Dissertations / Theses on the topic 'Aquatic metabolism'

This thesis explored the uptake into the freshwater shrimp (Gammarus pulex) and the water boatman (Notonecta glauca) of key pharmaceuticals drawn from different therapeutic classes and covering a range of physico-chemical properties. For one compound, uptake was also assessed using the freshwater snail Planobarius corneus. In G. pulex, bioconcentration factors (BCFs) ranged from 4.6 – 185900 and increased in the order moclobemide < 5-fluoruracil < carbamazepine < diazepam < carvedilol < fluoxetine. In N. glauca BCFs ranged from 0.1 – 1.6 and increased in the order 5-fluorouracil < carbamazepine < moclobemide < diazepam < fluoxetine < carvedilol. For P. corneus, the BCF for carvedilol was 57.3. The metabolism of the study pharmaceuticals in the shrimp was investigated. Diazepam was found to be metabolized by G. pulex and a metabolite was detected and tentatively identified as nordiazepam. For the other five study compounds no metabolites were observed and it was inferred that metabolism in G. pulex may not influence the BCF. The influence of dietary uptake was explored in the test organisms with carvedilol and fluoxetine. It was found that uptake from water was the predominant route of exposure for G. pulex but the data for N. glauca was contrasting and the exposure from the food was predominant. In both organisms a combination of food and water exposure resulted in a higher uptake of the compounds. The differences in degree of uptake from water across the organisms may be due to differences in mode of respiration, behaviour and the pH of the test system. The differences in degree of uptake from food across the organisms may be due to differences in feeding strategies. The degree of uptake of pharmaceuticals within an organism was related to the hydrophobicity of the pharmaceuticals.

Pharmaceuticals which are used worldwide are designed to facilitate the life for the human society and have an important role in treatment and prevention of disease for both humans and animals. They are ubiquitous in the aquatic environment and are mainly derived from municipal wastewater treatment plants (WWTPs) due to their low removal rate. Therefore, their presence in the environment is directly linked to the human impact. Various biological and abiotic processes in the environment can transform them to transformation products (TPs). In many cases, transformation is already initiated in the human body by a variety of drug-metabolizing enzymes. The metabolites formed through human metabolism present some modifications in their chemical structures that can differ in physicochemical properties to their parent compound. Once they are excreted from the human body, both the unmetabolised parent drug and their metabolites enter WWTPs by means of the sewer system. Since the WWTPs are not designed to remove completely pharmaceutical residues, the fraction not removed after the treatment will eventually end up in the receiving water bodies. Consequently, due to pharmaceutical transformations in the human body, biotransformations in WWTPs and phototransformations in surface water, they can potentially produce a high number of TPs in real world samples which makes their identification a challenge. In this thesis, two different approaches (TPs profiling and suspect screening) based on high resolution mass spectrometry (HRMS) for the detection and identification of TPs of pharmaceuticals were investigated. TPs profiling approach was applied for the identification of phototransformation products of an antiviral zanamivir (ZAN) in batch reactors filled with surface water. On the other hand, suspect screening approach was applied for evaluation of transformation, prioritization and identification of photoTPs of six iodinated contrast media in surface water. Finally, a combination of suspect and TPs profiling approach was applied for the detection of TPs of an anticonvulsant lamotrigine and its main human metabolite lamotrigine N2-glucuronide which were formed as the result of their degradation in both activated sludge and pH dependent hydrolysis. The TPs profiling approach for evaluation of these transformations is illustrated in the example of photodegradation of an antiviral ZAN with identification of its TPs in surface water (Chapter 3.). Here a set of lab- scale experiments was performed in order to determine the susceptibility of ZAN towards photodegradation under simulated and natural sunlight. The identification of the TPs was performed using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry (HILIC-HRMS) where four photoTPs were tentatively identified and their proposed structures were rationalized by photolysis mechanisms. Kinetic experiments showed that photodegradation kinetics of ZAN in surface waters would proceed with slow kinetics since upon exposure of aqueous solutions of surface water (20 μg L-1) to simulated sunlight, ZAN was degraded with t1/2 of 3.6 h. Under natural sunlight irradiating surface water, about 30 % of the initial concentration of the antiviral disappeared within 18 days. However, when ZAN and its TPs were retrospectively screened from surface water extracts, neither the parent nor the TPs were detected. The results of this TP profiling used for the identification of TPs of ZAN, although straightforward, suggests that it is not suitable when dealing with a considerably elevated number of TPs formed in batch experiments. However, time and effort needed to be optimised for the structure elucidation of 108 photoTPs of six iodinated contrast media (ICM) (Chapter 3.). Again, the photodegradation study was performed in surface water spiked with the ICMs using a sunlight lab-scale simulator. 108 TPs were generated and each photoTP was characterised by its unique exact mass of the molecular ion and retention time and added to a suspect list. Once the suspect list was generated, the photoTPs were searched in thirteen surface water samples which were extracted using a generic solid- phase extraction method (four cartridges of different chemistries in order to retain ample number of compounds with different chemical properties). Based on their detection frequency (those TPs with the frequency higher than 50 % were deemed important), eleven TPs were prioritized and their structures elucidated by HRMS and NMR (when possible). Out of the eleven prioritised TPs, ten were formed as the result of deiodination (either by deiodination, oxidative deiodination or intramolecular elimination). In the real surface water samples, median concentration of parent compounds was 110 ngL-1 reaching up to 6 µgL-1 for iomeprol while TPs were found at median concentration of 8 ngL-1, reaching up to 0.4 µgL-1 for iomeprol TP651-B. Here detection-based prioritization served as a crucial step to reduce the number of TPs to be identified and thereby reducing costs and time for the subsequent target analysis. This time-effective approach not only guaranties that the degradation products elucidated would be found, but also that they are environmentally relevant. In summary, the proposed screening approach facilitates the evaluation of the degradation of polar compounds at a real scale with a fast detection of TPs without prior availability of the standards. Approach used for detection and identification of TPs of ICM in Chapter 3 was an example of suspect screening where the suspect list of TPs was generated at lab-scale, In Chapter 4, the work started with a suspect screening of lamotrigine (LMG) and related compounds (its human metabolites, synthetic impurities and photoTPs) which were listed from the literature and searched in wastewater and surface water samples. As the result of suspect screening, LMG, three human metabolites and a LMG synthetic impurity (OXO-LMG) were detected in the screened samples. Preliminary results showed significantly higher concentrations of OXO-LMG in wastewater effluent, suggesting its formation in the WWTPs. However, biodegradation reactors amended with mixed liquor at neutral pH showed that LMG is resistant to biodegradation with only about 5 % elimination after 6 days. Since LMG is extensively and predominantly metabolised by phase II metabolism to its N2-glucuronide, this metabolite (LMG-N2-G) was degraded following the same experimental setup. Results showed that this metabolite was the actual source of the TP detected. Additionally, in batch experiments, LMG-N2-G was transformed, following pseudo-first kinetics, to three TPs as a result of i) deconjugation (to LMG), ii) oxidation of the glucuronic acid (to LMG-N2-G-TP430) and iii) amidine hydrolysis in combination with deconjugation (to OXO-LMG). In order to further rationalize the formation of the TP OXO-LMG, the stability of LMG-N2-G and related compounds was studied as a function of pH in the range of 4 – 9. Same as during biodegradation, LMG was stable across the entire pH range tested. However, LMG-N2-G was transformed to three TPs at neutral – basic pH. They were identified as TPs formed after hydrolysis of amidine and guanidine moieties. The third TP detected was an intermediated in the guanidine hydrolysis reaction. Kinetic experiments in wastewater samples at different concentration (20 and 200 nM) and pH (pH 6.5, 7, 8, 8.5 and 9) demonstrated that while the degradation constants were concentration independent, at higher pH, LMG-N2-G degraded at higher rate. The pH-dependent stability experiments of related compounds with different nitrogen N2-substituents on the 1,2,4-triazine ring showed that reaction of amidine and guanidine hydrolysis depends on imine tautomer equilibrium whose formation depends directly on the N2-supsitutent. LMG- N2-G major abiotic TP (amidine hydrolysis TP) was detected in hospital effluent and WWTP influent samples. Having in mind the concentrations of both biotic and abiotic TPs detected, a total mass balance at two- concentration levels batch reactors was closed at 86% and 102%, respectively. In three WWTPs total mass balance of LMG-N2-G ranged from 71-102%. Finally, LMG-N2-G and its TPs were detected in surface water samples with median concentration ranges of 23–186 ngL-1. The work presented in this chapter gives a new insight into the behaviour of glucuronides of pharmaceuticals, suggesting that they might also be sources of yet undiscovered, but environmentally relevant TPs.
Els productes farmacèutics, l'ús dels quals s'estén a nivell mundial, estan dissenyats per millorar la qualitat de vida de la societat i juguen un paper clau en el tractament i la prevenció de malalties, tant en homes com en animals. Aquests compostos químics es troben de forma ubíqua en el medi ambient. Això es deu principalment a les estacions depuradores d'aigua residual (EDARs), les quals no són capaces d'eliminar de manera eficient aquest tipus de compostos, ja que no estan dissenyades amb aquesta finalitat. Per tant, la presència de fàrmacs en el medi ambient està directament relacionada amb l'activitat humana. Un cop al medi ambient, l'estructura d'aquests compostos pot ser modificada per diferents processos biològics i abiòtics, generant-se així els que es coneixen com a productes de transformació (PTs). De fet, la transformació dels fàrmacs pot iniciar-se en alguns casos en el cos humà, després de la seva administració a causa de l'activitat metabòlica dels diferents enzims que posseeix l'home. Els metabòlits formats en aquests processos presenten algunes modificacions en les seves estructures químiques pel que fa al compost original, i, en conseqüència, unes propietats fisicoquímiques diferents. Un cop excretats, tant el fàrmac original no metabolitzat com els seus metabòlits arriben a les EDARs mitjançant la xarxa de sanejament municipal d'aigües residuals. La fracció d'aquests compostos que no s'elimina en els diferents tractaments realitzats en l'EDAR, es descarrega juntament amb l'efluent de la planta als aigües receptores. El gran nombre de transformacions que poden experimentar els fàrmacs en el seu cicle de vida a causa del seu metabolisme en el cos humà, la seva biotransformació per microorganismes i la seva fototransformació per llum solar, pot generar un nombre molt elevat de PTs en el medi ambient, i, per tant, la identificació dels mateixos, necessària per avaluar el destí dels fàrmacs en el medi ambient, és un desafiament. En el desenvolupament d'aquesta tesi es van aplicar dues aproximacions analítiques diferents: a)avaluació de perfils de PTs generats en experiments a escala de laboratori i b) anàlisi qualitativa dirigida suspect screening en mostres reals, tots dues basades en espectrometria de masses d'alta resolució (HRMS) per a la detecció i identificació de PTs de productes farmacèutics. L'aproximació d'avaluació de perfils de PTs en reactors a escala de laboratori es va aplicar per identificar productes de fototransformació (fotoPTs) de l'antiviral zanamivir (ZAN) en aigua superficial. L'aproximació de suspect screening es va aplicar per prioritzar i identificar fotoPTs de sis mitjans de contrast radiològics iodats (ICM) en aigua superficial. Finalment, una combinació de les dues aproximacions es va aplicar per detectar PTs de l’anticonvulsiu lamotrigina (LMG) i del seu principal metabòlit humà, el lamotrigina-N2-glucurònid (LMG- N2-G), resultants de la seva degradació tant en fangs activats com a reaccions d'hidròlisi a diferents valors de pH.

This research explored fate of organic contaminants in aquatic plant systems through (i) experimental development of relationships to describe sorption, uptake and enzymatic processing of contaminants by plants and inhibition of aquatic plants by contaminants and (ii) incorporation of experimental relationships into a conceptual model which describes contaminant fate in aquatic plant systems. This study focused on interactions of aquatic plants L. minor and M. aquaticum with halogenated phenols. 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4-dichlorophenol (2,4-DCP) are precursors for the highly toxic and heavily applied herbicides 2,4,5-T and 2,4-D and were examined in detail. Chlorophenols are generally resistant to microbial degradation, a property which may limit microbial remediation options as effective alternatives for clean up of contaminated sites. Relationships for fundamental interactions between plants and contaminants that dictate uptake, enzymatic processing and sequestration of contaminants by aquatic plants were established. An assay which quantified production of oxygen by plants was developed to quantify plant metabolic activity and inhibition. Uptake of chlorinated phenols depended on plant activity and aqueous phase concentration of contaminant in the protonated form. Therefore, plant activity, contaminant pKa and media pH were established as critical parameters controlling rate of contaminant uptake. A conceptual model was developed which incorporated plant activity and inhibition into a mathematical description of uptake of organic contaminants by aquatic plants. The conceptual model was parameterized using experimental data delineating effect of plant activity, inhibition and speciation on contaminant uptake and the model was verified using independently gathered data. Experimentation with radio-labeled chlorinated phenols established that contaminants were sequestered internal to plants by plant enzymatic processing. 19F NMR was established as a technique to quantify transformation and conjugation products internal to plants and contaminant assimilation by plants and demonstrated that multiple metabolites containing the parent compound were present and quantifiable internal to plants. Finally, fate of plant-sequestered contaminants in an anaerobic bioassay was examined using Desulfitobacterium sp. strain Viet1. The results of this study address the role of aquatic plants in sequestration of contaminants in surface waters that indicate the potential and limitations of use of aquatic plants in natural and engineered treatment systems.

Dominant genera of bacteria were isolated from three river waters during anthracene and pentachlorophenol biotransformation studies. The genera Pseudomonas, Acinetobacter, Micrococcus, Chromobacterium, Alcaligenes, Azomonos, Bacillus, and Flavobacterium were capable of biotransforming one or both of these compounds. These isolates were subjected to further biotransformation tests, including river water and a basal salt medium with and without additional glucose. The results of these experiments were evaluated statistically. It was concluded that only a limited number of the bacteria identified were able to transform these chemicals in river water. The addition of glucose to the growth medium significantly affected the biotransformation of these chemicals. It was also determined that the size of the initial bacterial population is not a factor in determining whether biotransformation of anthracene or pentachlorophenol can occur.

Diel dissolved oxygen (DO) data were used to characterize seasonal, inter-annual, and longitudinal variation in production and respiration for the James River Estuary. Two computational methods (Bayesian and bookkeeping) were applied to these data to determine whether inferences regarding DO metabolism are sensitive to methodology. Net metabolism was sensitive to methodology as Bayesian results indicated net heterotrophy (production < respiration) while bookkeeping results indicated net autotrophy (production > respiration). Differences in net metabolism among the methods was due to low seasonal variation in respiration using the Bayesian method, whereas bookkeeping results showed a strong correlation between production and respiration. Bayesian results suggest a dependence on allochthonous organic matter (OM) whereas bookkeeping results suggest that metabolism is dependent on autochthonous OM. This study highlights the importance in considering the method used to derive metabolic estimates as it can impact the assessment of trophic status and sources of OM supporting an estuary.

I investigated how anthropogenic acidification influences stream metabolism and nitrogen (N) cycling by considering the stress response of microbial compartments responsible for these ecosystem processes. Microcosm incubations of leaf biofilms from streams of differing pH revealed greater rates of fungal biomass-specific respiration (i.e. the stress metric qCO₂) and biomass-specific N uptake (i.e. qN) with increasing acidity. The positive relationship between qCO₂ and qN indicated alternate fates for N other than structural biomass, possibly related to increased exoenzyme production as part of the stress response. Whole-stream ¹⁵N experiments and measurements of respiration and fungal standing crop across the pH gradient resulted in similar patterns in qCO₂ and qN found in microcosm experiments, supporting qCO₂ as an ecosystem-level stress indicator and providing insight towards controls over N cycling across the pH gradient. Fungal biomass and ecosystem respiration declined with increasing acidity while N uptake metrics were not related to pH, which suggested qN in acid streams was sufficiently high to counteract declines in fungal abundance. During spring, chlorophyll a standing crops were higher in more acidic streams despite lower nutrient concentrations. However, N uptake rates and gross primary production differed little between acid and circumneutral streams. Reduced heterotrophy in acid streams was apparent in lower whole-stream respiration rates, less ability to process organic carbon, and little response of N uptake to added carbon resources. Overall, acid-induced stress in streams was found to impair decomposer activity and caused a decoupling of carbon and nitrogen cycles in these systems.
Ph. D.

The rocky intertidal zone, experiencing fully marine and fully terrestrial conditions, has become increasingly investigated as a model ecosystem for studying the future implications of climate change. The barnacle, Balanus glandula, a common rocky intertidal inhabitant, plays an important role as a key prey item for many organisms. Low tide can be particularly challenging for barnacles as they are marine organisms subjected to the abiotic conditions of a terrestrial environment. The most stressful of these are increased temperature and decreased oxygen availability. This study aimed to investigate how low tide impacts the energy budget, specifically the digestion, of B. glandula. Barnacles are unable to feed at low tide however, if they were able to digest at low tide, they could maximize their energy intake by emptying their stomach to prepare to feed at the next high tide. However, digestion is a metabolically costly activity, which could make it less energetically favorable to digest when there’s less oxygen available. To test for an effect of low tide on digestion, barnacles were fed, and the time to first fecal production measured as a ‘baseline’. This was repeated, but barnacles were exposed to either a 16ºC or 35 ºC low tide immediately after being fed. The change in digestion time was calculated by comparing these two times for each barnacle. It was found that regardless of temperature, barnacles delayed their digestion by about 50-60 minutes after exposure to a one hour low tide. To determine the energetic cost of digestion, the rate of oxygen consumption was compared between starved and digesting barnacles. I was unable to detect any evidence of elevated metabolic activity during digestion. Additional testing is needed to confirm these results as the barnacles may have not fed during the trial, thus had no food to digest. While it appears that increasing temperatures associated with climate change will have little impact on the digestion of barnacles at low tide, if climate change alters the duration of low tide, there could be an energetic impact to barnacles due to the slowing of their metabolism as indicated by the delay in their digestion.

Over the past few decades, there has been increased research focus on carbon cycling within aquatic systems, especially with the changing global climate. Inland waters play a major role in the global carbon cycle, but the fundamental features remain poorly understood, particularly the large lakes of the world. Our experimental approach assessing the carbon budget of Lake Superior, the largest freshwater lake by area, provides spatial and temporal variability that has been previously overlooked but may be critical to our understanding on the biogeochemical processes controlling the lake. Multiple stations were chosen across the lake, both nearshore and offshore, to evaluate the variability in physical mixing regimes and biogeochemical processing. Short and long-term carbon consumption measurements were coupled to assess the heterotrophic activity relative to the lability of dissolved organic carbon. Partial pressure of carbon dioxide (pCO2) was directly measured to determine the metabolic nature of the lake and the amount of carbon dioxide (CO2) that fluxes across the air-water interface. The pCO2 results were further coupled with an isotopic approach measuring oxygen-18 (δ18O) to evaluate how the metabolism of Lake Superior has changed over a decadal scale. A range of environmental factors, including temperature, photodegradation and source/quality of organic carbon, influenced short and long-term carbon consumption. In-situ pCO2 observations supported a temporal switch in metabolism from the lake being a source of CO2 in the spring to being a sink in the summer driven by biological components of the system. When the pCO2 results were coupled with the isotopic measurements over the past decade (1999-2011), Lake Superior was dominated by respiration during isothermal conditions and production during stratification. In the past decade, Lake Superior has experienced increased surface water temperatures, shifting the metabolic state to a shorter net heterotrophic period in the spring and a longer net autotrophic period in the summer. This research highlights fundamental aspects of Lake Superior’s metabolism that have been previously understudied, as well as providing key information about processes controlling its carbon budget, and giving a better understanding of how climate change will continue to impact Lake Superior.

The main diet of herbivorous waterfowl, such as swans, coots, and ducks, consists of submerged macrophytes. The grazing by waterfowl below the surface may affect the biomass and species composition of the submerged plants. The vegetations is, however, also affected by e.g. water turbidity, periphyton growing on the plants and water temperature. This literature study aimed to assess the impact of waterfowl on the biomass and production of submerged vegetation in shallow, eutrophic lakes and how this may vary with the season. The review showed that the density of submerged vegetation biomass varies during the seasons but tends to be highest in the summer. Waterfowl density is usually highest during autumn migration. According to experimental field studies in which waterfowl were excluded from protected areas, showed that protected plants had longer shoots and fewer stumps and, in several studies, also higher biomass, than in areas where waterfowl resided free. Although several studies have shown that waterfowl affect the biomass of macrophytes in different ways, is still uncertain to what degree and whether this has a negative or positive impact on the species composition of vegetation in a longer time perspective. However, alternative state shifts in shallow eutrophic lakes from a clear to a turbid water state have not been shown to be caused by waterfowl. With climate change, the time and place of waterfowl migration and wintering will most probably change. This could lead to altered pressure and seasonality of the grazing by waterfowl on submerged vegetation.
Växtätande sjöfåglars diet består huvudsakligen av undervattensvegetation. Fåglarna betar av växterna oftast under ytan, men undervattensvegetationens biomassa och artsammasättning påverkas inte bara av sjöfåglar, utan också av en rad andra faktorer, till exempel vattnets grumlighet, påväxtalger och vattentemperatur. Syftet med denna litteraturstudie var att undersöka under vilka förutsättningar betning från sjöfåglar har betydelse för undervattensvegetation. Växternas biomassa har visats minska i samband med sjöfåglars födointag, om den huvudsakligen består av vattenväxlighet. Vegetationens täthet varierar under året men uppnår störst täthet under sommaren. Sjöfåglar har visats vara i störst täthet under höstmånaderna, i samband med flyttning. Experimentella studier har genomförts genom att jämföra skyddade områden där sjöfåglar utestängts med öppna områden där sjöfåglar kunnat beta. I de skyddade områdena var växterna generellt längre och hade flera skott. Där fanns också färre stubbar än i de öppna områdena. Flera studier har visat att sjöfåglarna påverkar vegetationens biomassa på olika sätt men det är fortfarande osäkert i vilken grad och om detta över längre tid har en negativ eller positiv påverkan på vegetationens artsammansättning. I och med nutida klimatförändringar kan sjöfåglars tidpunkt och plats för migration och övervintring att förändras. Detta kan leda till att sjöfåglarna inte emigrerar från sjöar och kommer därav med största sannolikhet påverka undervattensvegetationen.

Fish may be exposed to an array of pharmaceuticals that are discharged into the aquatic environment, paralleling advances in medical knowledge, research and technology. Pharmaceuticals by their nature are designed to target specific receptors, transporters, or enzymes. Nuclear receptors (NRs) are often a key component of the therapeutic mechanism at play, and many of these are conserved among vertebrates. Consequently, fish may be affected by environmental pharmaceutical exposure, however there has been relatively little characterisation of NRs in fish compared with in mammals. In this thesis common carp (C. carpio) were exposed to selected pharmaceuticals in vitro and in vivo to investigate effects centred on the pregnane X receptor (PXR) and peroxisome proliferator-activated receptor alpha (PPARα), two key NRs involved in organism responses to pharmaceutical exposure. The PXR acts as a xenosensor, modulating expression of a number of xenobiotic metabolising enzymes (XMEs) in mammals. In a primary carp hepatocyte model it was shown that expression of a number of XMEs was altered on exposure to rifampicin (RIF), as occurs in mammals. This response was repressed by addition of ketoconaozle (KET; PXR-antagonist), indicating possible PXR involvement. The genes analysed showed up-regulation on exposure to ibuprofen (IBU) and clofibric acid (CFA), but not clotrimazole (CTZ) or propranolol (PRP). The lack of response to mammalian PXR-agonist CTZ was unexpected. In contrast, the same XME genes were found to be up-regulated in vivo after 10 days of exposure of carp to CTZ, although this response occurred only for a relatively high exposure concentration. CTZ was found to concentrate in the plasma (with levels up to 40 times higher than the water). Development and application of a reporter gene assay to measure PXR activation in carp (cPXR) and human PXR showed CTZ activation of cPXR, supporting data from the in vivo studies. Furthermore, activation was seen at concentrations as low as 0.01 μM. Interestingly RIF did not induce a response in the cPXR reporter gene assay, contrasting with the hepatocyte culture work. Taken together, the data presented here suggests divergence in the PXR pathway between mammals and fish in terms of ligand activation and downstream gene targets. PPARα was investigated in carp in vivo using CFA as a mammalian PPARα-agonist. Overall the resulting data suggested a broadly similar role for this NR in lipid homeostasis in fish as for mammals, with a number of PPARα-associated genes and acyl-coA oxidase (ACOX1) activity up-regulated in response to CFA exposure. A number of XMEs were also up-regulated by CFA (in vivo and in vitro), potentially extending the role of PPARα in fish (carp) to regulation of xenobiotic metabolism. The work presented has provided further characterisation of PXR and PPARα in fish. Elucidation of these pathways is vital to provide meaningful data in terms of establishing toxicity and mechanism-of-action data for pharmaceuticals and other compounds in fish, to allow validation of read-across approaches and ultimately aid in their environmental risk assessment. In vitro approaches are attractive ethically, financially and can provide useful mechanistic characterisation of compounds and the primary hepatocyte model and reporter gene assays used here show potential for the screening of pharmaceutical compounds in fish. However, further understanding of the metabolism of drugs and chemicals in fish is required to establish the true value of these methods for informing on possible effects in fish, in vivo.

Aquatic eutrophication is a serious global problem, associated with phytoplankton blooms, hypoxia, and loss of species. The objective of this thesis was to advance understanding of stream and lake eutrophication within Canterbury (South Island, New Zealand). I investigated three key questions: 1) How do riparian characteristics control stream trophic state, 2) how does stream trophic state in the Canterbury region compare to stream trophic state nationally and internationally, and 3) what factors control trophic state in Te Wairewa/Lake Forsyth. I measured rates of stream community metabolism in 21 Canterbury streams over a gradient of riparian canopy cover, and conducted a literature review of national and international studies of stream metabolism. I also examined the occurrence of cyanobacterial blooms in Te Wairewa in relation to water quality and weather from 17 years of measurements, and performed series of nutrient addition assays on the lake to assess nutrient limitation. I found that riparian characteristics strongly controlled stream trophic state by shading, thereby reducing photosynthetic productivity. This overwhelmed the effects of high nitrate concentrations, which increased primary production. Compared to national and international rates of stream metabolism, Canterbury streams were strongly heterotrophic, with low rates of autotrophic production. Catchment streams draining into Te Wairewa were unlikely to be the main source of nutrients supporting large cyanobacterial blooms. Instead, internal lake nutrient loading mechanisms associated with calm weather were likely to supply blooms. My results emphasize the importance of light limitation, nitrogen and heterotrophy in controlling stream trophic state, and nutrient supply and weather in controlling lake trophic state.

This research examined the effects of acclimation to lowered salinity, elevated temperature, and hypoxia on aerobic metabolism of the Gulf killifish, Fundulus grandis, a common estuarine resident of the Gulf of Mexico. Standard metabolic rate (SMR), maximum metabolic rate (MMR), absolute aerobic scope (AAS), and critical oxygen tension (Pcrit) were each influenced by one or more acclimation treatments. Assessing the consistency of these traits measured in the same individuals over time, all were found to be significantly repeatable with no indication that the repeatability of any traits was affected by acclimation conditions. Significant correlations were found between SMR and Pcrit (positively correlated), between SMR and AAS (negatively correlated), between MMR and AAS (positive), and between AAS and Pcrit (negative). This study, therefore, documents the effects of acclimation on these traits, their repeatability, and correlations among them. It further suggests that repeatability of these traits is not context dependent.

As águas superficiais da Amazônia, que englobam, dentre outros, rios das mais variadas ordens, desde pequenos igarapés, até o rio Amazonas, um dos maiores do mundo, são um componente importante do funcionamento deste ecossistema de escala continental. Um paradigma acerca dos sistemas fluviais é que estes integram os processos que ocorrem em suas bacias de drenagem e, portanto, alterações na cobertura vegetal e nos usos da terra, podem interferir diretamente nos parâmetros físicos e químicos dos compartimentos aquáticos. Algumas áreas da Amazônia, como o estado de Rondônia, por exemplo, têm sofrido alterações substanciais nas suas bacias de drenagem e as conseqüências destas nos sistemas fluviais ainda são pouco compreendidas. Sabe-se que a conversão de florestas em pastagens, o tipo de alteração mais comum nos ecossistema de Rondônia, resulta na compactação dos solos e no aumento da erosão. Ao mesmo tempo, diversos estudos têm demonstrado que os sedimentos em suspensão, carreados nos rios, podem ser importantes fontes de carbono e outros nutrientes limitantes para o metabolismo aquático. Com base nestas premissas, este estudo visou avaliar a importância dos sedimentos em suspensão grossos (maiores que 63 µm) e finos (menores que 63 µm e maiores 0,1 µm) no metabolismo de alguns rios de Rondônia, sob diversas coberturas e usos da terra. Para tal, as concentrações destes sedimentos foram artificialmente aumentadas em amostras incubadas no escuro, nas quais os consumos de oxigênio indicaram as taxas respiratórias. No caso dos sedimentos em suspensão finos, observaram-se aumentos nas taxas respiratórias em praticamente todos os casos nos quais suas concentrações foram aumentadas. Os sedimentos em suspensão grossos, por outro lado, somente favorecem aumentos nas taxas respiratórias nos períodos de maiores precipitações. Estes resultados relacionam-se com a composição dos sedimentos. Sedimentos em suspensão finos, por sua maior capacidade de adsorver substâncias orgânicas e nutrientes, constituem uma fonte permanente de substrato para o metabolismo aquático. Sedimentos em suspensão grossos, por outro lado, somente carreiam fração substancial de material orgânico durante o período de maiores precipitações, quando os solos são “lavados” pelas enxurradas. Apesar destas diferenças no papel relativo destas duas frações do material particulado no metabolismo destes rios, fica evidente que o aumento da erosão pode acarretar mudanças significativas no metabolismo dos sistemas fluviais da Amazônia.
Amazonian surface waters, encompassing rivers of distinct orders, from small streams to one of the largest of the world, the Amazon, are important components of the functioning of this continental-scale ecosystem. A paradigm about fluvial systems is that they integrate the processes that occur at their watersheds and, therefore, changes in land use/cover may directly interfere on physical and chemical parameters of the aquatic compartments. In some areas of the Amazon, such as in the state of Rondonia, for example, the landscape has been significantly altered, and the consequences of these changes are still poorly understood. It is well know that the conversion of forests into pastures, a common type of land use change in Rondonia, can cause the compactation of soils and increased erosion rates. At the same time, several studies have demonstrated that suspended sediments can be important sources of carbon and other nutrients to river metabolism. Based on those assumptions, this study aimed the evaluation of the importance of coarse (larger than 63 µm) and fine (smaller than 63 µm and larger than 0,1 µm) suspended sediments on the metabolism of some rivers of Rondonia. This assessment was made through incubations in the dark of samples enriched in sediments, in which the consumption of oxygen indicated respiration rates. In the case of fine suspended sediments, most incubated samples showed increased respiration rates as a result of the concentration of these particles. Coarse suspended sediments, on another side, only favor the increase in respiration rates during the high water period. These results are related to the composition of these sediments. Fine suspended sediments constitute a constant substract for the aquatic metabolism, as a result of their larger surface area and capacity for absorving organic substances and nutrients. Coarse sediments, however, only favor respiration during the high water period, when overland flow carries organic debris from land into the water systems. Although these two fractions of suspended sediments have different relative roles in the metabolism of rivers, it becomes evident from this data that increasing the erosion in the Amazon may affect significantly the metabolism of fluvial systems in this region.

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As evidências da contaminação das águas por resíduos de medicamentos e seus subprodutos levou esse grupo de resíduos a compor a lista de poluentes orgânicos emergentes, como consequência da expansão do uso de medicamentos, como o antidepressivo cloridrato de fluoxetina e o anti-inflamatório diclofenaco. Diversos Processos Oxidativos Avançados vêm sendo aplicados para a degradação destes compostos. Dentre eles, o processo de irradiação com feixe elétrons obteve bons resultados na remoção de toxicidade e degradação de fármacos. O presente estudo consistiu em aplicar radiação ionizante como uma possível tecnologia para degradar os fármacos em águas. A irradiação de solução aquosa contendo os fármacos foi aplicada usando acelerador de elétrons, cuja eficiência foi discutida mediante análises químicas (Cromatografia Líquida Ultra Rápida e Carbono Orgânico Total (COT)), ecotoxicológicas (ensaios de toxicidade com Vibrio fischeri e Daphnia similis) e biológicas (Ensaios Respirométricos). Os resultados de COT indicaram mineralização não significativa dos compostos, mesmo sendo observada degradação máxima de 99,9% para o diclofenaco e 55% para o cloridrato de fluoxetina na mistura (1:1) em 5.0 kGy. Foi observada toxicidade aguda dos fármacos, sendo mais acentuada para a fluoxetina, seguido do diclofenaco e, finalmente, da mistura para V. fischeri. Quando D. similis foram empregadas nessa avaliação, a ordem de toxicidade foi de fluoxetina, a mistura de ambos os medicamentos e do diclofenaco. Além disso, foi observada remoção de toxicidade nas amostras irradiadas em todas as doses aplicadas para a bactéria V. fischeri, com maior eficiência de remoção de toxicidade de 55%, em 5 kGy, na mistura dos dois fármacos. Para a D. similis, foi observada remoção significativa de toxicidade da mistura apenas na dose 2,5 kGy. Os ensaios respiroétricos não indicaram biodegradabilidade após o tratamento.
Dissertação (Mestrado em Tecnologia Nuclear)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Global biodiversity is declining as a direct result of anthropogenic climate change. Ectothermic species have become focal organisms for studying the ecological effects of altered climates due to the clear relationship between environmental temperatures and ectotherms’ basic physiological functions. Historically, examinations of these effects have focused heavily on heliothermic lizards, and most others have tended to focus on single populations or sympatric species within a single community. Addressing the longterm energetic implications of environmental temperature variation will provide valuable insight into the cascading physiological effects that certain populations or species may experience as a result of altered climates. In this study, we used thermal and behavioral data collected between 2010 and 2017 from four distinct populations of Pacific rattlesnakes (Crotalus oreganus) on the Central Coast of California. Two of these populations occupy thermally mild, coastal habitats while the other two occupy more thermally dynamic, inland habitats. Using operative temperature models, surgically implanted temperature loggers, and radiotelemetry, we collected data on the thermal microhabitats available within each of these study sites as well as field active body temperatures for 85 individual snakes. With the addition of a lab-derived preferred body temperature range, we determined the thermal quality of each site and the thermoregulatory accuracy of snakes from each population. Field behavioral observations, gathered from snakes at all four sites simultaneously during the year 2017, revealed how snakes utilize the thermal landscape and adjust thermoregulatory behavior to mitigate the effects of different climates. Although overall thermal quality was best at coastal sites, thermal quality of the microhabitats within each site varied greatly. Consistent with findings in other squamate reptiles, inland snakes thermoregulated more accurately, despite being in more thermally constrained environments. Despite the fact that coastal snakes had lower mean field active body temperatures, the preferred body temperatures of snakes were the same across all four sites. However, field active body temperatures were consistently lower than the preferred range, suggesting there are additional variables that influence thermoregulatory behavior. Using established equations estimating the resting metabolic rates of snakes based on body mass and temperature, we calculated resting metabolic rate and annual baseline maintenance energy expenditure for each population. Coastal snakes, which had lower field active body temperatures, had overall lower metabolic rates than inland snakes, but upon correcting for mass, snakes at neither coastal nor inland sites differed in metabolic rates. Therefore, the majority of the differences observed in metabolic rates are driven by body size and not field-active body temperature. Inland snakes need, on average, approximately 1.6x more food annually than coastal snakes. Due to overall low resting metabolic rates, this translates to snakes at all sites needing less than one ground squirrel (their most common food item on the Central Coast) per year to fuel basic physiological functions. Finally, we used conservative predictive climate change models allowing either 1°C or 2°C increases to predict changes in the thermal quality of each site and ensuing changes in snake metabolic rates and maintenance energy expenditure. Due to the relatively high preferred body temperature of C. oreganus, thermal quality of the environment will actually increase under these climate models; due to an increase in ambient temperature, the proportion of hourly temperatures that fall within the preferred body temperature range will increase. If snake body temperature were to increase as the climate warms, a theoretical increase in body temperature of 1 and 2°C would have a low impact on the overall energetic needs of snakes, still allowing them to meet baseline maintenance energetic needs with only one large meal a year. Furthermore, we expect small increases in ambient temperature to have little impact on rattlesnakes because they are fairly precise thermoregulators, maintaining fairly constant body temperatures regardless of their thermal surroundings. Overall, our results show that studying the thermal ecology of multiple populations of a single species can reveal fine-scale information about the relationship between the thermal landscape and both ectotherm behavior and physiological processes. Additionally, our findings show that some species of large-bodied reptiles may be robust to modest thermal perturbations under conservative climate change predictions.

Pesquisas sobre diferentes modelos de treinamento no meio aquático já demonstraram seus efeitos benéficos em diferentes capacidades físicas, bem como para a população com doenças metabólicas. Entretanto, para sujeitos portadores de síndrome metabólica (SM) são escassos os estudos que investigaram qual modelo de treinamento no meio aquático poderia gerar melhoras físicas e metabólicas. Assim, o objetivo do presente estudo foi comparar os efeitos neuromusculares e metabólicos de 12 semanas de treinamento aeróbio, treinamento de força e treinamento combinado no meio aquático em mulheres com SM. Foram selecionadas 51 mulheres, pós-menopáusicas, sedentárias e portadoras de SM que foram randomicamente divididas em três grupos de intervenção: hidro-aeróbica (HA; n=18; 63,77±5,03 anos), hidro-força (HF; n=16; 61,01±4,93 anos) e hidro-combinada (HC; n=17; 60,52±6,91). Uma sub-amostra participou de oito semanas sem prática de exercício físico para caracterizar um período controle. Os três grupos de intervenção realizaram duas sessões semanais de 60 minutos durante 12 semanas. Antes e após o período de treinamento foram realizadas análises sanguíneas, testes de força muscular, testes funcionais e questionário de qualidade de vida Para análise estatística foi utilizada o modelo de Equações de Estimativas Generalizadas (GEE) com post hoc de Bonferroni (α = 0,05). No período controle, nenhuma das variáveis avaliadas apresentou alteração significativa (p>0,05). Após o período de intervenção houve uma redução significativa da glicemia de jejum (HA: -7,6%; HF: -14,4%: HC: -14,0%), da pressão arterial sistólica (HA: -2,9%; HF: -8,5%: HC: -4,0%) e na contagem total dos fatores da SM (HA: -1,7%; HF: -6,7%: HC: -8,7%) sem diferença entre os grupos. Para a força muscular, houve um aumento significativo na força muscular dinâmica máxima de extensores de joelho (EJ) (HA: 29,6%; HF: 14,6%: HC: 26,7%) e flexores de cotovelo (FC) (HA: -0,1%; HF: 3,1%: HC: 7,4%), para a força resistente de EJ (HA: 25,2%; HF: 18,9%: HC: 23,8%) e FC (HA: 24,3%; HF: 16,6%: HC: 27,8%), para a contração isométrica voluntária máxima de EJ (HA: 11,0%; HF: 8,4%: HC: 26,4%), sem diferença entre os grupos de treinamento Para a atividade eletromiográfica (EMG) do reto femoral (RF) houve um aumento significativo apenas para o grupo HA (35,3%). A EMG de vasto lateral (VL) apresentou efeito significativo ao longo do tempo sem diferença entre os grupos (HA: 33,2%; HF: 40,4%: HC: 52,6%). Os testes funcionais apresentaram melhora significativa ao longo do tempo: Sentar e levantar (HA: 34,8%; HF: 29,0%: HC: 25,1%), Time-up-and-go (HA: -13,5%; HF: -11,8%: HC: -13,7%) sem diferença entre os grupos. A qualidade de vida apresentou aumento significativo no domínio físico (HA: 3,66%; HF: 3,88%: HC: 3,81%) sem diferença entre os grupos. Dessa forma, conclui-se que não houve diferenças expressivas entre os modelos de treinamento aeróbio, de força muscular ou combinado nas variáveis analisadas. Todos os treinamentos de hidroginástica foram eficientes para diminuir fatores da síndrome metabólica como a glicemia de jejum e a pressão arterial e para aumentar a força muscular dinâmica máxima, a força resistente e a força isométrica. Além disso, os treinamentos melhoraram a qualidade de vida e a capacidade funcional das mulheres.
Research about different training models in the aquatic environment has already demonstrated its beneficial effects on different physical capacities as well as for the population with metabolic diseases. However, for subjects with metabolic syndrome (MS) there are few studies that investigated which training model in the aquatic environment could generate physical and metabolic improvements. Thus, the aim of the present study was to compare the neuromuscular and metabolic effects of 12 weeks of aerobic training, strength training and combined training in the aquatic environment in women with MS. Fifty-one women, postmenopausal, sedentary and SM carriers were randomly divided into three intervention groups: hydro-aerobic (HA, n = 18, 63.77 ± 5.03 years), hydro-power (HP; N = 16, 61.01 ± 4.93 years) and hydro-combined (HC; n = 17; 60.52 ± 6.91). A subsample participated in eight weeks without physical exercise to characterize a control period. The three intervention groups performed two weekly sessions of 60 minutes for 12 weeks. Before and after the training period, blood tests, muscle strength tests, functional tests and a quality of life questionnaire were performed For statistical analysis, the Generalized Estimates Equations (GEE) model with Bonferroni post hoc (α = 0.05) was used. In the control period, none of the evaluated variables presented significant alteration (p> 0.005). After the intervention period there was a significant reduction of fasting blood glucose (HA: -7.6%, HP: -14.4%: HC: -14.0%), systolic blood pressure (HA: -2.9 HP: -8.5%: HC: -4.0%) and in the total counting of the factors of the SM (HA: -1.7%, HP: -6.7%: HC: -8.7% ) without difference between groups. For muscle strength, there was a significant increase in maximal dynamic muscle strength of knee extensors (KE) (HA: 29.6%, HP: 14.6%: HC: 26.7%) and elbow flexors (EF) (HA: -0.1%, HP: 3.1%: HC: 7.4%), for maximal repetitions of KE (HA: 25.2%, HP: 18.9%: H: 23.8% ) and EF (24.3%; HF: 16.6%: HC: 27.8%), for the maximum voluntary contraction of KE (HA: 11.0%, HP: 8.4% 26.4%), without difference between the training groups. For the electromyographic (EMG) activity of the rectus femoris (RF) there was a significant increase only for the HA group (35.3%). The vastus lateralis (LV) EMG showed significant effect over time with no difference between the groups (HA: 33.2%, HP: 40.4%: HC: 52.6%) The functional tests showed significant improvement over time: sitting and lifting (HA: 34.8%, HP: 29.0%: HC: 25.1%), Time-up-and-go (HA: 5%, HP: -11.8%: HC: -13.7%) without difference between groups. The quality of life showed a significant increase in the physical domain (HA: 3.66%, HP: 3.88%: HC: 3.81%) without difference between the groups. Thus, it was concluded that there were no significant differences between the aerobic training, muscle strength or combined models in the analyzed variables. All water-based training was efficient to decrease metabolic syndrome factors such as fasting blood glucose and blood pressure and to increase maximal dynamic muscle strength, endurance strength and isometric strength. In addition, training improved the quality of life and functional capacity of women.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Ecologia, Florianópolis, 2011
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Espécies bênticas podem afetar a distribuição de outros organismos e o funcionamento do sistema pelas mudanças em variáveis físicas, químicas e biológicas introduzidas por suas atividades. A presença de poliquetas do gênero Diopatra e da fauna associada aos seus tubos foi manipulada experimentalmente para verificar seus efeitos na estrutura da comunidade e no metabolismo do entorno em baixios de maré nãovegetados. Para tanto, experimentos de campo foram realizados em três locais comparando três tratamentos: i) tubos com Diopatra; ii) tubos sem Diopatra e iii) ausência de tubos e Diopatra (controle). Para avaliar os efeitos sobre o metabolismo do ecossistema, câmaras bênticas foram empregadas para medidas dos fluxos de oxigênio e nutrientes entre o sedimento e a coluna de água. Sedimento, meiofauna e macrofauna foram coletados em locais com e sem a presença de Diopatra para verificar seus efeitos na estrutura das comunidades bênticas. De modo geral, a presença dos tubos favoreceu maiores densidades e número de espécies da macrofauna e, para alguns táxons, a presença do próprio tubícola foi importante. Ao contrário, a meioinfauna não discriminou as estruturas físicas e ocorreu em densidades menores em tratamentos com a presença de Diopatra, provavelmente devido à bioturbação e atividades de alimentação. Embora tenha havido variabilidade nas respostas dos fluxos dependentes de cada local, tanto a presença de Diopatra quanto as altas densidades da macrofauna influenciaram o fluxo de nutrientes nitrogenados e de oxigênio. Processos como nitrificação, denitrificação, amonificação e redução do nitrato parecem ser resultantes da excreção faunal, da bioirrigação e do microambiente disponibilizado pelos tubos.
Benthic species may affect distribution of other organisms and system functioning by changes in physical, chemical and biological variables caused by their activities. The presence of polychaete of genera Diopatra and fauna associated to their tubes was experimentally manipulated in the field to access their effects on the surrounding community structure and ecosystem metabolism in intertidal plains. For that, experiments were set up in three sites, comparing three treatments: i) tube with Diopatra; ii) tubo without Diopatra e iii) no tube and no Diopatra (control). To evaluate the effects of Diopatra and associated fauna over ecosystem metabolism, benthic chambers were employed to measure, oxygen and nutrient fluxes between sediment and water column. Sediment, meiofauna and macrofauna were collected in points with and without Diopatra to verify their effects on the structure of benthic communities. In general, the tubes favored higher densities and species numbers of macrofauna and, for some taxa, the presence of Diopatra was important. On the other hand, the meioinfauna had similar densities between control and treatments with tube without Diopatra and occurred even in lower densities in treatments with Diopatra presence. Probably it was due to bioturbation and feeding activities. Although the fluxes responses have been variable dependig on the site, either Diopatra presence or high densities of macrofauna affected nitrogenous nutrients and oxygen fluxes. Processes like nitrification, idenitrification, amonification and nitrato reduction seem to be resultant of faunal excretion, bioirrigation and microhabitat provided by the tubes.

Running injuries are common, usually causing athletes to cease or significantly reduce participation in a particular sport. The recent development of aquatic treadmills (ATM), an alternative to land treadmill (LTM) running, provides another option. This study sought to examine the metabolic (VO2) relationship between varying jet resistances and running speed on an ATM versus LTM. This was accomplished by developing two linear regression equations and a prediction equation. One linear regression represented the predicted VO2 from a given speed and jet resistance setting in the water, the other linear regression predicted VO2 on land from a given speed and the prediction equation was designed to match land speed to a VO2 score derived from ATM running conditions. This study examined experienced runners (N = 18). Each subject completed an initial VO2 peak test, three LTM trials, and 18 ATM trials. Each ATM trial consisted of running for three minutes at either a relatively slow, moderate, or somewhat fast speed while one of six ATM jet settings ranging from 0 to 100% jet capacity in 20% increments were assigned to the trial. Oxygen consumption (VO2) and heart rate (HR) were measured during each trial while ratings of perceived exertion (RPE) were solicited immediately following each trial. Resulting analysis produced an ATM linear regression for each jet resistance setting and a LTM linear regression equation of VO2 = 4.16 * speed + 7.39. A prediction equation for each jet resistance setting was then determined from the linear regression equations for both the ATM and LTM conditions. Results showed that at and between 0-40% jet resistances that there is not a marked difference in metabolic cost but from 40-100% jet resistances the VO2 is influenced more strongly. These results demonstrate that ATM metabolic costs are not only influenced by jet resistance settings but at jet resistances of 40% or greater provide an intensity of exercise that mimics running faster on LTM. This provides an added benefit for those individuals who may be limited due to acute overuse-type injuries or returning to full LTM activity following lower extremity surgery.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Ecologia
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Lagos costeiros são ambientes que apresentam características particulares quanto a sua dinâmica, estado trófico, morfologia e interação das comunidades biológicas. Em geral, são ambientes rasos submetidos à intensa ação dos ventos. A lagoa do Peri localizada na ilha de Santa Catarina (27°44#S e 48°31#W) é um sistema costeiro subtropical de água doce com 5,7 km2 de área e 4,2 m de profundidade média. De acordo com a literatura, apresenta homogeneidade horizontal e vertical para clorofila a e nutrientes, característica oligotrófica para a concentração de nutrientes e presença de cianobactérias. Com o objetivo de verificar a ocorrência de autotrofia e heterotrofia na lagoa do Peri, determinaram-se as taxas de produção primária (bruta e líquida) e de respiração fitoplanctônica através de incubações em garrafas claras e escuras durante 24 horas no outono, inverno, primavera e verão. Incubações em garrafas claras e escuras, também foram conduzidas bimensalmente, em quatro profundidades em uma estação central da lagoa, para avaliar a variação vertical das taxas de produção primária (bruta e líquida) e de respiração bem como sua relação com a comunidade fitoplanctônica. Condições de heterotrofia foram encontradas no outono, quando as maiores taxas de respiração ocorreram devido ao aumento da disponibilidade de matéria orgânica e fósforo total. Condições de autotrofia ocorreram no verão devido às maiores taxas de produção primária líquida, favorecida pelas maiores temperaturas e luz. As taxas de produção primária, respiração e a densidade dos grupos fitoplanctônicos, em geral, não apresentaram variação vertical, em função da homogeneização promovida pelos ventos incidentes na lagoa do Peri. Condições limitantes de luz e nutrientes, para o crescimento fitoplanctônico, foram os fatores atribuídos as menores taxas de produção primária encontradas neste ambiente em relação a outros ambientes tropicais e subtropicais. A densidade fitoplanctônica e condições de temperatura e luz favoreceram a produção primária na primavera e no verão. Cyanobacteria e Chlorophyta foram os grupos mais importantes da comunidade fitoplanctônica, em termos de densidade e diversidade, com destaque para Cylindrospermopsis raciborskii que dominou na maior parte do período de estudo. Assim, a lagoa do Peri apresentou alternância entre períodos autotróficos e heterotróficos em função das diferentes condições ambientais e as taxas de produção primária foram menores que em outros ambientes, variando em função das variáveis bióticas e abióticas.
Coastal lakes are environments with distinct characteristics when it comes to dynamics, trophic state, morphology, and the interaction of biological communities. They are generally shallow environments subjected to intense winds. Peri lagoon located on the island of Santa Catarina (27°44'S and 48°31'W) is a subtropical coastal system of freshwater with an area of 5.7 km2 and average depth of 4.2 m. According to literature, it is horizontally and vertically homogenous for chlorophyll a and nutrients, oligotrophic characteristics for nutrient concentration and presence of Cyanobacteria. In order to verify the occurrence of autotrophy and heterotrophy in the Peri lagoon, primary production (gross and net) and respiration rates were determined by incubating in light and dark bottles over a period of 24 hours in the fall, winter, spring and summer. Incubations in light and dark bottles were also conducted every two months, at four depths in the central part of the lagoon, in order to evaluate the vertical variation in primary production rates (gross and net) and respiration and the relationship to the phytoplankton community. Heterotrophic conditions were found in autumn, where the highest rates of respiration were found due to an increased availability of organic matter and total phosphorus. Autotrophic conditions occurred in the summer due to higher rates of net primary production, favored by higher temperatures and light availability. In general, the primary production rates, respiration and phytoplankton group density, did not show vertical variation due to the homogenization promoted by winds in the Peri lagoon. Light and nutrients limitation were attributed to the lower rates of primary production found in this environment than other ones. However, phytoplankton density, temperature and light conditions favored the primary production rates in spring and summer. Cyanobacteria and Chlorophyta were the most important groups of the phytoplankton community in terms of density and diversity, especially Cylindrospermopsis raciborskii, which was dominant during the majority of the study period. Thus, the Peri lagoon had alternating autotrophic and heterotrophic periods due to different environmental conditions and the primary production rates were lower than other tropical environments, depending on biotic and abiotic variables.

This diploma thesis deals with the biodegradation of whey on selected microbial products (carbohydrates, lactic acid, acetic acid and ethanol) thermophilic bacteria of genus Thermus aquaticus and mesophilic bacteria of genus Lactobacillus casei and Bacillus coagulans. For cultivation was used as medium whey, from which the proteins were removed and which was enriched with nutrients. On the basis of culture in the fermentor were determined growth curve and the HPLC method were determined individual bioremediation products.

Populations that utilize deep water running (DWR) are described in Chapter I. A review of the literature concerning maximal and submaximal responses during DWR, shallow water running and swimming is presented in Chapter II. The protocols to elicit maximal responses during DWR and treadmill running (TMR), subject characteristics, and statistical methods employed are described in Chapter III. The results, presented in Chapter IV, indicate that the DWR protocol is a reliable test for eliciting peak oxygen consumption and heart rate. Furthermore, the metabolic responses during DWR are lower than TMR. Chapter V discusses factors which might limit maximal responses during DWR. Chapter VI contains suggestions for further research. Raw data are presented in Appendix A.

Orientador: Eduardo Alves de Almeida
Coorientador: Daniel Schlenk
Banca: Claudia Bueno dos Reis Martinez
Banca: Gisela de Aragão Umbuzeiro
Banca: Larissa Paola Rodrigues Venancio
Banca: Igor Dias de Medeiros
Resumo: O Brasil é o principal produtor de cana-de-açúcar do mundo e, para que esta demanda seja suprida, muitos compostos químicos são aplicados nas culturas, visando controlar o surgimento e proliferação de pragas. Assim, faz-se o uso dos praguicidas, dentre eles, o diuron (N-(3,4-diclorofenil)-N,N-dimetiluréia), que é aplicado em plantações ao redor do mundo. O diuron pode ser biodegradado em outros três principais compostos o 3,4-dicloroanilina (DCA), 3,4-diclorofenilureia (DCPMU) e 2,4-diclorofenil-N-metilureia (DCPMU). Normalmente, o diuron é aplicado nas plantações juntamente com os alquilfenóis etoxilatos (APE), como o nonilfenol etoxilato (NPE) e o octilfenol etoxilato (OPE), compostos que facilitam a dispersão do diuron. Alguns estudos têm demonstrado que tanto o diuron quanto os alquilfenóis podem causar alterações enzimáticas e/ou estrogênicas em diversos organismos. Dentre as enzimas que podem ser alteradas, estão as enzimas de biotransformação, tanto de fase I (7-etoxirresorufina-O-deetilase - EROD, 7-pentóxiresorufinaO-desalquilase - PROD, 7-benzilóxiresorufina-O-desalquilase - BROD e a P450 aromatase), quanto as de fase II (glutationa-S-transferase - GST) e as proteínas de fase III (resistência a multixenobioticos - MXR). Outros parâmetros que podem sofrer alterações são os antioxidantes (superóxido dismutase - SOD, catalase - CAT, glutationa peroxidase - GPx, glutationa redutase - GR, glutationa-6-fosfato desidrogenase - G6PDH, aldeído desidrogenase - ALDH e...
Abstract: Brazil is the main sugar cane producer in the world, and to support these productions many chemical compounds have being apply in agriculture, aiming to control the appearance and proliferation of pests. Therefore, the use of pesticide, as diuron, (N- (3,4-dichlorophenyl) -N,N-dimethylurea), in some crops in the world is common. Diuron can be biodegraded in three others compounds, 3,4-dichloroaniline (DCA), 3,4-dichlorophenylurea (DCPU) and 2,4-dichlorophenyl-N-methylurea (DCPMU). Normally, diuron have being applied associated with alkylphenols ethoxylates (APE), like nonilphenol (NP) and octylphenol (OP), which increase the solubility and dispersion of the herbicide. Some studies have shown that diuron and alkylphenols can cause enzymatic and/or estrogenic changes, in various organisms. Therefore, biotransformation enzymes, phase I (7-ethoxirresorufin-O-deethylase - EROD, 7- pentaxirisorufin-O-dealkylase - PROD, 7-benzyloxysorufin-O-desalkylase - BROD and P450 aromatase), phase II (glutathione-S-transferase-GST) and phase III proteins (multixenobiotic resistance - MXR), and antioxidant parameters (superoxide dismutase - SOD, catalase - CAT, glutathione peroxidase - GPx, glutathione reductase - GR, glutathione-6-phosphate dehydrogenase - G6PDH, aldehyde dehydrogenase - ALDH and lipid peroxidation), which are responsible for the control between production and degradation of reactive oxygen species (ROS). The endocrine parameters that can be altered and used as biomarkers are ...
Doutor

Les agents tensio-actifs de synthese constituent, a cause de leur large diffusion, une forme de pollution preoccupante atteignant tout le milieu naturel. Ce memoire vise a completer les donnees concernant le devenir, dans differents compartiments du milieu aquatique (eau, sediment), d'une variete de produits tensio-actifs tres utilises actuellement: les alkylbenzenesulfonates lineaires (las). Dans la premiere partie de cette etude sont presentees les connaissances existantes a propos des agents-tensio actifs et plus particulierement les las. Par la suite sont detaillees les diverses etapes de l'elaboration d'une methode analytique basee sur la chromatographie liquide haute performance (hplc), permettant de detecter simultanement les las et les metabolites qui sont susceptibles de se former au cours de leur biodegradation. Des techniques d'extraction ont ete mises, au point dans le but de concentrer et de purifier les echantillons environnementaux en vue de l'analyse chromatographique. En utilisant la methode de l'experimentation in-vitro, nous avons pu mettre en evidence la decomposition, en presence d'oxygene, d'un las et l'apparition de residus de degradation dans des echantillons d'eau et de sediment provenant d'un etang du milieu mediterraneen (l'etang de bolmon). Les metabolites de degradation observes ont ete identifies et ne sont pas persistants. Apres une investigation concernant le degre de pollution du bolmon par les detergents nous avons place un dispositif dans l'etang contenant un sediment artificiellement contamine par du las pour savoir si la biodegradation peut se produire dans le milieu naturel au sein meme du sediment. Les resultats obtenus montrent l'importance preponderante des phenomenes de ramaniement (hydrodynamisme, bioturbation) sur la regeneration du sediment

A thesis submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, Republic of South Africa, in fulfillment of the requirements for the degree of Doctor of Philosophy in Engineering. Johannesburg, 2016
The process of photosynthesis is of great importance as it is the reaction of carbon dioxide (CO2) and water with the help of light, ’free’ energy from the sun, to form useful carbohydrates and oxygen. Photosynthesis is therefore useful both in carbon dioxide mitigation and growing bio-feedstocks towards making biofuel. This thesis aims to address two areas for analysing the photosynthesis process: 1. Looking at the physical limits of the growth; and 2. Improving the production rate of some aquatic plants, such as duckweed and microalgae. To address the first aim, the fundamental concepts of thermodynamics were used to analyse the photosynthetic process. It was found that the theoretical minimum number of moles of photons (NP) required (9–17) is less than the values reported by other researchers, suggesting that the photosynthesis process is highly irreversible and inefficient (operating at 35% efficiency or less). This is because the number of moles of photons will increase with greater process irreversibility (when the entropy generated is greater than zero). If the photosynthesis process is indeed that irreversible then the removal of heat (the heat not used by other cellular processes) by the plant becomes a major problem. It is suggested that transpiration, and other cellular processes, are the processes by which that is done, and it is shown that the water needs of the plant for transpiration would dwarf those needed for photosynthesis. Knowing the fundamental limits to growth could also be of use because if an organism was growing at a rate close to this value there would be no advantage to try to do genetic modification to improve its rate. Following the ideas presented above a spectrophotometer was used not only to obtain the absorption spectrum of algae, but it was also used to grow small samples at specific light wavelengths. The algae species researched was Desmodesmus spp., which, for example, is used to remediate waste water or as a source of feedstock for biofuel production. It also tolerates high CO2 concentrations. This simple experimental method demonstrated that a specific light wavelength (in particular the Secomam Prim spectrophotometer) 440 nm was preferred for the algae growth. It was recommended that this specific light wavelength would be best for growth. It might also be useful to know this fact particularly when designing photobioreactors, as this could reduce the amount of heat released into the surroundings and thus make the process more energy efficient. Interestingly, the wavelength for maximum growth corresponded to one of the peaks in the absorption spectra but there was no increase in growth rate corresponding to any of the other peaks. To address the second aim, the author determined how well predictions on improving the growth of algae (Desmodesmus spp. for example), based on a theoretical model, would work when tested experimentally. What the researcher found was that the method improved algae production, using the same set of equipment. The production was improved by a factor of 1.28 and 1.26 (at product concentrations 1000 mg/L and 600 mg/L respectively) when retaining 40% of the algae suspension. The method may be particularly useful when large amounts of biomass are required as there is no extra cost of purchasing additional equipment. The same model was applied to a growth profile of duckweed (Spirodela polyrhiza 8483, which is convertible into biofuel or a source of food), and the author showed that the model could work if the duckweed was provided with an added carbon source. In order to find an economical and reliable alternative to bridge the scale gap between laboratory and industrial production, the author checked if duckweed species (Spirodela polyrhiza 8483, Spirodela polyrhiza 9509, Lemna gibba 8428, Lemna minor DWC 112, Wolffia cylindracea 7340 and Wolffia globosa 9527) could be cultivated in media less expensive than the basal laboratory medium (Schenk and Hildebrandt). The author found that duckweed can be cultivated more efficiently, and in a more cost-effective manner, in the alternative media types, while maintaining growth rates, RGR 0.09 day-1, and starch contents, 5– 17%(w/w), comparable with that obtained with the conventional laboratory media. Thus, by looking at the photosynthesis process thermodynamically and experimentally, it is shown to be possible to improve the process by using concepts presented in this thesis.
MT2017

This thesis considers the implications of changes in the supply of resources for photosynthesis, with regard for modes of carbon acquisition employed by aquatic plants of the River Murray. Carbon supplies are inherently more variable for aquatic plants than for those in terrestrial environments, and variations are intensified for plants in semi-arid regions, where water may be limiting. In changeable environments the most successful species are likely to be those with flexible carbon-uptake mechanisms, able to accommodate variations in the supply of resources. Studies were made of plants associated with wetland habitats of the Murray, including Crassula helmsii, Potamogeton tricarinatus, P. crispus and Vallisneria americana. The aim was to elucidate the mechanisms of carbon uptake and assimilation employed, and to determine how flexibility in carbon uptake and/or assimilation physiology affect survival and distribution. Stable carbon isotopes were used to explore the dynamics of carbon uptake and assimilation, and fluorescence was used to identify pathways and photosynthetic capacity. The studies suggest that physiological flexibility is adaptive survival in changeable environments, but probably does not enhance the spread or dominance of these species. V. americana is a known bicarbonate-user, and it is shown here that it uses the Crassulacean Acid Metabolism (CAM) photosynthetic pathway under specific conditions (high light intensity near the leaf tips) concurrently with HCO[subscript]3 - uptake, while leaves deeper in the water continue to use the C[subscript]3 pathway, with CO₂ as the main carbon source. However, V. americana does not use CAM when under stress, such as exposure to high light and temperature. The diversity of carbon uptake and assimilation mechanisms in this species may explain its competitive ability in habitats associated with the Murray. In this way it is able to maximise use of light throughout the water column. In shallow, warm water, where leaves are parallel to the surface, CAM ability is likely to be induced along the length of the leaf, allowing maximal use of carbon and light. The amphibious C. helmsii is shown to use CAM on submergence, even where water levels fluctuate within 24 hours. This allows continued photosynthesis in habitats where level fluctuations prevent access to atmospheric CO₂. It appears that stable conditions are most favourable for growth and dispersal, and that the spread of C. helmsii is mainly by the aerial form. Carbon uptake by P. tricarinatus under field conditions is compared with that of P. crispus to demonstrate differences in productivity associated with aqueous bicarbonate and atmospheric CO₂ use. P. tricarinatus uses HCO[subscript]3 - uptake to promote growth toward the surface, so that CO₂ can be accessed by floating leaves. Atmospheric contact provides access to light and removes the limitation of aqueous diffusive resistance to CO₂, thereby increasing photosynthetic capacity above that provided by submerged leaves.
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Thesis (Ph.D) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

碩士
東海大學
生命科學系
101
Recently, the rising in water temperature and the eutrophication caused by global warming, and anthropogenic discharge of the nitrogenous wastes, lead to a decrease in aquatic oxygen solubility. Serious consequences for aquatic life could be expected if the hypoxic condition prolongs for a period of time. To balance the O2 and metabolism demands, organisms may change from aerobic to anaerobic respiration by utilizing glycogen as the emergency fuel in glycogenolysis, and the function of sodium-potassium adenosine triphosphatase (Na+/K+-ATPase, NKA) may be depressed to preserve energy consumption. In the process of glycogenolysis, glycogen phosphorylase (GP) degrades glycogen in muscle and liver into glucose-1-phosphate for glycolysis. According to previous studies, the glycogen-rich cells (GR cells) in fish gills located right next to mitochondria-rich cells (MR cells). The degrading of glycogen in GR cells could, therefore, cope with high ATP demand from NKA in MR cells when fish is under stress. In addition to the metabolic suppression, organisms produce free radicals harmful to cells in the hypoxic and recovery conditions. Therefore, how organisms adapt to hypoxia is an important issue for survival. The aquatic air-breathing anabantoid fish Helostoma temminckii, with the accessory air-breathing organ labyrinth organ connected with the gills, can live in the hypoxic environment. From the previous studies, it is known that no inhibition of NKA protein abundance and activity in hypoxia was observed within the first three days of the experiment in the other air-breathing fish Trichogaster lalius which has similar gill morphology with H. temminckii. In the present study, it is hypothesized that GP in GR cells degrades glycogen to generate ATP so that NKA in the gills of H. temminckii will not be inhibited. H. temminckii has better antioxidant ability and this will help to prevent from free radical damages in hypoxia and recovery in normoxia. The result indicates that the aquatic air-breathing H. temminckii increased the air-breathing frequency under hypoxia. The protein abundance of GP increased during hypoxic treatment, but expression of NKA did not change accordingly. Glycogen contents degraded during hypoxic condition in the gills and liver. For the antioxidant mechanism in the gills, the superoxide dismutase (SOD) activity in recovery group was higher than that in the hypoxic group and the glutathione peroxidase (GPx) increased under hypoxic and recovery groups. Moreover, glutathione-s-transferase (GST) activity increased in the recovery group. In the liver, the SOD increased under hypoxia and, catalase (CAT) activity in recovery group was higher than that in the hypoxic group. Furthermore, glutathione reductase (GSR) activity increased in the recovery group. In conclusion, H. temminckii regulates its behavioral, biochemical and physiological conditions for surviving under stress condition.

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardant chemicals that are added to plastics, electronic components, furniture foam, and textiles to reduce their combustibility. Of the three commercial mixtures historically marketed, only DecaBDE, which is constituted almost entirely (~97%) of the fully brominated congener decabromodiphenyl ether (BDE-209), continues to be used in the U.S. today. While decaBDE is scheduled for phase-out in the U.S. at the end of 2013, exposures to BDE-209 and other PBDEs will continue into the foreseeable future as products that contain them continue to be used, recycled, and discarded. In addition, decaBDE use continues to be largely unrestricted across Asia, although restricted from use in electronic equipment in Europe.

Despite limits placed on PBDE uses, they are ubiquitous contaminants detected worldwide in humans and wildlife. Major health effect concerns for PBDEs come largely from evidence in laboratory rodents demonstrating neurotoxicity, reproductive and developmental impairments, and thyroid disruption. The potential for PBDEs, particularly BDE-209, to disrupt thyroid regulation and elicit other toxic outcomes in fish is less clear. Thus, the overall objective of this thesis research was to answer questions concerning how fish, as important indicators of overall environmental health, are metabolizing PBDEs and whether and how PBDEs are disrupting thyroid hormone regulation. The central hypothesis was that PBDE metabolism in fish is mediated by iodothyronine deiodinase (dio) enzymes, which are responsible for activating and inactivating thyroid hormones, and that PBDE exposures are causing thyroid system dysfunction across fish life stages.

Under the first research aim, in vitro experiments conducted in liver tissues isolated from common carp (Cyprinus carpio) suggested a role for dio enzymes in catalyzing the reductive debromination of PBDEs. Carp liver microsomes efficiently debrominated BDE-99 to BDE-47, and enzymes catalyzing this reaction were associated predominantly with the endoplasmic reticulum (i.e., microsomal fraction) where dio enzymes are located. Competitive substrate experiments in carp liver microsomes also demonstrated that rates of BDE-99 debromination to BDE-47 were significantly inhibited upon challenges with 3,3',5'-triiodothyronine (rT3) and thyroxine (T4). This finding supported the hypothesis that enzymes involved in the metabolism of PBDEs may have high affinities for thyroid hormones. Indeed, experiments to determine apparent enzymatic kinetics (apparent Vmax and Km values) of BDE-99 hepatic metabolism suggested that enzymes responsible for the catalytic activity appeared to have a higher affinity for native thyroid hormone than BDE-99.

The second and third research aims were focused on evaluating BDE-209 accumulation, metabolism, and thyroid toxicity in juvenile and adult life stages of fish using the fathead minnow (Pimephales promelas) as a model. BDE-209 bioaccumulated and was debrominated to several reductive metabolites ranging from penta- to octaBDEs in both juvenile and adult fish exposed to BDE-209. In addition, thyroid hormone regulation in juvenile and adult male fathead minnows was severely disrupted by BDE-209 at low, environmentally relevant exposures. In juvenile minnows, the activity of dio enzymes (T4-outer ring deiodination; T4-ORD and T4-inner ring deiodination; T4-IRD) declined by ~74% upon oral doses of 9.8 ± 0.2 µg/g wet weight (ww) food at 3% body weight (bw)/day for 28 days, compared to controls. Declines in dio activity were accompanied by thyroid follicle hypertrophy indicative of over-stimulation and injury. In addition to thyroid disruption, a distinctive liver phenotype characterized by vacuolated hepatocyte nuclei was measured in ~48% of hepatocytes from treated fish that was not observed in controls.

Under the third research aim, adult male fathead minnows received dietary treatments of BDE-209 at a low dose (95.3 ± 0.41 ng/g-food at 3% bw/day) and a high dose (10.1 ± 0.10 µg/g-food at 3% bw/day) for 28 days followed by a 14-day depuration period to evaluate recovery. Compared to negative controls, adult male fish exposed orally to BDE-209 at the low dose tested for 28 days experienced a 53% and 46% decline in circulating total T4 and T3, respectively, while fish at the high BDE-209 dose tested had total T4 and T3 deficits of 59% and 62%, respectively. Depressed levels of plasma thyroid hormones were accompanied by a 45-50% decline in the rate of T4-ORD in brains of all treatments by day 14 of the exposure. The decreased T4-ORD continued in the brain at day 28 with a ~65% decline measured at both BDE-209 doses. BDE-209 exposures also caused transient, tissue-specific upregulations of relative mRNA transcripts encoding dio enzymes (dio1, dio2), thyroid hormone receptors (TR&alpha, TR&beta), and thyroid hormone transporters (MCT8, OATP1c1) in the brain and liver in patterns that varied with time and dose, possibly as a compensatory response to hypothyroidism. In addition, thyroid perturbations at the low dose tested generally were equal to those measured at the high dose tested, suggesting non-linear relationships between PBDE exposures and thyroid dysfunction in adult fish. Thus, mechanisms for BDE-209 induced disruption of thyroid regulation can be proposed in adult male minnows that involve altered patterns of thyroid hormone signaling at several important steps in their transport and activation.

A growing body of evidence describing PBDE toxicity in biota, including data generated here, along with studies showing continued and rising PBDE body burdens, raises concern for human and wildlife health. Long delays in removing PBDEs from the market, their ongoing presence in many products still in use, and their active use outside the U.S. and European Union will leave a lasting legacy of rising contamination unless more concerted regulatory and policy actions are taken to reduce future exposures and harm.

Dissertation

碩士
國立臺灣師範大學
生物研究所
90
The aim of this study was to investigate the impact of white rust on the morphology and photosynthetic physiology of Ipomoea aquatica, a very common vegetable in Taiwan, and to examine the hypothesis that increasing the activity of invertase was the primary factor responsible for the reduction of photosynthesis in diseased leaves. According to the morphology and size of sporangia and oospores examined by light and electronic microscopes, the studied pathogen was identified to be Albugo ipomoeae-aquaticae. Changes in morphology and physiology of infected leaves occurred 7 days after infection. Yellow-brown patches and white rust pustules appeared respectively on the upper and lower surfaces of locally infected cotyledons and leaves. Elevation of respiratory rates and the activities of invertase, ADP-glucose pyrophosphorylase and b-amylase, increase in the amounts of glucose, fructose, starch and trehalose, decrease in the contents of chlorophyll a, carotenoids and sucrose, and in the activity of sucrose synthase, and decline in the rates of assimilation, transpiration, and stomatal conductance were observed in the infected tissues during the infection (7-14 days after infection). The timing of the changes in the photosynthetic and carbohydrate metabolism of the infected tissues described above suported the hypothesis.

碩士
臺灣大學
環境工程學研究所
98
Licorice is one of common traditional Chinese medicines. Glycyrrhizin (GLY), the principle active ingredient of licorice, is the ingredient of antitussive and mucolytic-expectorant for the treatment of chronic hepatitis or allergies. GLY has also been widely used in food additives industry. After oral administration, GLY and its metabolite glycyrrhetic acid (GA) enter the aquatic environment via septic systems. The aims of this research were to: 1) develop analytical method of GLY and GA based on a solid phase extraction (SPE) procedure followed by liquid chromatography tandem mass spectroscopy and 2) investigate GLY and GA in Sindian river and two hospital effluents. In this research, the optimized SPE procedure that used Oasis MCX cartridges were pre-conditioned by rinsing with 6 mL methanol (MeOH) and 6 mL deionized water (DI water). After the conditioning step, water samples were percolated through MCX cartridges. Water samples were adjusted at pH 2 with hydrochloric acid. Then, the elute solution selected 6mL MeOH-NH4OH (95:5, v/v). Finally, the extract solutions are complete evaporated by nitrogen gas at 37 °C and reconstituted to 400 μL by MeOH-DI water (25:75, v/v). All standards were spiked at a concentration 100 ng/L into DI water and blank water samples. Recoveries ranged from 95-103% for DI water, river and hospital effluents by using the optimized SPE method. The method detection limits (MDLs) defined by signal-to-noise ratio of at least 10:1. MDLs of GLY and GA were 10 ng/L in DI water. MDLs varied between GLY and GA and were 10 ng/L and 50 ng/L for surface water, and 20 ng/L and 200 ng/L for hospital effluents, respectively. The result indicated the ubiquitous occurrence of GLY and GA in Sindian river. The detected concentration of GLY and GA ranged from 11 to 82 ng/L and 77 to 327 ng/L in Sindian river, respectively. The detected frequency of GLY and GA was 83% and 67%. The detected concentration of GLY and GA in hospital ranged from 1443 to 1750 ng/L and 226 to 7737 ng/L, respectively

Climate changes affect species interactions which can have cascading effect up to the ecosystem level. This work investigates the effects of temperature and predator size on predator prey interactions by measuring the feeding rates of predators and metabolic rates of both predator and prey, using dragonfly larvae Aeshna cyanea and toad tadpoles Bufo bufo as a model system. Possible consequences of the findings for the impacts of climate change and predation on amphibian populations are discussed.

Dans les océans, les procaryotes sont des acteurs clés dans le cycle du carbone puisqu’ils consomment une fraction importante de la matière organique dissoute (MOD) relâchée par les producteurs primaires. Puisque cette matière organique est très complexe et de biodisponibilité variable, les communautés de procaryotes qui la consomme sont très diversifiées et spécialisées pour certains types de composés organiques. En utilisant cette matière organique, les procaryotes contribuent à réintroduire ce carbone dans le réseau trophique, une source d’énergie essentielle dans les gyres oligotrophes de l’océan. Toutefois, puisque cette consommation n’est pas parfaite, une quantité importante de carbone est relâchée sous forme de CO2 lors de la respiration, mais aussi sous forme de MOD récalcitrante, contribuant à séquestrer du carbone dans les océans. Le but de cette thèse est d’une part, de dresser un portrait global de la biodisponibilité de la MOD et d’autre part, de déterminer l’influence de la biodisponibilité de cette dernière sur la composition et le métabolisme des procaryotes dans la mer du Labrador, une mer dont le rôle est critique dans la régulation du climat. Plus spécifiquement, nous identifions pour la première fois comment la distribution spatiale des procaryotes influencent leur métabolisme et est influencée par leur préférence alimentaire dans les eaux de surface de la mer du Labrador. Finalement, nous regardons comment la matière organique produite en surface est transformée et séquestrée en profondeur suite à la convection hivernale dans la mer du Labrador. Le budget de carbone dans les océans n’est toujours pas balancé. Afin de mieux connaître les sources et la biodisponibilité du carbone dans les différents milieux aquatiques, nous avons évalué la biodisponibilité de la MOD à travers le continuum aquatique, des lacs jusqu’à l’océan. En menant une méta-analyse sur le sujet, nos résultats montrent que la proportion de matière organique labile, c’est-à-dire facilement utilisable par les procaryotes, est d’environ 6% dans tous les environnements aquatiques. Toutefois, la proportion de matière organique semi-labile, celle qui nécessite plus de transformation par les procaryotes, est grandement liée à la proximité au milieu terrestre. Les seuls écosystèmes aquatiques déviant de ces deux constats sont ceux en période d’efflorescence algale: ils contiennent beaucoup plus de carbone labile et semi-labile que ceux à l’équilibre. Nous avons estimé que le carbone semi-labile peut soutenir 62% de la biomasse de procaryotes dans les lacs et les milieux côtiers. Dans un deuxième temps, nous évaluons l’influence de la MOD sur le métabolisme et les communautés de procaryotes. Nous avons fait trois missions océanographiques sur la mer du Labrador à bord du navire Hudson pour déterminer la composition de la MOD et la communauté des procaryotes ainsi que leur métabolisme. En utilisant une approche novatrice, la modélisation de la distribution spatiale de l’abondance des procaryotes, nous avons montré à quel point celle-ci est importante pour déterminer leur préférence alimentaire ainsi que leur métabolisme. Nous avons également proposé un nouveau cadre conceptuel qui vise à faciliter la recherche à l’interface de la biogéochimie, de l’écologie microbienne et du métabolisme microbien. Dans un dernier temps, nous avons comparé la capacité des procaryotes venant de différentes profondeurs océaniques à séquestrer le carbone. Lors de la consommation de la MOD, les procaryotes en relâche une petite fraction sous forme plus récalcitrante. En répétant ce processus, le carbone résiduel devient très récalcitrant et peut résister à la consommation par les procaryotes durant des centaines d’années. Nous avons montré que les procaryotes de l’océan profond sont plus efficaces pour séquestrer le carbone de cette façon. Nos résultats montrent que ce sont les taxons rares des procaryotes qui sont les éléments clés dans cette suite de transformation qui mène à la séquestration du carbone appelée pompe microbienne. Cette thèse contribue à la compréhension du cycle du carbone dans la mer du Labrador et dans les écosystèmes aquatiques en général. Nous avons proposé une approche novatrice permettant de lier la qualité de la MOD à la composition des communautés de procaryotes qui la dégrade, un défi qui perdure depuis des dizaines d’années. De plus, nous montrons pour la première fois la que la pompe microbienne de carbone est un processus itératif fortement relié à la succession de la communauté de procaryotes. Nous montrons également que la pompe microbienne est active dans chaque strate océanique, mais que les procaryotes rares issus de l’océan profond sont plus efficaces à séquestrer le carbone. Mieux comprendre comment la composition de la MOD influence les procaryotes est primordial puisqu’ils sont centraux au cycle du carbone océanique.
Oceanic prokaryotes are key players in the carbon cycle by consuming dissolved organic mat-ter (DOM) produced by primary producers. As this organic matter is highly complex with varying degree of bioavailability, prokaryotic communities are highly diverse and different taxa target certain types of organic compounds. By consuming this organic matter, prokary-otes reintroduce this carbon into the food web, a critical energy flow in oligotrophic gyres. However, this consumption is not perfect and they release a lot of carbon as CO2 through respiration, but also as recalcitrant DOM. Thus, they contribute to carbon sequestration in aquatic ecosystems. The objective of this thesis is to characterize DOM bioavailability and its influence on the composition and metabolism of prokaryotic communities in the Labrador Sea, described as one of the Earth’s climate system tipping elements. More precisely, we quantify for the first time how the spatial abundance distribution of prokaryotes influences ecosystem metabolism and organic matter association in the surface waters of the Labrador Sea. Lastly, we look at how DOM produced at the surface is transformed and sequestered following the Labrador Sea winter convective mixing. The oceanic carbon budget is still unbalanced. In order to better understand its carbon sources and bioavailability, we characterize DOM bioavailability across the aquatic contin-uum, from lakes to the open ocean. Using a meta-analysis, our results show that the propor-tion of labile organic matter, i.e. readily available for prokaryotes, is similar at around 6% in all aquatic ecosystems. However, the proportion of semi-labile organic matter, i.e requiring transformations to be consumed by prokaryotes, is highly related to terrestrial connectivity. The only ecosystems that did not follow these patterns were in a phytoplankton bloom pe-riod and had a high proportion of labile and semi-labile organic matter as their counterparts at equilibrium. Finally, we estimated that semi-labile organic matter could sustain 62% of prokaryotic biomass in lakes and coastal zones. Second, we evaluated the influence of DOM on prokaryotic metabolism and community composition. In order to determine organic matter composition, prokaryotic community composition and metabolic rates, we did three oceanic cruises in the Labrador Sea onboard the Hudson ship. By using spatial abundance distribution modelling of prokaryotes, we identified strong associations between groups of this novel approach and organic matter composition. We also proposed a framework to bridge the gap between prokaryotic diversity, microbial ecology, and biogeochemistry among methods and across scales. Lastly, we compared how prokaryotic communities from different oceanic strata could se-quester carbon. When they consume organic matter, prokaryotes release a small amount in recalcitrant forms. Through this iterative process, called the microbial carbon pump, prokaryotes contribute to carbon sequestration by creating highly recalcitrant compounds that resist further degradation for hundreds of years. We have shown that all prokaryotes enable the microbial carbon pump, but that prokaryotes from deeper strata are more effi-cient. Our results also conclusively show that the rare prokaryotic taxa are key players in the microbial carbon pump. This thesis contributes to better understand the carbon cycle in the Labrador Sea and in all aquatic ecosystems. We proposed a novel framework to relate biogeochemistry, prokaryotic diversity and microbial ecology which has been a challenge for decades. Moreover, we con-clusively showed for the first time that the iterative process of the microbial carbon pump is related to prokaryotic succession. We also show that it happens in all oceanic strata, but that rare prokaryotes from the deep ocean are more efficient to sequester carbon. Better understanding how DOM composition influences prokaryotes is of prime importance as they are the main drivers of the oceanic carbon cycle.

According to recent reports, increases in water acidification and changes in seawater salinity are predicted to occur in the next 100 years. The increase of atmospheric CO2 already caused a pH reduction in surface waters by more than 0.1 units below the pre-industrial average of 8.1, and it is expected to decrease between -0.13 and -0.43 units by the end of this century. Climate change can also occur through alterations in seawater salinity. Warmer temperatures and reduced rainfall increase seawater salinity, while extreme rainy events decrease seawater salinity. Both situations will promote species responses. Therefore, identifying the effects of predicted climate change in aquatic ecosystems must be a priority in order to maintain their biodiversity. Aside from climate change, there is an increasing concern about the large number of emerging pollutants that have been released into the environment without yet being regulated. Among these emerging pollutants are Engineered nanoparticles (ENPs). One of the types of ENPs that are most commonly used in recent years are Carbon Nanotubes (CNTs). Due to their unique chemical and nanotoxicological properties, it is expected that CNTs enter aquatic environments and accumulate in aquatic biota. As a matter of fact, CNTs toxicology in aquatic systems is complex. In the first instance particle size, shape, chemistry and capping agents will all play a role regarding the stability, and thus bioavailability. However, nanomaterial toxicity not only has been attributed to core structure and surface modification/functionalization, but also by the physico-chemical parameters of the media where the CNTs are presented, altering their dispersion and consequently their detection: aggregation/disaggregation, adsorption/desorption, sedimentation/resuspension and dissolution. Several works have described their impacts in the aquatic environment; however, no information is known on how predicted Climate Change could alter the CNT’s toxicity and their effects on marine organisms. Benthic species are a good model to evaluate the impacts of Climate Change and ENPs as they are sensitive to several environmental constrains. Essentially due to their life-history characteristics, as well as their relatively rapid response to pollution, several studies have been using benthic species as bioindicators for anthropogenic and natural stresses. Thus, the evaluation of the impacts of CNTs, under salinity changes and pH reduction on aquatic organisms is an urgent issue needing attention. Particularly, Ruditapes philippinarum (bivalve), Hediste diversicolor and Diopatra neapolitana (polychaetes) have been identified by several authors as a group of marine invertebrates that respond quickly to environmental disturbances, with a wide spatial distribution and economic relevance, namely in Portugal. Thus, the present proposal evaluated the toxic effects in terms of biochemical (energy reserves and metabolic activity, oxidative and neuro status) and physiological responses (regenerative capacity) in the cited species of salinity shifts and pH variation and the presence of CNTs acting along and in combinations. The two CNT materials selected in the present study were the pristine multi walled carbon nanotubes (Nf-MWCNTs) and the chemically functionalized MWCNTs, by introducing polar groups such as carboxyl groups (-COOH) increasing their stability and dispersibility in the water media. As a starting point we evaluated the possible effects of the carboxylation/functionalization of the surface of MWCNTs in organisms for each exposure concentration. In all invertebrate species it was possible to observe a dose-dependent increased of the toxicity, especially in terms of oxidative status, which is in line with the information provided by the literature. Moreover, comparing the toxic effects of both CNTs, in all invertebrate species major cellular damage was induced by carboxylated forms of MWCNTs in comparison to the pristine one. Subsequently we selected the two most deleterious concentrations of Nf-MWCNTs and f-MWCNTs, and we exposed the three invertebrate species to the combination of CNT materials with salinity shifts and pH variations assessing if both climate change factors altered the toxicity of both MWCNT materials as well as the sensitivity of all these species exposed to these contaminates. The present findings underlined that Nf-MWCNTs and f-MWCNTs under control salinity and low pH generated major toxic impacts in the organisms compared to individuals maintained under low salinity and control pH, confirming that salinity shifts and pH variations may alter the chemical behaviour of both MWCNTs and consequent fate in exposed individuals. Moreover, we observed species-dependent sensitivity to contaminants confirming that the higher susceptibility observed in some species would however be expected not only to depend on the characteristics of the compounds, but also on the physiology of that particular species. For a better environment protection, the Ecological Risk Assessment of the mentioned stressors must include ecologically relevant endpoints and exposure scenarios to drive accurate safety levels towards biodiversity conservation.
De acordo com publicações recentes, nos próximos 100 anos prevê-se um aumento na acidificação da água do mar e alterações na sua salinidade. Nas águas superficiais, o aumento do CO2 atmosférico já causou uma diminuição do pH em mais de 0,1 unidades comparando com 8,1, a média referente à época pré-industrial. Está previsto que até ao final deste século, esta redução do pH possa atingir valores na ordem das 0,13 e 0,43 unidades. As alterações climáticas podem também resultar em alterações na salinidade da água do mar. A salinidade é mais alta quando as temperaturas são mais altas e os períodos de chuva são reduzidos enquanto que, eventos de chuva intensa diminuem a salinidade da água do mar. Em qualquer dos cenários, estas alterações irão promover respostas por parte das espécies. Portanto, é imperativo identificar os efeitos das alterações climáticas nos ecossistemas aquáticos de modo a conservar a sua biodiversidade. Para além das alterações climáticas, há uma preocupação crescente com o grande número de poluentes emergentes que têm sido descartados no meio ambiente sem serem devidamente regulamentados. Entre estes poluentes emergentes estão as nanopartículas artificiais (Engineered nanoparticles - ENPs). Um dos tipos de ENPs mais usados nos últimos anos são os Nanotubos de Carbono (Carbon nanotubes - CNTs). Devido às suas propriedades químicas e nano-toxicológicas únicas, é expectável que os CNTs entrem nos ambientes aquáticos e se acumulem na fauna que lá vive. De facto, a toxicologia dos CNTs em sistemas aquáticos é complexa. Numa primeira análise, o tamanho, a forma, a estrutura química e os agentes de revestimento desempenharão um papel no que diz respeito à estabilidade e, portanto, à biodisponibilidade da partícula. No entanto, a toxicidade dos nano-materiais tem sido atribuída não só à sua estrutura central e modificação/funcionalização da sua superfície, mas também aos parâmetros físico-químicos do meio em que os nanotubos se apresentam e que podem alterar a sua dispersão e consequentemente a sua deteção: agregação/desagregação, adsorção/dessorção, sedimentação/ressuspensão e dissolução. O impacto dos nanotubos no meio aquático já foi descrito por vários autores; no entanto, ainda não se sabe de que forma as alterações climáticas podem alterar a toxicidade dos CNTs e subsequentemente os efeitos sobre os organismos marinhos. As espécies bentónicas são um bom modelo para avaliar os impactos das Alterações Climáticas e ENPs, uma vez que são sensíveis às mudanças ambientais. Principalmente devido às características do seu ciclo de vida, bem como à sua resposta relativamente rápida à poluição, há vários estudos que usam espécies bentónicas como bioindicadores para fatores de stress antropogénicos e naturais. Sendo assim, é urgente avaliar os impactos dos CNTs, sob alterações de salinidade e redução do pH em organismos aquáticos. Ruditapes philippinarum (bivalve), Hediste diversicolor e Diopatra neapolitana (poliquetas) são invertebrados marinhos que respondem rapidamente a perturbações ambientais e são caracterizados por uma ampla distribuição espacial e relevância económica, nomeadamente em Portugal. Pelo que, esta tese pretendeu avaliar os efeitos tóxicos de desvios de salinidade, variação de pH e presença de CNTs (atuando isolados ou em combinação) ao nível da resposta bioquímica (reservas de energia e atividade metabólica, estado oxidativo e neurotoxicidade) e da resposta fisiológica (capacidade regenerativa) das espécies acima citadas. Neste estudo, os dois materiais de CNT selecionados foram os nanotubos de carbono de parede múltipla, não funcionalizados (pristine multi walled carbon nanotubes - Nf-MWCNTs) e os MWCNTs quimicamente funcionalizados através da introdução de grupos polares como grupos carboxilo (-COOH), que aumentam sua estabilidade e capacidade de dispersão no meio aquoso. Como ponto de partida, para cada concentração de exposição, avaliamos os possíveis efeitos da carboxilação/funcionalização da superfície dos MWCNTs nos organismos. Em todas as espécies de invertebrados foi possível observar uma relação positiva entre o aumento da dose e a toxicidade, principalmente no que diz respeito ao estado oxidativo, o que está de acordo com a informação disponível na literatura. Além disso, comparando os efeitos tóxicos de ambos os CNTs, em todas as espécies de invertebrados, verificaram-se maior dano celular induzido pela forma carboxilada da MWCNT em comparação com a forma não funcionalizada. Posteriormente, selecionamos as duas concentrações de Nf-MWCNTs e f-MWCNTs mais perniciosas, e expusemos as três espécies de invertebrados à combinação destes materiais CNT com variações de salinidade e variações de pH, avaliando desta forma se estes fatores relacionados com as alterações climáticas modificavam a toxicidade de ambos os materiais MWCNT bem como a sensibilidade das espécies expostas a esses contaminantes. Os resultados obtidos salientam que Nf-MWCNTs e f-MWCNTs sob salinidade controlo e pH baixo, geraram grandes impactos tóxicos nos organismos em comparação com indivíduos mantidos em condições de salinidade baixa e pH controlo. Confirmou-se desta forma que, alterações de salinidade e variações de pH podem alterar o comportamento químico de ambos os MWCNTs e consequentemente o efeito em indivíduos expostos. Além disso, observamos que a sensibilidade ao contaminante é dependente da espécie o que confirma que a maior suscetibilidade observada em algumas espécies não é apenas um resultado das diferentes características dos compostos usados, mas também da fisiologia dessas espécies em particular. Para uma melhor proteção do meio ambiente, a Avaliação de Risco Ecológico dos fatores de stress mencionados, deve incluir objetivos e cenários de exposição ecologicamente relevantes para impulsionar medidas de segurança corretas e adaptadas com respeito à conservação da biodiversidade.
Programa Doutoral em Biologia e Ecologia das Alterações Globais

Dissertation submitted in partial compliance with the requirements for the Masters Degree in Technology, 2010.
Water storage dams play an important part in the collection and purification of water destined for human consumption. However, the nutrient rich silt in these dams promotes rapid growth of aquatic plants which tend to block out light and air. Glyphosate is universally used as the effective non-selective herbicide for the control of aquatic plants in rivers and dams. Invariably there is residual glyphosate present in water after spraying of dams and rivers with glyphosate herbicide. The amount of residual glyphosate is difficult to determine on account of high solubility of glyphosate in water. Thus a method of sample preparation and a sensitive HPLC method for the detection of trace amounts of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA) in water is required. A crucial step in sample preparation is pre-column derivitization of glyphosate with 9-fluorenylmethyl chloroformate (FMOC-Cl). For sample pretreatment, water samples were derivatized with FMOC-Cl at pH 9, extracted with ethyl acetate and sample clean-up was carried out by passing a sample through the SPE cartridge. For SPE, recovery studies were done to choose a suitable cartridge for glyphosate and AMPA analysis. The following cartridges were compared, namely, C18, Oasis HLB and Oasis MAX SPE cartridges. Best recoveries (101% for glyphosate and 90% for AMPA) were obtained using 500 mg of C18 solid-phase extraction cartridge. The eluent from SPE cartridge was injected into HPLC column. Three types of separation columns (namely; C18 column, silica based amino column and polymeric amino column) were compared for the separation of glyphosate and AMPA. The best separation of glyphosate and AMPA in water samples was achieved using a polymeric amino column and a mobile phase at pH 10 which contained a mixture of acetonitrile and 0.05 M phosphate buffer (pH 10) 55:45, (v/v) respectively. The method was validated by spiking tap water , deionized water and river water at a level of 100 μg/l. Recoveries were in the range of 77% -111% for both analytes. The method was also used in determining the levels of glyphosate and AMPA in environmental samples. This method gave detection limits of 3.2 μg/l and 0.23 μg/l for glyphosate and AMPA respectively. The limits of quantification obtained for this method were 10.5 μg/l and 3.2 μg/l for glyphosate and AMPA respectively.