This dissertation consists of five papers concerning nitrification in chloraminated drinking water distribution systems in a one and a half year field study. Seven finished waters were produced from different treatment processes and distributed to eighteen pilot distribution systems (PDSs) that were made pipes taken from actual distribution systems. Unlined cast iron (UCI), galvanized steel (G), lined cast iron (LCI), and PVC pipes were used to build the PDSs. All finished waters were stabilized and chloraminated before entering the PDSs. This dissertation consists of five major parts. (1) System variations of nitrates, nitrites, DO, pH, alkalinity, temperature, chloramine residuals and hydraulic residence times (HRT) during biological nitrification are interrelated and discussed relative to nitrification, which demonstrated Stoichiometric relationships associated with conventional biochemical nitrification reactions. Ammonia is always released when chloramines are used for residual maintenance in drinking water distribution systems, which practically insures the occurrence of biological nitrification to some degree. Biological nitrification was initiated by a loss of chloramine residual brought about by increasing temperatures at a five day HRT, which was accompanied by DO loss and slightly decreased pH. Ammonia increased due to chloramine decomposition and then decreased as nitrification began. Nitrites and nitrates increased initially with time after the chloramine residual was lost but decreased if denitrification began. Dissolved oxygen limited nitrifier growth and nitrification. No significant alkalinity variation was observed during nitrification. Residual and nitrites are key parameters for monitoring nitrification in drinking water distribution systems. (2) Using Monod kinetics, a steady state plug-flow kinetics model was developed to describe the variations of ammonia, nitrite and nitrate-N concentrations in a chloraminated distribution system. Active AOB and NOB biomass in the distribution system was determined using predictive equations within the model. The kinetic model used numerical analysis and was solved by C language to predict ammonia, nitrite, nitrate variation. (3) Nitrification control strategies were investigated during an unexpected episode and controlled study in a field study. Once nitrification began, increasing chloramine dose from 4.0 to 4.5 mg/L as Cl2 and Cl2:N ratio from 4/1 to 5/1 did not stop nitrification. Nitrification was significantly reduced but not stopped, when the distribution system hydraulic retention time was decreased from 5 to 2 days. A free chlorine burn for one week at 5 mg/L Cl2 stopped nitrification. In a controlled nitrification study, nitrification increased with increasing free ammonia and Cl2:N ratios less than 5. Flushing with increased chloramine concentration reduced nitrification, but varying flush frequency from 1 to 2 weeks had no effect on nitrification. (4) HPC variations in a chloraminated drinking water distribution system were investigated. Results showed average residual and temperature were the only water quality variables shown to affect HPC change at a five day distribution system hydraulic residence time was five days. Once nitrification began, HPC change was correlated to HRT, average residual and generated nitrite-N in the distribution system. (5) Biostability was assessed for water treatment processes and distribution system pipe by AOCs, BDOCs, and HPCs of the bulk water, and by PEPAs of the attached biofilms. All membrane finished waters were more likely to be biologically stable as indicated by lower AOCs. RO produced the lowest AOC. The order of biofilm growth by pipe material was UCI > G > LCI > PVC. Biostability decreased as temperature increased.

The purpose of this study was to identify the effect that water quality, pipe material, pipe size, flow conditions and the use of corrosion inhibitors would have on the rate of free chlorine and chloramine decay in distribution systems. Empirical models were developed to predict the disinfectant residual concentration with time based on the parameters that affected it. Different water treatment processes were used to treat groundwater and surface water to obtain 7 types of finished waters with a wide range of water quality characteristics. The groundwater was treated either by conventional treatment by aeration (G1) or softening (G2) or high pressure reverse osmosis (RO) and the surface water was treated either by enhanced coagulation, ozonation and GAC filtration (CSF-O3-GAC or S1) or an integrated membrane system (CSF-NF or S2). The remaining two water types were obtained by treating a blend of G1, S1 and RO by softening (S2) and nanofiltration (G4). A pilot distribution systems (PDS) consisting of eighteen (18) lines was built using old pipes obtained from existing distribution system. The pipe materials used were polyvinyl chloride (PVC), lined cast iron (LCI), unlined cast iron (UCI) and galvanized steel (G). During the first stage of the study, the 7 types of water were blended and fed to the PDS to study the effect of feed water quality changes on PDS effluent water quality, and specifically disinfectant residual. Both free chlorine and chloramines were used as disinfectant and the PDSs were operated at hydraulic retention times (HRT) of 2 and 5 days. The PDSs were periodically tested for free and combined chlorine, organic content, temperature, pH, turbidity and color. The data obtained were used to develop separate models for free chlorine and chloramines. The best fit model was a first-order kinetic model with respect to initial disinfectant concentration that is dependent on the pipe material, pipe diameter and the organic content and temperature of the water. Turbidity, color and pH were found to be not significant for the range of values observed. The models contain two decay constants, the first constant (KB) accounts for the decay due to reaction in the bulk liquid and is affected by the organics and temperature while the second constant, KW, represents the reactions at the pipe wall and is affected by the temperature of the water and the pipe material and diameter. The rate of free chlorine and chloramine decay was found to be highly affected by the pipe material, the decay was faster in unlined metallic pipes (UCI and G) and slower in the synthetic (PVC) and lined pipes (LCI). The models showed that the rate of disinfectant residual loss increases with the increase of temperature or the organics in the water irrespective of pipe material. During the second part of the study, corrosion control inhibitors were added to a blend of S1, G1 and RO that fed all the hybrid PDSs. The inhibitors used were: orthophosphate, blended ortho-polyphosphate, zinc orthophosphate and sodium silicate. Three PDSs were used for each inhibitor type, for a total of 12 PDSs, to study the effect of low, medium and high dose on water quality. Two PDSs were used as control, fed with the blend without any inhibitor addition. The control PDSs were used to observe the effect of pH control on water quality and compare to the inhibitor use. One of the control PDSs (called PDS 13) had the pH adjusted to be equal to the saturation pH in relation to calcium carbonate precipitation (pHs) while the pH of the other control PDS (PDS 14) was adjusted to be 0.3 pH units above the pHs. The disinfectant used for this part of the study was chloramine and the flow rates were set to obtain a HRT of 2 days. The chloramine demand was the same for PDS 14 and all the PDSs receiving inhibitors. PDS 13 had a chloramine demand greater than any other PDS. The lowest chloramine demand was observed in PDS 12, which received silicate inhibitor at a dose of 12 mg/L, and presented the highest pH. The elevation of pH of the water seems to reduce the rate of decay of chloramines while the use of corrosion inhibitors did not have any effect. on the rate of chloramine decay. The PDS were monitored for chloramine residual, temperature, pH, phosphate, reactive silica, and organic content. Empirical models were developed for the dissipation of chloramine in the pilot distribution systems as a function of time, pipe material, pipe diameter and water quality. Terms accounting for the effect of pH and the type and dose of corrosion inhibitor were included in the model. The use of phosphate-based or silica-based corrosion inhibitors was found to have no effect on the rate of chloramine dissipation in any of the pipe materials. Only the increase of pH was found to decrease the rate of chloramine decay. The model to best describe the decay of chloramine in the pilot distribution systems was a first-order kinetic model containing separate rate constants for the bulk reactions, pH effect and the pipe wall reactions. The rate of chloramine decay was dependent on the material and diameter of the pipe, and the temperature, pH and organic content of the water. The rate of chloramine decay was low for PVC and LCI, and more elevated in UCI and G pipes. Small diameter pipes and higher temperatures increase the rate of chlorine decay irrespective of pipe material. Additional experiments were conducted to evaluate the effect of flow velocity on chloramine decay in a pilot distribution system (PDS) for different pipe materials and water qualities. The experiments were done using the single material lines and the flow velocity of the water was varied to obtain Reynolds' numbers from 50 to 8000. A subset of experiments included the addition of blended orthophosphate corrosion inhibitor (BOP) at a dose of 1.0 mg/L as P to evaluate the effect of the inhibitor on chloramine decay. The effect of Reynolds' number on the overall chloramine decay rate (K) and the wall decay rate constant (W) was assessed for PVC, LCI, UCI, and G pipes. PVC and LCI showed no change on the rate of chloramine decay at any flow velocity. UCI and G pipes showed a rapid increase on the wall decay rate under laminar conditions (Re ≤ 500) followed by a more gradual increase under fully turbulent flow conditions (Re ≥ 2000). The use of the BOP inhibitor did not have an effect on the rate of chloramine decay for any of the pipe materials studied. Linear correlations were developed to adjust the rate of chloramine decay at the pipe wall for UCI and G depending on the Reynolds' number.

Opportunistic pathogens (OPs) can naturally colonize premise (i.e., building) plumbing and are the leading cause of disease associated with potable water in the U.S. and many other countries. While secondary disinfectant is added by utilities prior to water distribution through pipes, the residual in water at the property line is sometimes insufficient to suppress OP growth. Conditions encountered in premise plumbing can further diminish disinfectant in water after it crosses the property line. This dissertation examines how multiple factors at play in drinking water distribution systems and premise plumbing influence OP growth in order to inform development of rational guidance to reduce incidence of waterborne illness. Operating an at-scale cross-linked polyethylene (PEX) plumbing system with one water flush per day, influent chloramine always decayed within four hours in stagnant pipes containing mature biofilms, which is 2-3 orders of magnitude faster than in the same water not contacting pipes. Chloramine often followed second order decay kinetics, though decay rate coefficients were highly variable with some taps eventually transitioning from second to first order decay over time or with increasing influent chloramine concentration. The rate of chloramine decay was unexpectedly reduced in the water heater tank compared to room temperature pipes, possibly due to lower surface-area-to-volume ratio and higher temperature within the tank. A complementary glass jar experiment confirmed that, contrary to expectations, chloramine could decay slower at the higher temperature of 37-39°C maintained in the water heater, compared to the cooler 19-30°C typical of the pipes. These findings demonstrate the need for disinfectant decay models specific to conditions encountered in premise plumbing. Nitrification, a key microbial process that can catalyze chloramine decay, was typically complete within 24 hours after water entered the stagnant pipes. Counterintuitively, the water heater had a relatively lower rate of nitrification along with some detectable denitrification. This work also showed that oxygen, essential for aerobic microbial growth, can permeate through walls of PEX pipe and enter into the water from the atmosphere of the building. Considering the unique array of conditions that were found to influence the persistence of disinfectants in premise plumbing, a new approach was proposed for managing OP risk, referred to herein as the "CT framework." CT was defined as the integral of the chlorine concentration (C) at a point in the premise plumbing versus water retention time (T). Legionella pneumophila was not detectable in pipes with a CT > 78 mg*min/L over a 24 hour period, which is comparable to reported CT thresholds for 3-log inactivation of biofilm-associated L. pneumophila in batch experiments. There was a tradeoff between control of L. pneumophila and Mycobacterium avium in the water heater, as M. avium increased by >1 log as influent chloramine and CT increased, while L. pneumophila decreased by >1.5 logs. Further research is needed to elucidate the influence of factors such as water storage tank hydrodynamics and sediment on the persistence of different OPs. Building water retention time was also found to be an overarching variable that governs microbial growth in some circumstances in premise plumbing. Total cell counts and L. pneumophila occurrence mirrored expected trends based on the classic microbial growth curve with phases of lag, exponential growth, stationary growth, and decay. The location in the plumbing system where each phase dominated depended on water retention time, disinfectant level, and temperature. The microbial growth curve considerations add an additional dimension to the CT framework for predicting L. pneumophila growth potential in premise plumbing. Specifically, elevated heat or chloramine, was able to temporarily suppress or even eliminate growth, but the phases of classic microbial growth could be restarted once disinfectant or very high temperatures were absent. Total cell counts and L. pneumophila typically peaked at a building water retention time of 7 days, demonstrating that once a week flushing guidance to protect public health may not be advantageous in all situations. Collectively, this work offers fundamental and practical insights into factors driving disinfectant decay and microbial proliferation in premise plumbing, offering a modified CT and microbial growth concept framework to help guide the management of OPs in premise plumbing. / Doctor of Philosophy / Access to safe drinking water is fundamental to human health and wellbeing and is considered to be a human right by some agencies. Opportunistic pathogens (OPs) can grow in some drinking water systems and cause deadly diseases, such as Legionnaires' Disease. Legionnaires' Disease and illnesses caused by other OPs are now the leading cause of drinking water-associated disease in the U.S. and many other countries. Chlorine or chloramine are disinfectants required to be present in treated drinking water in the U.S. before it is piped through the distribution systems to consumers. This helps to limit growth of OPs and other microbes in the distribution systems. However, the concentration of disinfectant that remains in water as it crosses the property line is sometimes inadequate to suppress OP growth. Even if the amount of disinfectant entering a building is boosted, there are some plumbing materials and circumstances that can quickly reduce the disinfectant. These challenges are sometimes worsened by water and energy conservation efforts, which extend the time water spends in a building and presents tradeoffs with preventing OP growth. This dissertation examines how multiple factors at play in drinking water distribution systems and building plumbing individually and collectively influence OP growth, with a goal of developing rational guidance to reduce incidence of waterborne illness. Experiments were conducted using a large at-scale building plumbing system. These experiments revealed new insights into the relationship among factors such as how long the water stagnates in pipes, water temperature, the disinfectant concentration at each tap, and the level of specific OPs of concern. Chloramine was gone within four hours of stagnation in plastic cross-linked polyethylene (PEX) pipes containing a mature biofilm, which is 100-1000× faster than observed in the same water that did not contact pipes. The rate at which chloramine disappeared changed with conditions from tap to tap, or with time at a given tap, in ways that were unexpected based on prior assumptions. Further, the hydraulic characteristics and low temperature of the water heater influenced chloramine decay in the tank in a way that increased survival and release of OPs. We found that other microbes residing in pipes, such as nitrifying microbes, can also play a role in decay of disinfectant and their activity also is controlled by the water retention time and temperature in the system. These findings reinforce the need to thoroughly understand how chemical, biological, and hydraulic factors combine to influence OP growth in buildings. To account for the array of factors that contribute to the decay of disinfectant, we introduce premise plumbing "CT" as a new integrative framework to guide management of OPs. We define CT as the integral of the disinfectant concentration (C) at a stagnant point in the building plumbing verses the time (T) water has resided at that point, to characterize the ability of the water to kill or suppress growth of bacteria. If the calculated CT values in the at-scale plumbing system were high enough, Legionella pneumophila, the OP that causes Legionnaires' Disease, was never detected in pipes. However, if CT was too low, L. pneumophila was not controlled. Oddly, M. avium, another problematic OP, exhibited a contradictory trend within the water heater. This indicates that the CT concept may not control M. avium in chloraminated water heaters with complex water flow patterns and sediment. Higher chloramine caused lower L. pneumophila and higher M. avium in the water heater, but this tradeoff did not occur in cold water pipes when the room temperature was below that required for OP growth, indicating that room temperature setpoint could be a significant factor for OP control in buildings. Building water retention time, which is the time that water takes to move through the plumbing before it is consumed from a tap, was identified in this research to be a key driver of microbial growth that can be readily controlled by building managers. Trends of total microbial cell count and L. pneumophila in the premise plumbing system and complementary experiments followed all the phases of growth associated with bacteria in a simple glass jar, including a lag, rise, peak, and then decay of cells. Elevated heat or chloramine was able to temporarily suppress growth or even kill cells, but the phases of growth were again observed once the chemical or thermal disinfectant was removed. In any building, there is likely a frequency of flushing water at a given tap that is "worst case" for bacterial growth. In the absence of disinfectant, bacteria in pipes that are frequently supplied with nutrients through fresh water can be expected to have sustained growth, but if bacteria are starved of nutrients, there is some die off. In our system, total microbial cell counts and L. pneumophila peaked at a water retention time of about one week. Thus, this work suggests that current advice to flush building pipes once a week might sometimes create issues with microbial growth rather than solve them. Collectively, this research advances both fundamental and practical understanding of the factors driving disinfectant decay and microbial proliferation in premise plumbing. The premise plumbing CT and microbial growth concept framework is introduced to help inform better management of building water systems to prevent or remediate the growth of pathogens and reduce risk of human infection.

The aim of this thesis was to test and compare the effect of four selected and in present commonly used medical treatments on other species of fish in recirculating aquaculture system (RAS). This presented work was divided into two separate experiments dealing with curative baths for fry of pikeperch.The fry was raised in ponds, feeded by natural food, with the intention of adaptating for RAS after a bath. Baths were targeted against parasitic and bacterial diseases by using Bellasav, SAVO Original, formaldehyde and Chloramine T. The curative baths took 30 minutes after which the fish were transferred to clean water where the mortality within 24 hours and the efficacy of the preparation within 24 hours was monitored. In the first experiment were used fish with an average weight of 0.23+-0.05 g and a total lenght of 31.7+-2.5 mm at the age of 35 days. The tested doses were 0.5; 1; 1.5; 2; 5; 10 and 50 ml.m-3 for Bellasav, 1; 5; 10; 20 and 50 ml.m-3 for SAVO Original, 0.5; 1; 1.5; 2.5; 5; 10; 15; 30; 45 and 50 ml.100 l-1 for formaldehyde and 10; 20; 30; 40; 50; 100; 150 and 200 mg.l-1 for Chloramine T. The best efficacy was achieved at the concentration dose 1.5 ml.m-3 for Bellasav. The concentration dose 10 ml.m-3 of SAVO Original only reduced parasitic infection by one degree. An effective dose of formaldehyde was 1 ml.100 l-1 which reduced the parasitic infection by two degrees. The most effective preparation was Chloramine T at the concentration dose of 30 mg.l-1. In the second experiment were used fish with an average weight of 1.6+-0.4 g and a total lenght of 60.4+-3.56 mm at the age of 70 days. The tested doses were 10; 20 and 50 ml.m-3 for Bellasav, 10; 20 and 50 ml.m-3 for SAVO Original, 2.5; 5 and 10 ml.100 l-1 for formaldehyde and 40; 150 and 200 mg.l-1 for Chloramine T. The best efficacy was achieved at the concentration dose 20 ml.m-3 for Bellasav and 10 ml.m-3 for SAVO Original. The most effective dose for formaldehyde was 2.5 ml.100 l-1 which reduced the bacterial infection in fish skin by two degrees. The most effective preparation was Chloramine T again at the concentration dose 40 mg.l-1.

The protein folding problem involves understanding how the tertiary structure of a protein is related to its primary structure. Hence, understanding the thermodynamics associated with the rate-limiting steps for the formation of the earliest events in folding is most crucial to understanding how proteins adopt native secondary and tertiary structures. In order to elucidate the mechanism and pattern of protein folding, an extensively studied protein, Cytochrome-c (Cc), was chosen as a folding system to obtain detailed time-resolved thermodynamic profiles for the earliest events in the protein folding process. Cytochrome-c is an ideal system for understanding the folding process for several reasons. One being that the system can unfold and refold reversibly without the loss of the covalently attached heme group. A number of studies have shown that under denaturing conditions, ferrous Cc (Fe2+Cc) heme group in the presence of carbon monoxide (CO) results in a disruption of the axial heme Methionine-80 (Met80) bond ultimately unfolding the protein. CO-photolysis of this ferrous species results in the formation of a transient unfolded protein that is poised in a non-equilibrium state with the equilibrium state being that of the native folded Fe2+Cc complex. This allows for the refolding reaction of the protein to be photo-initiated and monitor on ns - ms timescales. While CO cannot bind to the ferric form, nitrogen monoxide (NO) photo-release has been developed to photo-trigger ferric Cc (Fe3+Cc) unfolding under denaturing conditions. Photo-dissociation of NO leaves the Fe3+complex in a conformational state that favors unfolding thus allowing the early unfolding events of Fe3+Cc to be probed. Overall the results presented here involve the use of the ligands CO and NO along with photoacoustic calorimetry (PAC) to photo-trigger the folding/unfolding reaction of Cc (and modified Cc). Thus, obtaining enthalpy and molar volume changes directly associated with the initial folding/unfolding events occurring in the reaction pathways of both Fe2+ and Fe3+Cc systems that are most essential to understanding the driving forces involved in forming the tertiary native conformation. The PAC data shows that folding of proteins results from a hierarchy of events that potentially includes the formation of secondary structures, hydrophobic collapse, and/or reorganization of the tertiary complex occurring over ~ns – tens of µs time ranges. In addition, the PAC kinetic fits presented in this work is the first to report Cc folding exhibiting heterogeneous kinetics (in some cases) by utilizing a stretched exponential decay function.

Increasing freshwater scarcity across the world means that wastewater reclamation is being considered as a key method in which to meet the growing demand. Evolution of water reuse schemes where high quality product is required such as for indirect potable reuse has led to the adoption in recent years of the integrated membrane scheme using a combination of microfiltration or ultrafiltration with reverse osmosis membrane. However, despite technological advancements, these membranes are still prone to fouling resulting in increased costs through cleaning or replacement. This thesis aims to look at pretreatment to reduce the fouling propensity of the microfiltration membranes via a 600m3 /d pilot plant which was commissioned to investigate indirect potable reuse. A range of pretreatments including pre-screening, pre-coagulation, powdered activated carbon and granular activated carbon were assessed based on fouling amelioration, water quality improvement and cost analysis. Results showed that ferric sulphate dosing was the most effective in terms of reducing the reversible fouling rate especially at high turbidity loads enabling higher flux to be realised leading to a small cost benefit. Activated carbon proved the most effective pretreatment in terms of organic removal and a significant reduction in the irreversible fouling rate. However, the cost involved in using this as a pretreatment is significant compared to possible cost savings through reduced requirement for chemical cleaning. This pretreatment is only viable if it obviates the need for a separate organic removal process.

Three advanced methodologies for the preparation of extremely sterically hindered tertiary alkyl amines have been developed. The syntheses of 28 novel tertiary alkylamines that accommodate unusual steric hindrance are detailed. The electrophilic amination of alkyl Grignard reagents with N-chlorodialkylamines, in the presence of N,N,N′,N′-tetramethylethylenediamine (TMEDA) as a key additive, gives a variety of unprecedentedly sterically hindered tertiary alkylamines in good yields. Alternative strategy to 1-adamantyl-substituted (1-Ad) sterically hindered tertiary amines, which involved instead an SN1 reaction between 1-Ad cation with various secondary amines, is described. A complementary strategy to 1-Ad-based sterically hindered tertiary amines, which involves an iminium salt intermediate, is also reported. Salient features of the three protocols that are detailed here include unusual tolerance of steric hindrance, mild reaction conditions employed, ease of product isolation-purification, and absence of catalysts/transition metals. The molecular structures of two faithful examples of extremely sterically hindered tertiary alkylamines were determined by single crystal X-ray diffraction, and the height “h” of nitrogen pyramid of these compounds were measured. The NMR spectra show a restriction in rotation at room temperature among many hindered tertiary amines, and some of them exhibit two complete sets of peaks for two non-equivalent rotamers at room temperature. 15N NMR has been applied to study the structural changes in highly sterically hindered tertiary amines. Most of these compounds have been shown to undergo Hofmann type elimination reaction upon thermolysis at 100 degree in inert solvents, like toluene. / In der vorliegenden Arbeit wurden drei Methoden zur Synthese von tertitären Aminen mit extremer sterischer Hinderung entwickelt und zur Synthese von 28 neuen tertiären Alkylaminen mit entsprechender sterischer Hinderung angewendet. Die elektrophile Aminierung von Grignard-Reagenzien mit N-Chlordialkylaminen, unter Zusatz von N,N,N′,N′-Tetramethylethylendiamin (TMEDA) als Schlüsselkomponente, ermöglicht einen einfachen Zugang zu einer Vielzahl von tertiären Aminen mit extremer sterischer Hinderung mit guten Ausbeuten. Eine alternative Synthesestrategie unter SN1-Bedingungen führt zu sterisch-gehinderten 1-Adamantyl-substituierten (1-Ad) tertiären Aminen durch die Reaktion eines 1-Ad-Kations mit unterschiedlichen sterisch-gehinderten sekundären Aminen. Angelehnt an die zuvor beschriebene Reaktion können auch sterisch gehinderte Imine über eine Iminium-Salz-Zwischenstufe zu sterisch-gehinderten 1-Ad-substituierten tertiären Aminen umgesetzt werden. Auch in diesen Fall zeichnet sich die Reaktion durch eine bemerkenswerte Toleranz gegenüber sterischer Hinderung, milden Reaktionsbedingungen, leichte Produktisolierbarkeit und die Abwesenheit von Übergangsmetallkatalysatoren aus. Die molekulare Struktur zweier repräsentativer tertiärer Alkylamine mit extremer sterischer Hinderung wurde mittels Röntgeneinkristallstrukturanalyse untersucht und die Höhe “h” ihrer Stickstoff-Pyramide bestimmt. Die NMR-Spektren zeigen bei RT eine Einschränkung der freien Rotation um die N-C-Bindungsachse, teilweise führt dies zu vollständig getrennten Signalsätzen für die einzelnen Rotamere. 15N-NMR-Spektroskopie wurde ebenfalls zur Untersuchung von Strukturveränderungen genutzt. In inerten Lösungsmitteln, wie Toluol, zeigen die Verbindungen bei 100 °C in den meisten Fällen eine Hofmann-Eliminierung.