chemical analyzes were collected as pH, conductivity, solids and moisture, and heavy metals contained in CONAMA Resolution 375/2006. Ecotoxicological tests were made on samples of sludge in natura using representative soil organisms (Lactuca sativa) and aquatic (Daphnia similis and Vibrio fischeri). The results of the tests to Sewage Sludge Hazard Index (SSHI) proposed by Da Matta (2011) were applied. In this study it can be concluded that the sludge had relevant agronomic potential. The most stable sludge showed less toxicity, and hence smaller value of iple. The results of metals from 5 stations were lower than the values recommended by CONAMA Resolution 375/2006, but even so, the samples showed high toxicity. The index, based on ecotoxicological tests, proved to be a tool that can assist in making the safest decision regarding the disposal of sludge to soil
Mestrado
Tecnologia e Inovação
Mestra em Tecnologia

A busca pelo homem por uma vida saudável tem impulsionado pesquisas por novas substâncias capazes de atender tal desejo. O composto fenólico resveratrol (3, 4\', 5- trihidroxiestilbeno) é uma dessas substâncias que apresenta uma variedade de ações farmacológicas, como potencial antioxidante, capacidade antiinflamatória, proteção contra doenças cardíacas e câncer. Apesar dos inúmeros estudos sobre os benefícios do resveratrol à saúde, há poucos dados na literatura sobre sua toxicidade em organismos aquáticos, e principalmente sua concentração no ambiente, tornando o presente estudo fundamental para a contribuição de informações sobre a ecotoxicidade do resveratrol no ambiente aquático. O presente estudo avaliou a toxicidade do resveratrol em embriões e larvas de Danio rerio (zebrafish). Para isso foi realizado o ensaio in vitro de citotoxicidade do resveratrol, ensaios de ecotoxicidade e ensaio de biomarcadores enzimáticos. A avaliação do resveratrol por cromatografia líquida de alta pressão (HPLC) também foi realizada. De acordo com os resultados obtidos, o índice de citotoxicidade (IC50), concentração do resveratrol que causou a morte de 50% das células da linhagem NCTC-L929 foi de 39 mg L-1. A concentração de resveratrol que causa mortalidade em 50% dos organismos expostos (CL50), nos ensaios de ecotoxicidade crônica de curta duração com larvas do peixe Danio rerio foi de 51,37 mg L-1. A CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 96 h de duração foi de 75,33 mg L-1 e a CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 168 h de duração foi de 50,87 mg L-1. Nas concentrações mais elevadas de resveratrol foram observadas deformidades em embriões e larvas. O resveratrol alterou as atividades das enzimas LDH e ChE no estágio embriolarval de Danio rerio. Na análise do resveratrol por HPLC não foi observado degradação do composto.
The concern about human being healthy life has driven researchers to study new compounds capable of reaching that desire. Resveratrol (3, 4\', 5 trihydroxystilbene) a phenolic compound, is one of these substances which presents a variety of pharmacological actions, as antioxidant potential, antiinflammatory capacity, protection against heart and cancer diseases. Despite the numerous studies on the benefits of resveratrol to health, there is little evidence in the literature of its toxicity to aquatic organisms, and especially its concentration in the environment, making the present study fundamental for the contribution of information on the ecotoxicity of resveratrol in the aquatic environment. The present study evaluated the toxicity of resveratrol in embryos and larvae of Danio rerio (zebrafish). For this purpose the in vitro cytotoxicity of resveratrol assay, ecotoxicity assays and enzyme biomarker assay were performed. The evaluation of resveratrol by high pressure liquid chromatography (HPLC) was also performed. According to the results, the cytotoxicity index (IC50), concentration of resveratrol that caused the death of 50% of the cells of the NCTC-L929 lineage was 39 mg L-1. The concentration of resveratrol that causes mortality in 50% of exposed organisms (LC50) in the short-lived chronic ecotoxicity assays with larvae of the Danio rerio fish was 51.37 mg L-1. The LC50 obtained in the embryo-active acute ecotoxicity test of the Danio rerio fish with 96 h duration was 75.33 mg L-1 and the LC50 obtained in the embryo-active acute ecotoxicity assay of the Danio rerio fish with 168 h duration was 50.87 mg L-1. At higher concentrations of resveratrol deformities were observed in embryos and larvae. Resveratrol altered the activities of LDH and ChE enzymes in the embryonal stage of Danio rerio. No degradation of resveratrol was observed in the HPLC analysis of compound.

O crescente desenvolvimento populacional, industrial e a expansão agrícola vêm contribuindo de forma significativa para o aporte de nutrientes e metais nos ecossistemas aquáticos, interferindo na composição e estrutura das comunidades, dentre elas as macrófitas, ocasionando desequilíbrio ambiental, com perda das funções e serviços ecossistêmicos. Entre os diversos elementos inseridos de forma direta e indireta nos corpos hídricos, destacam-se fósforo, cobre e zinco, os quais, apesar de serem fundamentais para o metabolismo biológico, quando em excesso tornam-se prejudiciais, requerendo avaliações mais efetivas, de forma a melhor compreender e prevenir os potenciais riscos da exposição biológica a estes elementos estressores. Considerando o acima exposto, o objetivo da presente pesquisa foi avaliar o efeito do metal cobre sobre o desenvolvimento da macrófita Pistia stratiotes e o efeito dos metais cobre e zinco sobre o desenvolvimento e capacidade bioacumuladora da macrófita Azolla filiculoides, em diferentes concentrações de fósforo, por meio de testes ecotoxicológicos. Para tanto, aspectos nutricionais (nitrogênio, fósforo, cobre total e clorofila a e total) e de desenvolvimento (biomassa, TCR, número de folhas nascidas, ganho de diâmetro, altura e largura foliar, número de raízes nascidas, comprimento das raízes principais, clorose e necrose foliar) foram avaliados em estudos laboratoriais para P. stratiotes e aspectos nutricionais (todos acima citados) e de desenvolvimento (biomassa e TCR) para A. filiculoides. Pelos resultados obtidos verifica-se que os metais cobre e zinco, nas concentrações testadas, ocasionaram interferência no desenvolvimento e nutrição das macrófitas, sendo a maior sensibilidade ao cobre observada para P. stratiotes, registrando-se efeitos de maior toxicidade do cobre em relação ao zinco em ambas as macrófitas. Verificou-se, ainda, que o fósforo proporcionou maior proteção a A. filiculoides contra o efeito dos metais conforme o aumento de sua concentração e já no teste ecotoxicológico com P. stratiotes não foram registradas diferenças significativas em função do aumento das concentrações de fósforo. Dessa maneira, conclui-se que o metal cobre causou maior toxicidade às macrófitas do que o zinco, principalmente em concentrações mais baixas de fósforo, verificando-se maior capacidade bioacumuladora da espécie A. filiculoides quando comparada com P. stratiotes.
The increasing population and industrial growth and the agricultural expansion have contributed significantly to the supply of nutrients and metals in aquatic ecosystems, affecting the composition and structure of communities, among them the macrophytes, causing environmental imbalance, with loss of ecosystem functions and services. Among the various elements inserted directly and indirectly on water bodies, phosphorus, copper and zinc stand out, which, despite being fundamental to the biological metabolism, when in excess become harmful, and assessments that are more effective are required to better understand and prevent the potential risks of biological exposure to these stressors. Considering the above, the objective of this research was to evaluate the effect of copper metal on the development of the macrophyte Pistia stratiotes and the effect of copper and zinc metals on the development and the bioaccumulation capacity of the macrophyte Azolla filiculoides, in different concentrations of phosphorus, through ecotoxicological tests. To do that, nutritional (nitrogen, phosphorus, copper total and chlorophyll a and total) and development (biomass, RGR, number of leafs born, diameter gain, leaf height and width, number of roots born, length of the main roots, leaf chlorosis and necrosis) aspects were evaluated in laboratory studies for P. stratiotes and nutritional (all above) and development (biomass and RGR) aspects for A. filiculoides. From the results obtained it appears that the metals copper and zinc, on the tested concentrations, caused interference in the development and nutrition of the macrophytes, where more sensitivity to copper was observed for P. stratiotes, and the effects of enhanced toxicity of copper relative to zinc in both macrophytes. It was also found that the phosphorus provided greater protection to A. filiculoides against the effects of metals as its concentration was increased and, on the ecotoxicological test with P. stratiotes, no significant differences where found with the increasing concentrations of phosphorus. Thus, it is concluded that the copper metal has caused more toxicity to macrophytes than zinc, especially at lower concentrations of phosphorus, and was verified a greater bioaccumulation capacity on the A. filiculoides species when compared to P. stratiotes.

Dentre as tecnologias que surgiram para impulsionar a produtividade agrícola estão os agrotóxicos, cujo consumo tem aumentado nos últimos anos em todo o mundo. Estes podem agir toxicamente no meio ambiente, podendo contaminar o solo, os recursos hídricos e os organismos quando expostos direta e indiretamente a esses contaminantes. O produto comercial Vertimec® 18 EC é um desses potentes agrotóxicos e foi desenvolvido como inseticida para o controle de artrópodes da agricultura e usos domésticos. Estudos realizados com o produto revelaram interações com o meio ambiente, indicando toxicidade mesmo em concentrações baixas a organismos aquáticos (microcrustáceos e peixes). Dentro desse contexto o presente estudo visou avaliar os efeitos indiretos e diretos do agrotóxico Vertimec® 18 EC a partir da caracterização física, química, biológica e ecotoxicológica de amostras de água e sedimento de mesocosmos artificialmente contaminados e testes de toxicidade com o produto comercial. As análises físicas, químicas e biológica (clorofila), mostraram um efeito indireto do Vertimec® 18 EC na recuperação dos tratamentos contaminados, em relação à dinâmica dos nutrientes na água. As amostras de água e sedimento dos mesocosmos contaminados apresentaram efeito tóxico para o cladócero Ceriodaphnia silvestrii durante todo o período do experimento e esse efeito esteve diretamente relacionado com a aplicação do Vertimec® 18 EC. No entanto esse efeito não foi observado no fitoplâncton, o que foi demonstrado pelos testes realizados com a água proveniente do experimento in situ com Pseudokirchneriella subcapitata. Os valores de CE50, 48h e CI25 do Vertimec® 18 EC para Ceriodaphnia silvestrii foram de 81,64 e 6,3 nL/L. Além do efeito do agrotóxico, foi observado um efeito de confinamento nos mesocosmos, que resultou em toxicidade nos tratamentos controles.
Among the technologies that have emerged to boost agricultural productivity are pesticides whose consumption has increased in recent years throughout the world. These can act toxically in the environment and contaminate the soil, water and organisms when exposed directly and indirectly to these contaminants. The commercial product Vertimec® 18 EC is a potent pesticide such as insecticides and has been developed for the agricultural arthropods control and household uses. Studies with the product revealed interactions with the environment, indicating toxicity even at low concentrations to aquatic organisms (fish and microcrustaceans). Within this context, the present study aimed to assess the direct and indirect effects of pesticide Vertimec ® 18 EC from the physical, chemical, biological and ecotoxicological assessment of water and sediment samples of artificially contaminated mesocosms and toxicity testing with the commercial product. The physical, chemical and biological (chlorophyll) analysis showed an indirect effect of Vertimec® 18 EC on contaminated treatments recovery, against nutrients dynamic in the water. Samples of water and sediment contaminated mesocosms showed toxic effects to zooplankton Ceriodaphnia silvestrii throughout the experimental period and this effect was directly related to the application of Vertimec ® 18 EC. However, this effect was not observed in phytoplankton, which was demonstrated by performed tests with water from the experiment in situ with Pseudokirchneriella subcapitata. The EC50 values, 48 and 18 CI25 of Vertimec ® EC to Ceriodaphnia silvestrii were 81.64 and 6.3 nL/L. In addition to the agrochemical effect, was observed a confinement effect of the enclosures that resulted in toxicity control treatments.

O objetivo geral do trabalho foi avaliar a distribuição e os efeitos tóxicos dos metais Cr e Cu sobre os ecossistemas aquáticos lênticos, incluindo seus componentes bióticos e abióticos, utilizando-se sistemas com diferentes níveis de complexidade e organização. Os sistemas utilizados na avaliação da toxicidade de Cr e Cu foram: a) testes uniespecíficos com Selenastrum capricornutum (microalga), Daphnia similis e Ceriodaphnia dubia (Cladocera), Chironomus xanthus (Diptera, Chironomidae), Oreochromis niloticus (peixe) e Pistia stratiotes (macrófita); b) experimentos em microcosmos (testes multiespecíficos) com as comunidades naturais da represa do Lobo; c) experimentos em mesocosmos, contando com água, sedimento, plâncton, bentos, peixes e macrófitas naturais da represa do Lobo. Nos testes de toxicidade todas as espécies foram mais sensíveis ao Cu que ao Cr, e a sequência de sensibilidade foi D. similis> C. dubia> S. capricornutum> C. xanthus > P. stratiotes > O. niloticus. Nos experimentos em microcosmos e mesocosmos, as concentrações-teste foram definidas como o valor máximo permitido pela Resolução nº 20/1986 do CONAMA para Cr6+ e Cu2+ para corpos dágua Classe 2 (50 µg.L-1 e 20 µg.L-1, respectivamente). Em todos os experimentos a concentração de clorofila foi reduzida significativamente após a adição dos metais. Os efeitos sobre a densidade e riqueza de espécies das comunidades zooplanctônicas foram mais severos para o Cr que para o Cu; porém Cladocera foi mais sensível ao Cu. Cromo provocou uma maior inibição na produção primária em abril de 2002, enquanto que em novembro/dezembro de 2002 a produção primária sofreu maior decréscimo após a adição de Cu. Ambos os metais estudados apresentaram efeitos significativos sobre um grande número de variáveis da água, como oxigênio dissolvido, amônio, distribuição das formas fosfatadas, clorofila e material em suspensão, sendo os efeitos do Cr mais severos que os do Cu, em relação ao número de variáveis afetadas e ao tempo de restauração do sistema. Os sedimentos não apresentaram alterações após a adição de metais. Foi observada a ocorrência de bioacumulação de Cr e Cu pelo plâncton, bentos, macrófitas e peixes. A toxicidade dos metais estudados foi reduzida após a inclusão de peixes e macrófitas, principalmente para Cu. Apesar das concentrações de Cr e Cu relativamente baixas empregadas, os metais provocaram efeitos nos compartimentos bióticos e abióticos do sistema. Os resultados obtidos no presente trabalho podem ser úteis como subsídio para a avaliação dos valores máximos permitidos pela Resolução nº 20/1986 do CONAMA para os metais estudados.
The general aim of this work was to evaluate the distribution and the toxic effects of the metals Cr and Cu on lenthic aquatic ecosystems, using systems of different complexity and organization levels. The systems used for Cr and Cu toxicity evaluations were: a) single-specific tests with Selenastrum capricornutum (microalgae), Daphnia similis e Ceriodaphnia dubia (Cladocera), Chironomus xanthus (Diptera, Chironomidae), Oreochromis niloticus (fish) and Pistia stratiotes (macrophyte); b) microcosms experiments (multi- specific tests) with the Lobos reservoir natural communities; c) mesocosms experiments, with natural water, sediment, plankton, benthics, fishes and macrophytes from Lobos reservoir. In the toxicity tests, all the species were more sensitive to Cu than to Cr, and the sensitiviness sequence was D. similis> C. dubia> S. capricornutum> C. xanthus > P. stratiotes > O. niloticus. In the microcosms and mesocosms experiments, the test-concentrations were the maximum value allowed by the CONAMA Resolution nº 20/1986 to Cr6+ and Cu2+ to Class 2 water bodies (50 µg.L-1 and 20 µg.L-1, respectively). In all experiments the chlorophyll concentration was significantly reduced after the metal addition. The effects on the Zooplankton density and richness were stronger to Cr than to Cu; however Cladocera was more sensitive to Cu. Chromium caused higher inhibition in primary production in April/ 2002, but in November/ December 2002 the primary production suffered higher inhibition by Cu addition. Both studied metals showed significant effects on several water variables, as dissolved oxygen, ammonium, phosphate forms distribution, chlorophyll and suspended material, being the Cr effects stronger than the Cu effects, in relation to the number of affected variables and the system restoration time. The sediments didnt show alterations after the metal addition. Chromium and Cu bioaccumulation was observed by plankton, benthic invertebrates, macrophytes and fishes. The toxicity of the studied metals was reduced by the fish and macrophyte inclusion, mainly to Cu. Although the concentration of Cr and Cu were low, toxic effects were observed on both biotic and abiotic system compartments. The results obtained in this work may be useful as subsidy to evaluation of the maximum value allowed by the CONAMA Resolution nº 20/1986 to the studied metals.

A água é de grande importância para a sobrevivência da vida, o rio Corumbataí abastece as cidades de Analândia, Corumbataí, Rio Claro, Piracicaba entre outras. A toxicologia ambiental vem sendo disseminada como ferramenta no monitoramento ambiental, pois através de bioensaios é possível avaliar a qualidade dágua. O objetivo deste estudo foi avaliar a qualidade do corpo hídrico em questão através de testes de toxicidade crônica, aplicação de protocolo rápido, determinação de metais em sedimento em suspensão e a toxicidade aguda de dois herbicidas (ametrina e glifosato) com Ceriodaphnia silvestrii. As coletas foram realizadas mensalmente no período de agosto/2009 à julho de 2010. Para as 12 coletas verificou-se que pelo menos um ou mais pontos apresentavam concentração de Fe acima do valor máximo permitido pelo CONAMA. A oscilação da pontuação do protocolo de avaliação rápida esteve diretamente relacionada com os aspectos visuais e olfativos. No ensaio crônico com Ceriodaphnia silvestrii houve variação na reprodução dos organismos e através de análises estatísticas foi possível observar que o pH, oxigênio dissolvido, condutividade, Zn, Ti e Ni influenciaram na toxicidade da amostra. Os herbicidas ametrina e glifosato apresentaram a CE50 (48h) respectivamente de 0,50 e 4,5 mg L-1. Conclui-se que a toxicidade crônica foi influenciada pelos parâmetros físico-químicos e metais durante os 12 meses de coleta.O protocolo de avaliação rápida complementa os resultados obtidos dos testes de toxicidade crônica. Através dos ensaios de toxicidade agudo verificou-se que a ametrina é nove vezes mais tóxica do que o glifosato
Water is very importance for survive of life, the Corumbataí river provides water for Analândia, Corumbataí, Rio Claro and Piracicaba and others cities. The environmental toxicology has been disseminated how a tool in environmental monitoring, because through the bioassays is possible evaluate the quality of water body. The objective of this study was assess the quality of the water body in focus through chronic tests, rapid assessment protocols, determination of metals in suspended matter and acute toxicity of two herbicides (ametryn and gliphosate) with Ceriodaphnia silvestrii. The water samples were collected monthly between august 2009 to july 2010. For the 12 samples verified at least one or more samples has more iron concentration than maximum value permitted of CONAMA. The oscillation of the rapid assessment protocols scores was directed associated with visual and olfactory aspects. In chronic assays with Ceriodaphnia silvestrii has the variation in the reproduction of the organisms and through the statics analyzes was possible observed that the pH, dissolved oxygen conductivity, Zn, Ti and Ni influenced in the toxicity of sample. The herbicides ametryn and glyphosate showed the EC50 (48h)respectively 0.50 and 4.5 mg L-1. It is conclude that the chronic toxicity was influenced by physics- chemistries parameters and metals during the 12 months. The rapid assessment protocol complete the results from chronic tests of toxicity. Through of acute toxicity assays verified that the ametryn is nine times more toxic than glyphosate.

A dengue é um dos principais problemas de Saúde Pública da atualidade. É transmitida por mosquitos do gênero Aedes, sendo a espécie Aedes aegypti o vetor responsável pela transmissão no Brasil. O principal alvo de controle da doença é seu vetor, sendo o controle químico amplamente utilizado em todas as regiões atingidas. Como forma de prevenção ou em períodos de epidemia, torna-se necessária a eliminação dos mosquitos adultos, sendo indicada a nebulização a ultrabaixo volume (UBV) a frio do organofosforado malation por meio do uso de equipamentos pulverizadores acoplados a veículos. Este inseticida utilizado pode sofrer desvios durante e após a aplicação a UBV, caracterizando-se como um potencial agente de desequilíbrio ecológico, podendo atingir e gerar efeitos danosos em organismos não alvos aquáticos e terrestres. Este trabalho teve como objetivo desenvolver e validar um método de análise para o malation em cromatografia líquida de alta eficiência e obter recuperação do inseticida em níveis aceitáveis em água e solo, avaliar seu período de dissipação e tempo de meia vida (t1/2) nestas matrizes e classificá-lo quanto à toxicidade aguda e ao risco ambiental de acordo com diferentes autores, para o microcustáceo Daphnia magna, para a minhoca Eisenia foetida, para o peixe mato grosso (Hyphessobrycon eques) e para a macrófita Lemna minor. O método foi considerado adequado para análise do malation. A recuperação obtida para água foi de 97 por cento e para solo, 96 por cento . Efeitos tóxicos decorrentes da exposição ao inseticida foram observados para todos os organismos, sendo a Daphnia magna a espécie mais sensível aos testes de toxicidade aguda, com alto risco de intoxicação ambiental oriundo do uso peridomiciliar do malation. Em solo, o composto permaneceu na superfície com t1/2 de 4,5 h e sofreu lixiviação após ocorrência de chuva, sendo detectado por até 29 dias após a aplicação. Em água, o t1/2 foi de 0,1 h em pH 9.0 não sendo mais detectado a partir do segundo dia após aplicação. Em pH 7.0 e 5.0, esse período estendeu-se por até 7 dias, sendo o t1/2 de 3,2 e 4,4 d, respectivamente.
Dengue is one of the major public health problems nowadays. It is transmitted by mosquitoes of the genus Aedes, and the Aedes aegypti specie is the vector responsible for transmission in Brazil. The main target to control the disease is its vector, and the chemical control is widely used in all affected regions. As a preventive measure or in epidemic periods, it becomes necessary to eliminate adult mosquitoes, being indicated the ultralow volume nebulization of malathion organophosphate in cold way through the use of sprinklers attached to vehicles. This used insecticide can suffer deviation during and after application ULV, characterizing itself as a potential agent of environmental imbalance, with the possibility of reach and generate harmful effects on aquatic and terrestrial non-target organisms. This work had the objective to develop and validate a method of analysis for malathion in high performance liquid chromatography and obtain recovery of insecticide in acceptable levels in water and soil, evaluate its dissipation period and half-life time in these matrices and classify it on acute toxicity and environmental risk according to different authors, to the microcrustacean Daphnia magna, to the earthworm Eisenia foetida, to the fish Hyphessobrycon eques and to the macrophyte Lemna minor. The method was considered appropriate for analysis of malathion. The obtained recovery for water was 97 per cent and for soil, 96 per cent . Toxic effects resulting from exposure to the insecticide were observed for all organisms, resulting that Daphnia magna is the most sensitive species to the acute toxicity tests, with high risk of environmental intoxication originated from malathion peridomicilary use. In soil, the compound remained on the surface with t1/2 of 4.5 h and suffered lixiviation after rain occurrence, being detected for up to 29 days after application. In water, the t1/2 was 0.1 h in pH 9.0 and was not detected from the second day after application. In pH 7.0 and 5.0, this period was extended for up to 7 days, being the t1/2 of 3.2 and 4.4 d, respectively.

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Capes
Apple crops have huge importance to South Brazil economic, which concentrate more than 95% production in country. There are use of many pesticides in apples, with emphasis to fungicide Mancozeb and insecticide Chlorpyrifos. Although these substances have been developed to acting in a specific group of organisms, they are potentially dangerous to all alive organisms direct or indirectly exposed. Depending on persistence and toxicity, pesticides could interfering in soil basics process, how respiration and fauna soils activity, that directly influence nutrients cycling, organic matter decomposition and improvement physical chemical characteristics, being this products a risk to soil. Not application only, but handle and cleaning of machines with residues are dangerous to environmental too, and could induce punctual contamination. In some countries, to complete degradation of this waste, there is a bed for biological degradation, called Biobeds. Ecotoxicoly is a tool that could be used to measure pesticide risks and from their wastes to environmental receptor – since microorganisms until superiors vertebrates, having international protocols to application of assays. The aim of this study, was investigate toxicity caused by Mancozeb and Chlorpyrifos in two different Oxisols, representatives to apple crops, and effluent of pulverizer tank with wastes, to establish risks doses to three organisms from soil - Folsomia candida (Colembolla), Enchytraeus crypticus (Enchytraedae) e Eisenia andrei (Lumbricidae), using ISO protocols, and analyses Biobeds about its toxicity to organisms after contamination, comparing to natural soils, observing use possibility in handle of pesticides with spill simulation. Chlorpyrifos show higher toxic than Mancozeb, especially to Collembola (LC < 1 mg kg-1). Earthworm, organism used in Brazil’s law to approve pesticides was less sensible than other 18 19 for both products. Lethal and reproduction assays showed different risk doses in soils. Biobeds reduced waste pesticide risks during time and this not happen in one natural soil, that indicate dissolving products its not enough to reduce or eliminate the risks for soil organisms. In spill simulation, Biobeds was efficient in eliminate toxicity to earthworms and enchytraeids, although, not to Collembola and long time assays are necessary to reduce Chlorpyrifos toxicity to this organism. Due to large number of active ingredients using in agriculture actually, deep impact pesticide studies in soil organisms are necessary and alternatives of this wastes discharge
O cultivo de maçã tem grande representatividade na economia da região Sul do Brasil e uma série de agrotóxicos é utilizada, destacando-se o fungicida Mancozebe e o inseticida Clorpirifós. Apesar de essas substâncias serem desenvolvidas para atuar em um conjunto de organismos alvo, são potencialmente danosas para todos os organismos vivos expostos aos produtos. Dependendo de sua persistência e toxicidade, os agrotóxicos podem interferir em processos básicos do solo, tais como a respiração e a atividade da fauna edáfica que reflete diretamente na ciclagem de nutrientes, decomposição da matéria orgânica e melhoria de atributos químicos e físicos, representando estes compostos um risco para o solo. O manuseio e limpeza de maquinário com resíduos o produto também são potencialmente perigosas ao ambiente, podendo causar contaminações pontuais. Em alguns países para a completa degradação deste resíduo, são utilizados leitos biológicos de descarte (Biobeds). A ecotoxicologia é uma ferramenta que pode ser utilizada para mensurar o risco dos agrotóxicos e seus resíduos a receptores ambientais. O objetivo desta dissertação foi avaliar a toxicidade causada pelos ingredientes ativos Mancozebe e Clorpirifós em dois solos representativos para a cultura da maçã no Sul do Brasil – Latossolo e Nitossolo, e do efluente do tanque pulverizador com resíduos destes produtos, a fim de estabelecer dosagens de risco a três organismos representantes da fauna edáfica – Folsomia candida (Colembolla), Enchytraeus crypticus (Enchytraedae) e Eisenia andrei (Lumbricidae), e analisar Biobeds quanto a sua toxicidade aos organismos após sua contaminação, quando comparado aos solos analisados, observando a possibilidade de uso dos Biobeds para descarte de efluentes de agrotóxicos e como contensão em caso de derrame acidental. Foram realizados ensaios laboratoriais utilizando 14 15 protocolos ISO para ensaios letais (14 dias) e subletais (28 dias para colêmbolos e enquitreídeos e 56 dias para minhocas) de ecotoxicidade com os organismos indicados em câmara a 20ºC e fotoperíodo com 8/16 horas de luz/escuro. O Clorpirifós mostrou-se mais tóxico que o Mancozebe, principalmente aos colêmbolos, que são afetados em ensaios letais e subletais por doses < 1 mg kg-1. As minhocas, únicos organismos utilizados na legislação brasileira para credenciamento de agrotóxicos no mercado mostraram-se menos sensíveis que os colêmbolos e enquitreídeos aos dois produtos testados para ensaios letais (CL50), e subletais de reprodução (CE50), que apresentaram doses de risco distintas, e o Latossolo apresentando maior toxicidade quando contaminado pelos agrotóxicos testados. Os Biobeds reduziram a toxicidade dos agrotóxicos ao longo do tempo para os organismos edáficos, o que não foi observado no Latossolo, mostrando que mesmo um resíduo muito diluído, como o da lavagem do equipamento de aplicação, pode oferecer risco a fauna do solo ao longo do tempo. Na simulação de derrame acidental, os Biobeds foram eficientes em eliminar a toxicidade para minhocas e enquitreídeos, mas não para os colêmbolos, sendo necessários ensaios a longo prazo para determinar a possibilidade de redução da toxicidade do Clorpirifós aos organismos nesta situação. Devido ao grande número de ingredientes ativos utilizados na agricultura atualmente, são necessários estudos mais profundos do impacto destes agentes químicos à fauna edáfica, e também das alternativas de descarte dos resíduos oriundos dessa atividade

The Severn Estuary and Bristol Channel have a long history- of metal contamination from anthropogenic sources. Nonetheless, the Estuary- supports a large and varied faunal community including a large population of the brown shrimp Crangon crangon, which is a key species in the ecosystem. The aim of this study was to detect the impacts of chronic metal contamination in the Severn Estuary by studying the effects of metal on shrimp at the individual and population level. Shrimp were collected from the Severn Estuary and Bristol Channel and, for comparison, from the Greater Thames Estuary and analysed for metals. Metals were highest in shrimp from the Severn Estuary where cadmium was very- bioavailable. Cadmium concentrations were up to 30 times higher in Severn shrimp (15 ug g-1) than in Thames shrimp (0.5 ug g-1 although the two sites had similar environmental conditions. Chronic environmental metal exposure of Severn Estuary shrimp may have produced a cadmium tolerant population. Severn shrimp had a significantly higher LC50 and LT50 than reference shrimp from an uncontaminated site and greater levels of metallothionein were induced by cadmium exposure. Metallothionein may act as a mechanism of tolerance by sequestering harmful metal ions. Prey consumption and burying success was reduced in crangon after exposure to environmentally realistic concentrations of cadmium that were 1O fold lower than the 96 h LC50' Impairment of these behaviours may alter fitness and have consequences for population increase. However, Severn Estuary shrimp were more successful than the reference shrimp indicating a greater tolerance of the sub-lethal effects of cadmium exposure. This study has shown that Severn Estuary shrimp are able to accumulate exceptionally high levels of cadmium and show increased tolerance to acute and sub-lethal effects of cadmium exposure. These adaptations exemplify the environmental impact of heavy metal contamination in the Severn Estuary.

As the nanotechnology industry grows rapidly, the release of nanoparticles (NPs) will increase in the environment. The special functional properties of NPs bring uncertainty on how they will affect exposed organisms. Aquatic organisms may be particularly at risk because this environmental compartment acts as a sink for most contaminants. Two major NPs used in the nano industry are silver (Ag) and titanium dioxide (TiO2). Ag is being exploited principally for its antimicrobial properties and TiO2 for its photocatalytic properties. There is evidence that both of these particles can induce harmful effects in exposed organisms but our understanding on the mechanisms for their effects, uptake and fate of these NPs is still very limited, especially for environmentally relevant exposure regimes. The studies conducted during this thesis investigated the potential for biological effects to Ag and TiO2 in the aquatic environment using the zebrafish (Danio rerio). Uptake and effects of a range of NPs of different sizes (Ag -10nm, 35nm and a bulk counterpart 600-1600nm, and TiO2 -7nm, 10nm, 35nm and a bulk counterpart 134nm) were assessed in zebrafish embryos exposed via the water column. It was established that TiO2NPs had no adverse effects on zebrafish embryos even at an exposure concentration of 25 000 µg/L. In contrast, AgNPs induced dose dependent lethality and the AgNP (35nm) was most toxic. At lower exposure concentrations Ag induced a number of morphological defects in embryos and apoptosis was seen to occur around 7 hours post fertilisation (hpf) in the extended yolk sac region of the embryo. Coating the AgNP materials with citrate and fulvic acid significantly reduced toxicity. Coherent Anti Raman Scattering (CARS) microscopy was applied to the exposed embryos and indicated that there was little, if any uptake of Ag/TiO2 NPs as particles into the embryo. CARS however showed very significant aggregation of both NPs at the chorion surface. It was concluded from this work that the toxicity observed was most likely mediated via silver ions dissociating from the AgNPs. In this work, the metallothionein (mt2) gene was activated (detected using whole mount in-situ hybridisation-WISH) at sublethal exposure concentrations (500 µg AgNP/L and 12 µg AgNO3/L) in the extended yolk sac region of the embryo (24hpf), which is known to be a site of metal detoxification. In a second series of studies to explore the mechanisms and target tissues for AgNPs, WISH was adopted in early life stages of zebrafish using various genes that were markers of stress responses. These genes were metallothionein (mt2), glutathionine S-Transferase Pi (gstp), glutathionine S-Transferase Mu 1 (gstm1), hemeoxygenase 1 (hmox1) and ferritin heavy chain 1 (fth1). In the first instance the ontogeny of expression of these genes was established for up to 12 days post fertilisation (dpf) to determine the optimal time point to test for responses of these genes for the particle exposures. Early life stages of the zebrafish were exposed to citrate coated AgNP (10nm, 500 µg/L), Ag bulk (160nm, 500 µg/L) and silver ions (20 µg/L). The Ag materials induced upregulation in mt2, gstp and gstm1 in various target tissues including the yolk sac, olfactory bulbs, lateral line neuromasts, ionocytes in the skin and in regions of the head. Silver ions affected the same target tissues and induced the same gene responses as AgNPs, albeit there were differences in the levels of these gene responses between these two treatments. In contrast, both hmox1 and fth1 were downregulated as a result of Ag exposure. To further explore the molecular mechanism by which AgNP toxicity occurs, an Nrf2 (a transcription pathway involved in oxidative stress) mutant zebrafish was included in exposures to Ag. These studies found that mt2 and gstp were both expressed at lower levels in the Nrf2 mutant zebrafish exposed to Ag materials compared with in the wild type zebrafish. This suggests that the Nrf2-Keap pathway plays a key role in controlling the expression of these genes that are responding to the AgNPs exposures. This work demonstrated that WISH provided a highly effective integrative approach for identifying target tissues exposure to the different silver materials and for exploring functional pathways of effects. In the final study of this thesis, adult breeding zebrafish were fed AgNP via the diet to investigate uptake and accumulation into target organs, the possibility of maternal transfer and the potential for subsequent effects in exposed offspring. Adult zebrafish were fed at a rate of 5µg Ag/g at 3% of their body weight to Ag materials (AgNP 10nm and AgB 600-1600nm) over a period of 26 days. No effects were seen in the adult fish on any of the measures taken of fitness (condition factor index, gonadsomatic index, hepatosomatic index, haematocrit index). Ag did not significantly affect fecundity (numbers of eggs spawned) or fertility (numbers of eggs fertilised). A detectable level of uptake of Ag in target organs occurred only in the AgNP treatments: liver (up to 2.1 µg/g in males) and gonads (up to 0.5 µg/g in males), suggesting particle size plays a role in the uptake and translocation through membranes into target organs and thus an enhanced bioavailability for AgNP compared with AgB. Maternal transfer was only detectable in the AgNP treatment (up to 0.43 ng Ag/per embryo). Gene responses of mt2 and gstp measured in the subsequent offspring via WISH analysis at 24 hpf indicated significant mt2 upregulation occurred after 2 weeks of adult exposure in both AgNP and AgB treatments. This demonstrates the potential for maternal exposure effects for exposure to AgNPs. Furthermore, challenging the subsequent embryos to the same Ag materials and measuring responses of mt2 via WISH indicated de-sensitisation to Ag in offspring where adults were treated with both nano and bulk forms of Ag. This finding has important considerations for risk assessments for silver base materials. Overall, the findings presented in this thesis have provided a body of evidence to show that silver material on a nanoscale may be of a greater hazard to fish and this warrants further investigation to consider more appropriately the potential risks associated with discharge of AgNPs into the aquatic environment.