A flow-through pond at White Sulphur Springs National Fish Hatchery was evaluated as culture environment for juvenile freshwater mussels of Villosa iris and Lampsilis fasciola. Survival did not differ significantly (p = 0.1910) over 93 d for V. iris cultured with silt (mean 49.8% ± SD 14.5) and without (mean 32.9% ± SD 11.7). Survival differed significantly (p <0.0001) between juveniles of V. iris (mean 49.8% ± SD 14.5 at age 93 d) and L. fasciola (mean 6.3% ± SD 4.5 at age 86 d). This may indicate that the pond failed to meet requirements of L. fasciola, or may have resulted from microhabitat variables. Growth did not differ significantly between species (p = 0.1315). Villosa iris reached a mean length of 1.81 mm ± SD 0.67, and L. fasciola 1.78 mm ± SD 0.78. Water quality parameters remained within suitable ranges, and planktonic algal densities were between 2850 - 6892 cells/mL. Survival of V. iris and growth of both species compares favorably to previous culture attempts. Juveniles of V. iris and L. fasciola were exposed to ammonium chloride solutions for 96 h in static renewal conditions at 12°C and 20°C. Calculating LC50 values with the Trimmed Spearman-Karber method, juveniles of L. fasciola (mean 96 h LC50 of 0.26 mg/L NH3-N) were significantly more tolerant of unionized ammonia than juveniles of V. iris (mean 96 h LC50 of 0.11 mg/L NH3-N). The only organisms with reported LC50 values lower than those seen for V. iris juveniles were Ceriodaphnia dubia and Hyella azteca. / Master of Science

There is a great deal of concern about the declining freshwater mussel fauna of North America. Although deteriorating water quality and habitat degradation may account for much of the decline, it has been suggested that the exotic Asian clam, Corbicula fluminea, may be having an effect on native unionids. Negative impacts may result directly from competition or indirectly, because of Corbicula population crashes that release ammonia and reduce dissolved oxygen in the sediment. Laboratory tests were conducted to determine the relative sensitivity of native mussel and Asian clam life stages to unionized ammonia, and mussel glochidia were the most sensitive (24-hr LC50 of 0.11 mg/L NH₃-N). Juvenile and adult mussels were similarly sensitivity, with average 96-hr LC50's of 0.49 and 0.52 mg/L NH₃-N, respectively. Adult C. fluminea were the least sensitive, having an average LC50 of 0.80 mg/L NH₃-N. The EPA standard test organism, Ceriodaphnia dubia, had one of the lowest LC50's (0.07 mg/L NH₃-N) of the five species, and the fathead minnow, Pimephales promelas, had the highest (1.18 mg/L). The differing sensitivities of the various life stages are important when trying to determine the impact of an Asian clam dieoff. If a dieoff occurs at a time of year when the more sensitive life stages, such as glochidia are present, then the impact on mussel recruitment may be greater. Two miniature artificial stream tests were used to determine the effect of clam density on dieoff rate, ammonia production and dissolved oxygen levels. Only clams at the highest density of 10,000/m2 experienced 100% mortality. Unionized ammonia levels exceeded 4.0 mg/L, and dissolved oxygen levels dropped below 1.0 mg/L during the dieoff. The amount of unionized ammonia produced was twofold greater than the concentration that produced an LC50 in adult C. fluminea and ~40 times greater than the LC50 for V. iris glochidia. Factors thought to have contributed to the C. fluminea dieoff were flow rate, low dissolved oxygen levels, temperature and perhaps ammonia. A complete dieoff did not occur until flow was stopped and dissolved oxygen concentrations began to drop. One-hundred percent mortality occurred in 38 days for the first test, and 21 days in the second test. Higher water temperatures in the first test (26° C) compared to the second test (average = 21.7°C) are thought to have resulted in the faster dieoff. / Master of Science

The Pacific white leg shrimp, Litopenaeus vannamei, is a potential species for low salinity inland aquaculture. Due to several independent variables, such as species, age, size, salinity and pH, that must be taken into account, there are gaps in the literature pertaining to the toxicity of ammonia and nitrite to shrimp. This study was conducted to investigate the individual and combined effects of ammonia and nitrite on L. vannamei postlarvae (25-45 days old) at 10 ppt salinity, 28 C and a pH of 7.8. The independent variables were salinity, total ammonia as nitrogen (TAN) and nitrite-N (NO₂-N), separately and combined. The TAN experiments were conducted at 18 and 10 ppt salinity while the NO₂-N test was conducted at 10 ppt salinity. Combined TAN and NO2 tests were also conducted at 10 ppt salinity. The LC50 values for TAN at 18 ppt salinity, TAN at 10 ppt salinity, and NO2-N at 10 ppt were observed to be 42.92, 39.72 mg/L (2.26 and 2.09 mg/L unionized ammonia-N), and 153.75 mg/L, respectively. When NO₂- N was adjusted to the LOEC level and TAN concentrations were varied, synergistic effects were observed, with an LC50 calculated to be 28.2 mg/L TAN (1.49 mg/L unionized ammonia-N). However, when the ammonia level was adjusted to the LOEC and nitrite was varied, antagonistic effects were observed with an LC50 calculated to be 163.3 mg/L NO₂-N. The results suggest that further investigations into the combined effects of ammonia and nitrite at varying concentrations and lower salinities will be important in developing "standard operating procedures" for the shrimp industry. / Master of Science

Phenotypic plasticity is hypothesized to facilitate colonization by enabling rapid adaptive responses to novel environments. The colonization of land exposed ancestrally aquatic animals to new ecological and physiological challenges, including toxic waste disposal in dry environments. The repeated evolution of terrestrial breeding in frogs creates opportunities to study developmental adaptations that may facilitate aquatic-to-terrestrial transitions. My dissertation examines the regulation of nitrogen excretion by early life stages in three anuran lineages that independently evolved terrestrial development. First, to assess developmental and environmentally cued changes, I measured N-waste accumulation over development in wet and dry environments in four species, then determined ammonia LC50 values to assess their risk of toxicity on land and the adaptive role of urea excretion. Ammonia accumulates developmentally in clutches or nests of all species and I found urea from both parental and embryonic larval sources. Embryonic larval urea excretion increased in response to dry conditions, and with ammonia accumulation, in the two species with longer terrestrial periods, and their urea excretion appears adaptive, preventing exposure to potentially lethal levels of ammonia. Where early life stages did not risk ammonia toxicity, they excreted no urea. Next, I examined biochemical mechanisms of ammonia detoxification. Urea excretion involves early onset of activity of two ornithine-urea cycle enzymes, arginase and carbamoyl phosphate synthetase, with regulatory plasticity in response to ammonia level during prolonged terrestriality and experimentally high aquatic ammonia. Glutamine synthetase activity provides another mechanism to detoxify ammonia during terrestrial development. Finally, I examined effects of prolonged terrestriality and the larval foam-making activity that supports it on larval physiology, development, and metamorphosis in Leptodactylus fragilis. Even young larvae effectively produced multiple foam nests. I found high ammonia concentrations in new larval nests, high urea excretion by developmentally arrested older larvae, and faster growth of larvae in water than while constructing nests. Larval foam-making extended terrestriality affected the aquatic larval period and age at metamorphosis, while metamorph size decreased with aquatic larval period, but increased with sibship size. Overall, my results suggest that, along with high ammonia tolerance, urea synthesis facilitates terrestrial development but carries physiological costs that may favor plasticity. Dehydration and ammonia accumulation are common, linked risks of terrestrial development. Along with parental adaptations, the evolved traits and plastic responses of early life stages are critical for transitions from aquatic to terrestrial breeding.

Anaerobic digestion (AD) is a sustainable waste management technology that primarily generates two products: biogas and digestate. The technology relies on the microorganisms' activity, which depends on several operational factors, such as pH, temperature, solid contents, and ammonia levels. Ammonia is an inorganic form of nitrogen resulting from the biodegradation of organic nitrogen. It is considered one of the major concerns for AD operations due to its inhibitory effects on some microorganisms, particularly methanogens. A common feedstock characterized by high nitrogen content is poultry manure (PM). PM is often avoided in anaerobic digesters due to the anticipated inhibition resulting from its high ammonia levels. However, since poultry manure is one of the most widely available organic wastes, researchers have extensively investigated ways to include PM as a primary feedstock for AD. One possible way to treat high ammonia levels in digestate is ammonia stripping, the physio-chemical separation of ammonia from a solution by introducing a stripping (carrier) gas. There are a few approaches to performing ammonia stripping in AD applications; the most commonly discussed in the literature are pre-hydrolysis and side-stream ammonia stripping. Pre-hydrolysis ammonia stripping is performed on raw feedstock after increasing pH and temperature and is usually not restricted in selecting the gas carrier. On the other hand, side-stream ammonia stripping is when a portion of the digester's working volume is filtered, and the filtrate is sent to a unit where pH and temperature are increased. The carrier gas in these systems is often limited to anaerobic gases such as biogas or steam. The third and most novel approach is post-hydrolysis ammonia stripping, conducted at an intermediate stage between hydrolysis and methanogenesis in a two-stage AD process. This configuration would address the shortcomings of the other two systems. However, there is minimal information on the feasibility and potential of this approach in the literature. This study aims to comprehensively investigate the post-hydrolysis ammonia stripping approach through the following four phases: Phase I) Proof of Concept; Phase II) Optimization; Phase III) Assessment of Alternative Carrier Gases; and Phase IV: Comparison of Different Ammonia Stripping Configurations. Phase I provided the proof of concept under the batch mode and compared the performance of post-hydrolysis ammonia stripping with two-stage AD and co-digestion to improve poultry manure's methane potential as the primary substrate. It was observed that ammonia stripping successfully improved methane potential by up to 150%, whereas improvements due to two-stage AD and co-digestion were limited to 41 and 9%, respectively. Phase II provided more insight into optimizing the ammonia stripping process. Different stripping conditions were tested (pH 7.8 (unadjusted), 9 and 10, temperature 25 (unadjusted), 40 and 55 °C, and flow rate 300 L/L/hour). The results showed that higher pH and temperature lead to higher removal efficiency. However, it was concluded that optimal conditions ultimately depend on the initial and target ammonia levels. Moreover, Analysis of Variance showed that pH and temperature were significant factors affecting the ammonia removal efficiency. In addition, it was observed that higher stripping temperatures (55 °C) enhanced the digestibility of PM and increased its methane potential more than stripping at 40 °C. It was concluded that the optimum stripping conditions were pH 9.5, temperature 40 or 55 °C, and flowrate of 100 L/L/hour to collectively increase ammonia removal while reducing the associated costs and material handling. In Phase III, renewable natural gas (RNG) was evaluated as a stripping medium in batch testing as a potential replacement for biogas and air. Ammonia stripping with RNG yielded promising results comparable to the application of air in terms of ammonia removal and enhancing biogas production from PM (60 and 69% ammonia removal for RNG and air, respectively). In addition, a metagenomic shotgun analysis showed that most biogas production was conducted through hydrogenotrophic methanogens instead of acetoclastic methanogens, which are more susceptible to high ammonia levels. Phase IV assessed the semi-continuous flow two-stage operation of mesophilic AD reactors coupled with different ammonia stripping configurations. Post-hydrolysis ammonia stripping successfully achieved a stable operation of PM mono-digestion at ammonia levels of 1700 and 2400 mg NH₃-N/L in the cases of stripping with air and RNG, respectively. In addition, post-hydrolysis ammonia stripping in semi-continuous flow mode may have promoted acetoclastic methanogens growth since volatile fatty acid concentrations were reduced in the digesters. Phase IV also proved that the performance of post-hydrolysis ammonia stripping is superior over pre-hydrolysis and side-stream ammonia stripping. In the semi-continuous flow reactors, post-hydrolysis ammonia stripping with air achieved on average 831 L biogas/ kg VS at an organic loading rate (OLR) of 2.6 g VS/L/day, whereas side-stream ammonia stripping resulted in average of 700 L biogas/ kg VS at OLR of 1.8 g VS/L/day and higher ammonia stripping requirements. Having said that, the base scenario (no ammonia stripping) was inhibited, indicating that both ammonia stripping configurations were considered successful in alleviating inhibitory effects of ammonia from poultry manure. Phase IV results also proved that air stripping repeatedly outperformed RNG as stripping mediums by having higher ammonia removal efficiencies resulting in higher methane production. However, stripping with RNG is believed to have more practical advantages than air due to avoiding the risk of oxygen infiltration into the reactor. Moreover, renewable natural gas has proven to be an efficient stripping medium that is available on-site. The final stage of Phase IV tested pre-hydrolysis ammonia stripping using air in batch mode and compared it with post-hydrolysis ammonia stripping. Pre-hydrolysis ammonia stripping provided little to no improvement to the methane potential of PM in batch mode and therefore was excluded from the semi-continuous flow experiment. The four phases of this study demonstrated the flexibility and the superiority of post-hydrolysis ammonia stripping over the other pre-hydrolysis and side-stream ammonia stripping. In addition, post-hydrolysis ammonia stripping was proven efficient and feasible for ammonia removal and enabling the mono- or co-digestion of poultry manure. The study also showed that using RNG instead of biogas can significantly reduce the operational costs of the treatment.

Contexte: L'encéphalopathie hépatique (EH) est une complication majeure de la maladie hépatique chronique (MHC) caractérisée par des symptômes débilitants, notamment des troubles cognitifs, psychiatriques et moteurs. Il est cru que l’EH, définie comme étant un syndrome métabolique, disparaît après une transplantation hépatique (TH). Cependant, des complications neurologiques persistantes ont été signalées chez jusqu'à 47 % des receveurs de TH. Plusieurs études rétrospectives ont démontré une association entre des antécédents d'épisodes d'EH pré-TH et une mauvaise condition neurologique après la TH. D’autre part, l’alcool est un facteur étiologique fréquent à l’origine de la MHC. Cependant, une consommation excessive d’alcool a également un impact sur le cerveau. À ce jour, l’impact des épisodes d’EH ainsi que de l’alcool sur le développement de l’EH et l’intégrité cérébrale reste indéfini. Par conséquent, nos objectifs étaient 1) d’évaluer l'impact de plusieurs épisodes d’EH (induits par l'ammoniac) et 2) d'évaluer l'effet d'une consommation constante d'alcool sur le déclin neurologique, l'intégrité et les lésions cérébrales chez les rats atteints de MHC induite par la ligature des voies biliaires (LVB). Méthodes: Pour le premier objectif, des rats LVB ont reçu une injection (s.c.) d'acétate d'ammonium (LVB-Ammoniac), précipitant un épisode sévère d'EH réversible (perte du réflexe de redressement) tous les 4 jours à partir de la semaine 3 post-chirurgie LVB ( total ; 4 épisodes). Des rats SHAM ont également reçu une injection d'ammoniaque et des rats témoins BDL/SHAM ont reçu une injection de solution saline. La coordination motrice (rotarod) et la mémoire à court et à long-terme (test de reconnaissance d'objets nouveaux) ont été évaluées une semaine après la dernière injection. Pour évaluer l'intégrité neuronale, une analyse d’immunobuvardage de type Western et d’immunofluorescence a été réalisée dans le cortex frontal, le cervelet et l'hippocampe. Pour le deuxième objectif, des rats LVB ont reçu de l'alcool (LVB-Alcool) deux fois par jour (51 % v/v, dose de 3 g/kg, par gavage) pendant 4 semaines. Les rats SHAM ont également reçu de l'alcool et les rats LVB/SHAM servant de contrôles ont reçu une solution saline. La coordination motrice (rotarod) et le comportement anxieux (champ ouvert et labyrinthe surélevé) ont été évalués une semaine après la dernière administration d'alcool. Des analyses d’immunobuvardage de type Western et d’immunofluorescence ont été effectuées pour étudier l'intégrité neuronale du cortex frontal et du cervelet. Résultats: Chez les LVB-Ammoniac, des niveaux protéiques plus élevés d’un marqueur astrocytaire (GFAP) et apoptotique (caspase-3 et Bax/Bcl2) ont été trouvés dans l'hippocampe, alors que les niveaux de marqueurs neuronaux (NeuN et SMI311) ont été réduits par rapport à tous les autres groupes expérimentaux. Les rats LVB-Ammoniac ont présenté des niveaux accrus de stress oxydatif plasmatique par rapport aux rats SHAM/BDL respectifs. Une diminution des niveaux de capacité antioxydante totale (CAT) et une augmentation des protéines modifiées par le 4-HNE ont été observées dans l'hippocampe (et non dans le cortex frontal ou le cervelet) du groupe LVB-Ammoniac par rapport aux rats SHAM/BDL respectifs. Les résultats d'immunofluorescence ont révélé la colocalisation du marqueur apoptotique (caspase-3 clivée) avec un marqueur neuronal (NeuN) dans la région CA1 de l'hippocampe des rats LVB-Ammoniac. Chez les rats LVB-alcool, il a été démontré une altération de la coordination motrice aux semaines 2, 3, 4 et 5 ainsi qu'une augmentation du comportement anxieux par rapport aux rats SHAM respectifs. Chez les rats BDL-alcool, il a été démontré une diminution des marqueurs neuronaux (NeuN et SMI311), une augmentation de l'activité enzymatique apoptotique (caspase-3 clivée), une augmentation des marqueurs de nécroptose (pRIP3 et pMLKL), une diminution de la CAT et une augmentation des protéines modifiées par 4-HNE dans le cervelet par rapport à tous les groupes. Les résultats d'immunofluorescence ont révélé la colocalisation du marqueur apoptotique (caspase-3 clivée) et du marqueur de nécroptose (pMLKL) dans les neurones de la couche granulaire du cervelet de rats LVB-alcool. Conclusion: De multiples épisodes d'EH sévère ont entraîné une perte de cellules neuronales dans l'hippocampe des rats LVB et qui est associée à une augmentation du stress oxydatif, à l'apoptose et à une diminution des cellules neuronales. Des niveaux élevés du marqueur astrocytaire (GFAP) dans l'hippocampe insinuent une gliose, pouvant être le résultat d'une perte neuronale. De plus, l'administration d'alcool aggrave les troubles de la coordination chez les rats LVB et qui ont été associés à une augmentation du stress oxydatif, à une diminution des marqueurs neuronaux (NeuN et SMI311) avec l’apoptose et la nécroptose dans le cervelet des rats LVB-alcool. Globalement, de multiples épisodes d’EH sévère ainsi qu'une consommation constante d'alcool, via le stress oxydatif, déclenchent une perte/lésion neuronale qui entraînera par conséquent une mauvaise condition neurologique après la TH. / Background: Hepatic encephalopathy (HE) is a major complication of chronic liver disease (CLD) characterized by debilitating symptoms, including cognitive, psychiatric, and motor disturbances. HE, defined as a metabolic syndrome, is believed to resolve following liver transplantation (LT). However, persisting neurological complications have been reported in up to 47% of LT recipients. Several retrospective studies have demonstrated an association between a history of HE episodes pre-LT and poor neurological outcome following LT. Furthermore, alcohol is a common etiological factor which causes CLD. However, excessive alcohol consumption also impacts the brain. To date, the impact of HE episodes as well as of alcohol on the development of HE and brain integrity remains undefined. Therefore, our aims were to 1) evaluate the impact of multiple episodes (induced by ammonia) and 2) evaluate the effect of constant alcohol consumption on neurological decline, brain integrity and injury in rats with CLD induced via bile-duct ligation (BDL). Methods: In the first aim, BDL rats were injected (s.c.) with ammonium acetate (BDLAmmonia), precipitating a reversible overt episode of HE (loss of righting reflex) every 4 days from week 3 post-BDL surgery (total; 4 episodes). SHAM rats were also injected with ammonia and BDL/SHAM rats were injected with saline as controls. To assess the neuronal integrity, western blot and immunofluorescence analysis were performed for frontal cortex, cerebellum, and hippocampus. Motor coordination (rotarod) and short- and long-memory (novel object recognition test) were assessed one week following last injection. In the second aim, BDL rats were administered alcohol (BDL-Alcohol) twice a day (51% v/v, dose of 3g/kg, via gavage) for 4 weeks. SHAM rats also received alcohol and BDL/SHAM rats received saline as controls. Motor coordination (rotarod) and anxiety-like behavior (open field and elevated plus maze) were assessed one week following last alcohol administration. Western blot and immunofluorescence analyses were performed to investigate neuronal integrity in frontal cortex and cerebellum. Results: In BDL-Ammonia, higher protein levels of astrocytic marker (GFAP) and apoptotic markers (caspase-3 and Bax/Bcl2) were found in the hippocampus, whereas neuronal markers (NeuN and SMI311) levels were reduced compared to all other experimental groups. BDLAmmonia rats showed increased levels of plasma oxidative stress compared to respective SHAM/BDL rats. Decreased levels of total antioxidant capacity (TAC) and increased 4-HNE modified proteins were found in the hippocampus (not in frontal cortex or cerebellum) of the BDL-Ammonia group compared to respective SHAM/BDL rats. Immunofluorescence results revealed the colocalization of apoptotic marker (cleaved caspase-3) with neuronal marker (NeuN) in the CA1 region of the hippocampus of BDL-Ammonia rats. BDL-Alcohol rats demonstrated impaired motor coordination at weeks 2, 3, 4, and 5 as well as an increase in anxiety-like behavior compared to respective SHAM rats. BDL-Alcohol rats showed a decrease in neuronal markers (NeuN and SMI311), an increase in apoptotic enzyme activity (cleaved caspase-3), an increase in necroptosis markers (pRIP3 and pMLKL), a decrease in TAC and an increase in 4-HNE modified proteins in the cerebellum compared to all groups. Immunofluorescence results revealed the colocalization of apoptotic marker (cleaved caspase3) and necroptosis marker (pMLKL) in granular layer neurons of the cerebellum of BDLAlcohol rats. Conclusion: Multiple episodes of overt HE led to neuronal cell loss in the hippocampus of BDL rats which was associated with increased oxidative stress, apoptosis and decreased neuronal count. Elevated levels of astrocytic marker (GFAP) in the hippocampus insinuate gliosis, possibly a result of neuronal loss. Moreover, alcohol administration worsens coordination impairments in BDL rats which were associated with increased oxidative stress, decreased neuronal markers (NeuN and SMI311) with apoptosis and necroptosis in the cerebellum of BDL-Alcohol rats. Overall, both multiple episodes of overt HE as well as continuous alcohol consumption, via oxidative stress, triggers neuronal loss/injury which consequently will lead to poor neurological outcome post-LT.