Eutrophication of lakes and streams are nowadays a well known environmental problem and implies an enrichment of the nutrients phosphorus (P) and nitrogen (N). Phosphorus is considered to be the most important component for the growth of aquatic plants and leads in too large quantities to an intensification of growth. Phosphate (PO4) is the fraction of phosphorus that can easiest be taken up by plants and thus have the greatest impact on eutrophication. Increased plant growth in lakes and unfavorable conditions for aquatic animals are two examples of negative consequences. A significant portion of the increased nutrient supply to nearby water can be derived to phosphate leaching from agricultural areas, where private sewers and agriculture is two main sources. How much of the diffuse leakage of phosphate derived from each source is uncertain. With an improved understanding of how the source and flow distribution of phosphate relationship works in an agricultural dominated catchment could a more cost efficient planning for choice of methods against leakage of the nutrient be achieved. The source and flow distribution of phosphate was therefore studied and a high resolution set of SMHI's hydrological model HYPE where set up over the area. Sampling of phosphate from different parts of the area where used together with modeled water flow and phosphate transport where calculated. Upstream and downstream dynamics of phosphate were compared during high water flow. Also sampling of the oxygen isotopic composition in phosphate ions from sewage and agricultural land were analyzed and used to estimate the source distribution at different situations of water flow. The collected information from these studies where used to identify from which source to phosphate and during which flow the leakage of phosphate where the most in the area. In addition to this an evaluation of the importance of the time and space resolution for the HYPE-model where made. Phosphate transports in space shows that the sewage-dominated basins where the areas that leak most phosphate per area unit and during low water flows. Agricultural areas account for more significant leakage during a high water flow. This is evident in autumn and winter and less evident during spring and summer. Oxygen isotopic composition of phosphate ions from wastewater and agricultural land could with a 99 % confidence be separated. This information was used for source separation of phosphate sources where sewage was the main source of water flow up to 23 l/s. At higher water flow increases agricultural land as a primary source and increases positively linearly with increasing water discharge. Both studies indicate that sewage accounts for the greatest leakage during low water. During a snow-melting period the phosphate leaching is highest at the beginning and gradually flushed out from the soil when the water flow remains high for several days in a row. The importance of a high resolution time and space step affected modeled data marginally positive in HYPE where time step had a more important role.

Stable isotope analysis is a tool employed in ecological studies to provide information on the movement of elements and energy through a system. The stable carbon and nitrogen isotope analysis of aquatic organisms has been commonly used to address questions related to energy transfer between organisms and to identify the reliance of aquatic organisms on different sources of organic matter within the system. Within the rivers, stable isotope analysis has been used to describe food webs and connect conditions within the watershed with the river. The Grand River watershed is a predominantly agricultural watershed which receives inputs from ~26 MWWTP and is managed for flow by multiple large reservoirs and weirs. The stable isotope values of aquatic organisms within this watershed were analyzed from samples collected between May and September, 2007. Sites were selected in relation to three different municipal waste water treatment plants (MWWTP) in the centre of the watershed and along a 200 km stretch of the main stem of the Grand River. Results show that stable isotope analysis can be used to differentiate organisms collected from different sites and which represent different trophic levels within the river system under select conditions. Sites which are influences by inputs from organic matter or nutrients within distinct isotope values can be distinguished easily if the input is large and the isotope values are significantly distinct from background values. For smaller inputs changes in stable isotope values were not observed relative the background variability in the system. In this case, sites should be selected to allow for the characterization the variation in isotope values already occurring within the river. Samples collected later in the growing seasons have more distinctive isotope values are between sites. At sites where seasonal variation is greater, the organisms collected may not show a clear separation between trophic levels. A lack of knowledge regarding the time period represented by the tissues of the organisms challenges interpretation these results. It is concluded that stable isotope values of aquatic organisms reflected the condition of this watershed. For nitrogen increasing loads from point sources were accompanied by increasing isotope values. Stable isotope values decreased over the river reach where recovery in river condition occurs as a result of ground water inputs. The influence of individual large MWWTP and reservoirs was observable and the management of the MWWTP and reservoir appears to affect the changes in isotope values which are observed.

An improved ability to assess whether individuals have been added through immigration or natality and lost through emigration or mortality could alleviate several problems in population ecology. Fortunately, advances in stable isotope techniques now allow the movements of individuals to be retraced from tissue values and provide an opportunity to link information about the origins of individuals with demographic rates so that questions about the significance of dispersal can be assessed. I used such an approach by combining feather isotope information with demographic rates derived from capture-mark-recapture of individual mallards (Anas platyrhynchos) breeding in the Canadian aspen parklands, at multiple spatiotemporal scales, to answer questions about population persistence, settling patterns by dispersers, and the fitness of immigrant birds relative to residents.<p> Feather isotope (ä34S, äD, ä15N, and ä13C) values from an independent sample of flightless mallard ducklings sampled from across the mid-continent breeding range was used to validate an existing model used for origin assignments. Spatial resolution analysis within the mid-continent mallard breeding range generally showed a loss in prediction when attempting to assign individuals to more narrowly separated geographic origins among boreal, aspen parkland and prairie regions. For feather äD, spatial resolution may be limited by temporal patterns of local climatic events that produce variability in consumer tissue values. Thus, the use of multiple feather isotope signals would provide more reliable information about the origin of individuals for addressing questions about long-distance dispersal in yearling mallards.<p> Demographic rescue in an apparent population sink near Minnedosa, Manitoba, Canada, was due to elevated survival rates from a highly productive group of nesting female mallards using nest tunnels (i.e., an artificial nesting structure) and recruitment of yearling females having natal origins within the aspen parklands. There was little evidence that immigration by yearling females dispersing long-distances was important to annual population growth rates. Consistently high annual survival rates of adult females using nest tunnels lowered the recruitment rates needed for population stability. While tunnel-origin and within-region recruitment of yearling females were nearly equally important to local population growth rate, fine-scale limitations of isotopic origin assignments prevented further assessment of where recruits originated from within the aspen parkland region.<p> Factors related to breeding area settling patterns of yearling females are not well understood despite implications to local population dynamics. The likelihood that immigrant yearling females would settle in a parkland breeding area was positively correlated with local breeding-pair density and the amount of perennial nest cover, but was negatively correlated with the amount of wetlands. Although these relationships were not well estimated, they are most consistent a hypothesis that females were attracted to breeding sites by conspecific cues rather than avoidance. Immigrants comprised an average of 9% (range: 0 39% over 22 sites) of yearling recruits; most had natal origins in the U.S. prairie pothole region but a non-trivial number originated from the boreal forest, indicating a high degree of connectedness among breeding regions resulting from long-distance natal dispersal.<p> One of the most frequent explanations for strong site fidelity in breeding female ducks is that females benefit from site familiarity. However, evidence for differential reproductive success between immigrant and resident yearling females was weak, On sites with favourable wetland conditions and low breeding-pair densities immigrant females were more likely to breed and nest successfully than were residents whereas under opposite wetland and pair conditions, resident females were favoured. Thus, the costs and benefits of a natal dispersal decision seemed to vary with social context and environmental conditions, and further work is needed to clarify these processes.

Despite a number of kinetic and spectroscopic studies, the chemical mechanisms of amine oxidation by flavoenzymes remain widely debated. The mechanisms of by Nmethyltryptophan oxidase (MTOX) and tryptophan 2-monooxygenase (TMO) were probed using a combination of pH and primary deuterium, solvent, and 15N kinetic isotope effects. Slow substrates were chosen for these studies; MTOX was characterized with N-methylglycine and TMO was characterized with L-alanine. Primary deuterium kinetic isotope effects of 7.2 and 5.3 were observed for sarcosine oxidation by MTOX and for alanine oxidation by TMO, respectively, independent of the substrate concentration and pH. Monitoring the reduction of flavin spectroscopically revealed no intermediate flavin species with both enzyme-substrate systems. Furthermore, the magnitudes of the 15N kinetic isotope effects observed with both systems suggest that nitrogen rehybridization and C-H bond cleavage are concerted. These results are consistent with both enzymes utilizing a hydride transfer mechanism for amine oxidation. The role of the iron ligands of tyrosine hydroxylase (TyrH) was also investigated. TyrH contains one iron per monomer, which is held by three conserved amino acid residues, two histidines and a glutamate. As a probe of the plasticity of the metal binding site, each of the metal ligands in TyrH was substituted with glutamine, glutamate, or histidine. The resulting proteins were characterized for metal content, catalytic activity, and dopamine binding. The H336E and H336Q enzymes retain substantial catalytic activity. In contrast, the E376Q enzyme retains about 0.4% of the wild-type catalytic activity, and the E376H enzyme has no significant activity. The H331E enzyme oxidizes tetrahydropterin in a tyrosine-independent manner. The position of the charge-transfer absorbance band for the H336E and H336Q enzyme-inhibitor complexes is shifted relative to that of the wild-type enzyme, consistent with the change in the metal ligand. In contrast, the E376H and E376Q enzymes catalyze dopamine oxidation. These results provide a reference point for further structural studies of TyrH and the other aromatic amino acid hydroxylases, and for similar studies of other enzymes containing this ironbinding motif.

Flavocytochrome b2 catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate OH and CH bonds by the wild type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The DVmax and D(V/Klactate) values are both 3.0, 3.6 and 4.5 for the wild type enzyme, flavin domain and D282N enzymes, respectively. The D20Vmax values are 1.38, 1.18, and 0.98 for the wild type enzyme, the flavin domain, and the D282N enzyme; the respective D20(V/Klactate) values are 0.9, 0.44, and 1.0. The Dkred value is 5.4 for the wild type enzyme and 3.5 for the flavin domain, whereas the D2Okred is 1.0 for both enzymes. The V/Klactate value for the flavin domain increases 2-fold at moderate concentrations of glycerol. The data are consistent with the lactate hydroxyl proton not being in flight in the transition state for CH bond cleavage and there being an internal equilibrium prior to CH bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium. Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide, carbon dioxide and water. Sequence alignments identified this enzyme as a member of the L-amino acid oxidase family. The tyrosine and arginine residues in L-amino acid oxidase that bind the carboxylate of o-aminobenzoate are conserved and correspond to Tyr413 and Arg98 in tryptophan 2-monooxygenase. Mutation and characterization of the Y413A, Y413F, R98K and R98A enzymes indicate that these residues are in the active site and interact with the substrate. Deletion of the OH group of Tyr413 increases the Kd for the substrate and makes CH bond cleavage totally rate limiting. The pH V/Ktrp rate profile for the Tyr413 mutant enzymes shows that this residue must be protonated for activity. For both the R98A and R98K enzymes flavin reduction is rate limiting. The Vmax and V/Ktrp pH profiles indicate that the unprotonated form of the substrate is the active form for activity.

Transition metal catalyzed reactions have an important place in synthetic chemistry, but the mechanistic details for many of these reactions remain undetermined. Through a combination of experimentally determined 13C kinetic isotope effects (KIEs) and density functional theory (DFT) calculations, some of these reactions have been investigated. The cyclopropanation of an olefin catalyzed by rhodium (II) tetrabridged complexes has been shown to proceed through an asynchronous, but concerted mechanism. DFT does not provide an accurate transition structure for the reaction of an unstabilized carbenoid with an olefin, but it does predict an early, enthalpically barrierless transition state which is consistent with the reactivity of unstabilized carbenoids. For the case of stabilized carbenoids, the theoretical structures predict the KIEs accurately and a new model is proposed to explain the selectivity observed in Rh2(S-DOSP)4-catalyzed cyclopropanations. The chain-elongation step of atom transfer radical polymerization (ATRP) has been shown to be indistinguishable from that of free radical polymerization (FRP) for the CuBr/2,2??-bipyridine system. While DFT calculations predict an earlier transition state than observed, the calculations suggest that with increasing levels of theory the predicted KIEs come closer to the observed KIEs. A recently proposed [2 + 2] mechanism for the cyclopropenation of alkynes catalyzed by Rh2(OAc)(DPTI)3 has been shown not to be a viable pathway. Rather, the experimental KIEs are predicted well by canonical variational transition state theory employing the conventional mechanism for cyclopropenation via a tetrabridged rhodium carbenoid. DFT calculations also suggest an alternative explanation for the observed enantioselectivity. The 13C KIEs for metal-catalyzed aziridination have been measured for three separate catalytic systems. While the KIEs do not completely define the mechanism, all of the reactions exhibit similar KIEs, implying similar mechanisms. A surprising feature of this system is the presumed nitrene intermediate??s triplet spin state. This complicates the DFT analysis of this system.

Tyrosine hydroxylase (TyrH) catalyzes the pterin-dependent hydroxylation of tyrosine to form dihydroxyphenylalanine. The enzyme requires one atom of ferrous iron for activity. Using deuterated 4-methylphenylalanine substrates, intrinsic primary and secondary isotope effects of 9.6 ± 0.9 and 1.21 ± 0.08 have been determined for benzylic hydroxylation catalyzed by TyrH. The large, normal secondary isotope effect is consistent with a mechanism involving hydrogen atom abstraction to generate a radical intermediate. The similarity of the isotope effects to those measured for benzylic hydroxylation catalyzed by cytochrome P-450 suggests that a high-valent, ferryl-oxo species is the hydroxylating species in TyrH. Uncoupled mutant forms of TyrH have been utilized to unmask isotope effects on steps in the aromatic hydroxylation pathway which also implicate a ferryl-oxo intermediate. Inverse secondary isotope effects were seen when 3,5-2H2-tyrosine was used as a substrate for several mutant enzyme forms. This result is consistent with a direct attack by a ferryl-oxo species on the aromatic ring of tyrosine forming a cationic intermediate. Rapid-freeze quench Mössbauer studies have provided preliminary spectroscopic evidence for an Fe(IV) intermediate in the reaction catalyzed by TyrH. The role of the iron atom in the regulatory mechanism has also been investigated. The iron atom in TyrH, as isolated, is in the ferric form and must be reduced for activity. The iron can be reduced by a number of one-electron reductants including tetrahydrobiopterin, ascorbate, and glutathione; however, it appears that BH4 (kred = 2.8 ± 0.1 mM-1 s-1) is the most likely candidate for reducing the enzyme in vivo. A one-electron transfer would require a pterin radical. Rapid-freeze quench EPR experiments aimed at detecting the intermediate were unsuccessful, suggesting that it decays very rapidly by reducing another equivalent of enzyme. The active Fe(II) form can also become oxidized by oxygen (210 ± 30 M-1 s-1); this increases the affinity of catecholamine inhibitors. Serine 40 can be phosphorylated to relieve the inhibition; however, results with S40E TyrH show phosphorylation does not have an effect on the rate constant for reduction of the enzyme but causes a 40% decrease in the rate constant of oxidation.

Over the past five years, gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses of phospholipid fatty acids (PLFAs) has been increasingly used to link organic matter (OM) sources with sedimentary bacteria. This technique has been applied across diverse estuarine and coastal sediments, including lower Laguna Madre, TX, an oligotrophic, coastal lagoon dominated by a single OM source, seagrasses; shelf stations, a eutrophic coastal region receiving multiple sources of OM, hypoxic regions that occur seasonally and deep slope and abyssal plain sediments of the Gulf of Mexico (GOM). Previous reports using the Laguna Madre data as examples, have been used to make comparisons of PLFA 16:0 and PLFA 15:0 isotope ratios and PLFA 16:0 and total organic carbon isotope ratios. Deviations from the 1:1 line in the former indicate living or recently senescent sources of organic matter are not predominantly bacterial. Deviations from the 1:1 line in the latter indicate living or recently senescent sources of organic matter differ isotopically from detrital or older OM in sediments. Prior to the work of Goni et al. (1998), carbon isotope ratios of OM in GOM sediments were interpreted as marine in origin. Based on a series of geochemical measurements, Goni et al. suggested that GOM sediments are largely composed of terrestrial organic carbon (OCterr). Furthermore, They went on to show that shelf and slope sediments were primarily C3 and C4 respectively. I report on the preferential utilization of autochthonous OM by sedimentary bacteria at the sediment surface and the shift to recalcitrant, terrestrially derived OM with depth.

Several organic reactions that could potentially involve coarctate transition states were investigated by a combination of experimental and theoretical studies. In the thermal fragmentation of ∆-1,3,4-oxadiazolines, the mechanism supported by kinetic isotope effects and theoretical calculations is a three-step process that does not demonstrate any special stabilization in coarctate transition states. Rather than undergoing a direct coarctate conversion to product, the mechanism avoids coarctate steps. The last step is a concerted coarctate reaction, but being concerted may be viewed as being enforced by the necessity to avoid high-energy intermediates. In the deoxygenation of epoxides with dichlorocarbene, the stabilization from the transition state aromaticity is not great enough to compete with the preference for asynchronous bonding changes. KIEs and calculations suggested that the reaction occurs in a concerted manner but with a highly asynchronous early transition state with much more Cα-O bond breaking than Cβ-O bond breaking. In the Shi epoxidation, a large β-olefinic 13C isotope effect and small α-carbon isotope effect indicated an asynchronous transition state with more advanced formation of the C-O bond to the β-olefinic carbon. The calculated lowest-energy transition structures are generally those in which the differential formation of the incipient C-O bonds, the "asynchronicity," resembles that of an unhindered model, and the imposition of greater or less asynchronicity leads to higher barriers. In reactions of cis-disubstituted and terminal alkenes using Shi's oxazolidinone catalyst, the asynchronicity of the epoxidation transition state leads to increased steric interaction with the oxazolidinone when a π-conjugating substituent is distal to the oxazolidinone but decreased steric interaction when the π-conjugating substituent is proximal to the oxazolidinone. Dynamic effects were studied in Diels-Alder reaction between acrolein and methyl vinyl ketone. This reaction yields two products in a ratio of 3.0 ± 0.5. Theoretical studies shows that only one transition structure is involved in the formation of both. Quasiclassical trajectory calculations on an MP2 surface give a prediction of a product ratio of 45:14 (3.2:1), which is in good agreement with the experimental observation.

The distribution of apple snail Pomacea canaliculata is islandwide but Pomacea scalaris is only found in southern Taiwan. In order to gain more information on the not well-known invasive species of P. scalaris, the growth and feeding of P. canaliculata and P. scalaris were studied. The hatching period of P. canaliculata (10.4¡Ó1.3 days) was shorter than that of P. scalaris (12.2¡Ó2.3 days). And, the hatching size of P. canaliculata was also smaller. Two weeks after hatching, a significant higher growth rate in P. canaliculata had been found. The 75-day growth equations for P. canaliculata and P. scalaris were y = 0.29x + 1.09 (p < 0.001) and y = 0.16x + 1.68 (p < 0.001), respectively. Feeding structures of mandible, radulae, central tooth and stomach in P. canaliculata were larger than that of P. scalaris. But, there was no significant difference in the length of gut between the two species. Food passing time in P. canaliculata (1.4¡Ó0.7 days) was less than that of P. scalaris (2.9¡Ó0.7 days). And, the approximate digestibility per day of P. canaliculata (44.41¡Ó24.02%) was higher than that of P. scalaris. Through the analyses of £_13C, £_15N, stomach content and feces, it is known that they have the same trophic level with similar food sources. Additionally, the values of £_13C and £_15N were significantly different between sites of JiouRu and Wugou-shui, Pingtung. In other words, the feeding environments were different between the two sites. Based on the results, it is concluded that the superiority of growth and feeding performance in P. canaliculata is important in shaping its wide distribution in Taiwan.