PHYTOREMEDIATION OF WEATHERED PETROLEUM IN GROUNDWATER BY ARROYO WILLOWS IN NUTRIENT AMENDED ON-SITE MESOCOSMS SARAH BRAGG-FLAVAN A large-scale mesocosm study was conducted to determine if vegetation with willow trees enhances biodegradation and to evaluate the mechanisms of natural biodegradation of weathered petroleum compounds under field conditions. The mesocosms were designed to model conditions at a former oil field where mid-range petroleum distillates were used as a diluent for pumping crude oil contaminated the soil and groundwater at the site with petroleum compounds. Ten mesocosms were constructed at the field site using un-impacted soil and diluent-impacted groundwater from the site. Five of the mesocosms were planted with Arroyo Willow trees native to the field site and the other five served as controls without trees. Since these willow trees are phreatophytes, their roots are capable of consuming water from the water table. A previous study was conducted using these mesocosms, however the willow trees then were in poor condition. In this study, fertilizer was added to the mesocosms to promote healthy growth of the willows. Fertilizer was added equally to mesocosms with and without willow trees to avoid introducing bias. Groundwater was circulated through the mesocosms for two 109 to 126 days runs, while the total petroleum hydrocarbon (TPH) concentrations of the groundwater were measured periodically. Dissolved oxygen concentrations were also monitored in each of the mesocosms to determine if the willow trees had any impact on oxygen transfer to the subsurface. In the first run without nutrient amendments the trees did not enhance biodegradation. All the mesocosms started with an average TPH concentration of 6.3 mg/L and ended with a concentration of 1.0 mg/L. After this first run, nutrient amendments were added to all the mesocosms, resulting in healthy trees with robust growth. With healthy willow trees, the planted mesocosms resulted in a statistically significant increase in long-term biodegradation of dissolved-phase petroleum compounds. The planted mesocosms resulted in 29 percent more degradation. These results agree with prior lab studies using bench-scale microcosms with media from the former oil field which indicated that TPH concentrations after 100 days were lower in containers with willows or lupines compared to controls without plants. Microtox® toxicity decreased for both planted and control mesocosms, showing no toxic root exudates or by-products. There are several potential mechanisms of the observed phytoremediation. Terminal restriction fragment analyses showed that the planted mesocosms had different microbial communities than the unplanted mesocosms. Thus, a possible mechanism of the phytoremediation is stimulation of a rhizobial microbial community that biodegrades petroleum compounds. The dissolved oxygen (DO) concentrations were actually lower in the planted mesocosms, possibly due to consumption of oxygen during biodegradation of root exudates. The reduced DO concentrations in the planted mesocosm discounts the possibility that the plants stimulated biodegradation by increasing oxygen transfer to the subsurface. It is not known from these experiments if the petroleum compounds were taken up by the plants or if the plants stimulated bacterial biodegradation. Since it is difficult for plants to uptake non-polar compounds with a high octanol-water coefficient (Kow), it is usually unlikely that plants could uptake petroleum compounds which usually have a Kow > 3. However, the log Kow of the dissolved phase diluent determined in this research was only 0.14. Although the mechanism by which the willow trees increased biodegradation was not elucidated, this study demonstrated that phytoremediation of the polar and hydrophilic weathered petroleum compounds was successful. Column chromatography was used to fractionate petroleum compounds extracted from the groundwater into aliphatic, aromatic and polar components so that biodegradation of each of these fractions could be determined independently. The first mesocosm experiments showed that regardless of the presence of trees, there was a decrease in TPH concentration for all three fractions. The overall unfractionated biodegradation rates averaged 41 ug/L/day over this experiment, and the biodegradation rate of the polar fraction was similar at 40 ug/L/day. In comparison, the biodegradation rates of the aliphatic and aromatic fractions were considerably lower at 1.2 and 2.6 ug/L/day, respectively.

Excess nutrients from nonpoint sources are an ongoing problem that is expected to worsen as population and fertilizer usage rise. Conventional centralized treatment systems are not well suited to address nonpoint source pollution. More distributed best management practices (BMPs) like constructed wetlands are a promising alternative and have been widely implemented in the US since the 1970's. Constructed wetlands are multi-functional systems that can effectively store and transform harmful contaminants using primarily natural processes. However, the removal of pollutants like nitrogen by wetlands is highly variable, likely due to a combination of factors such as plant species-specific assimilation behavior, the effects of plant communities on microbial diversity and function, and variable nitrogen inputs. In this study, the effect of plant species richness (i.e., number of plant species in a system) and seasonal nutrient loading (i.e., nitrogen fertilization) on the microbial community responsible for regulating nitrogen turnover in wetland mesocosm soils was investigated. The chip-based QuantStudio 3D digital PCR (QS3D dPCR) system was used to quantify ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), comammox, anammox, and denitrifiers. Principal component analysis (PCA) was used to identify dominant patterns in the microbial community and nitrogen species. Resampling-based analysis of variance (ANOVA) was used to assess statistical significance of any observed differences caused by nitrogen fertilization or plant species richness. Results indicated that fertilization or season, which was convolved with fertilization, was the dominant factor influencing the microbial community in the study environment (27% variance explained), as indicated by the disparate clustering of fall (fertilized) and spring (unfertilized) samples about principal component 1 (fall: negative PC1, spring: positive PC1). Because unplanted unfertilized controls sampled in November clustered within the season in which they were collected rather than with other unfertilized samples collected in May, season may have influenced microbial community shifts more than fertilization for unplanted systems. This finding should be interpreted cautiously, however, given the small number of unplanted unfertilized controls (N = 2) and the absence of similar controls in the planted systems. The most abundant bacterial groups detected in May (November) were AOB, nirK, anammox, and Nitrospira spp. NOB (AOB, anammox, Nitrospira spp. NOB, and nosZ). The effects of plant species richness were more nuanced, with greater richness significantly impacting the abundance of only a subset of bacterial groups (i.e., the nitrifying bacteria AOB, Nitrospira spp. NOB, and comammox, but not the denitrifying bacteria). Different relationships between richness and microbial abundance were observed in different seasonal nutrient loadings (i.e., interaction effects between richness and fertilization were detected for some bacterial groups). / MS / As global population continues to rise, fertilizer application is becoming more commonplace in order to meet increasing agricultural demand. Fertilizers supply nutrients like nitrogen that, in excess, can negatively affect water quality. Since conventional treatment systems are largely impractical to control such diffuse, nonpoint sources of pollution, more distributed best management practices (BMPs) like constructed wetlands are a promising alternative. Several important nitrogen transformations occur within wetlands, of which soil microbial communities have a significant influence over. For instance, nitrifying bacteria can transform ammonia into nitrate and denitrifying bacteria can transform nitrate into atmospheric nitrogen. Constructed wetlands are designed to mimic these complex, dynamic processes, and can be manipulated for more effective nitrogen pollution control. However, the removal of pollutants like nitrogen by wetlands is highly variable, likely due to a combination of factors such as plant species-specific assimilation behavior, the effects of plant communities on microbial diversity and function, and variable nitrogen inputs. In this study, the effects of plant species richness (i.e., number of plant species in a system) and seasonal nutrient loading (i.e., nitrogen fertilization) on several types of nitrifying and denitrifying bacteria in wetland mesocosm soils were investigated. Digital polymerase chain reaction (dPCR) was used to quantify bacterial abundance. Principal component analysis (PCA) was used to identify dominant patterns within the data and resampling-based analysis of variance (ANOVA) was used to assess statistical significance of any observed differences caused by fertilization, season, and/or plant species richness. Results indicated that fertilization or season, which was convolved with fertilization, wasthe dominant factor influencing the microbial community in the study environment. The effects of plant species richness were more nuanced, with greater richness significantly impacting the abundance of only a subset of bacterial groups (i.e., the nitrifying bacteria AOB, Nitrospira spp. NOB, and comammox, but not the denitrifying bacteria).

Marine ecosystems are highly dynamic and contain a diverse faunal assemblage that are subject to various natural and anthropogenic variability. Globally, seagrass ecosystems are located adjacent to coastal areas that are heavily impacted by human development and urbanization potentially altering the community structure within these ecosystem. Complex food webs connect many components of these systems, often in unexpected ways, and are ultimately based on one of two pathways: benthic (i.e., seagrass, epiphytes, microalgae, detritus) and planktonic (i.e., phytoplankton). Understanding the pathway which the food web is based gives further insight regarding the biological balance of the ecosystem; thus it is important to expand beyond bounds of single-species approaches for research and management. Observing what a predator consumes and its preference for any particular prey can be informative in regards to how a predator interacts and utilizes an ecosystem. Predatory fish are exposed to a wide range of potential prey with varying levels of mobility in their natural environment and may employ a wide range of feeding tactics in order to capture prey. Overall, prey availability and abundances are influenced by many factors (e.g., seasons, tides, habitat loss and gain) and many piscivorous fishes will undergo ontogenetic shift in diet in order to optimize their energetic return limiting the interpretation of electivity studies. Although much information can be obtained through stomach analysis of fish, some prey are more rapidly digested due to the lack of hard, external structures and may be overlooked in the analysis. Furthermore, prey preference cannot be based solely on the observed abundance in the diet of a predator because it takes time for a predator to find, consume, and digest prey (i.e., "handling time"). Therefore, multiple approaches, both observational and experimental, are necessary to fully understand trophodynamics of fishes and their ecosystems. To better understand the trophodynamics of the Spotted Seatrout, Cynoscion nebulosus, my thesis incorporates both an observational and experimental study. My observational study compares three stomach analyses datasets to address changes in the diet composition over a thirty-two year timespan during which natural and anthropogenic changes potentially altered the community structure of Tampa Bay. This is paired with an experimental study to address differently handling times of two morphologically- and behaviorally- different prey that were observed in the diet of juvenile Spotted Seatrout.

Four-month mesocosm experiment from June 1st to September 25th, 2009, was conducted to determine the effect of water level dierence in combination with fish predation pressure, on submerged macrophyte development, in an eutrophic shallow lake. Effect of water level fluctuation was simulated by placing enclosures to the different water depths that included 0.8 m, 1.6 m and 2.3 m on Lake Eymir. These enclosures having a cylindirical shape and 1.2 m diameter, were open to sediment and atmosphere interaction. The highest water level mesocosms were cancelled after fifth sampling due to rapture in the bags, hence this thesis does not include the results of 2.3 m. At each depth, half of the enclosures were stocked with planktivo-omnivorous fish (Tinca tinca, Alburnus spp.) which are natural fauna of Lake Eymir. Before stocking of fish, ten shoots of Potamageton pectinatus were added to all of the enclosures in order to observe submerged macrophyte development. Sampling for physico-chemical parameters, zooplankton, chlorophyll a, PVI% and periphyton was conducted weekly for the first five weeks, last six samplings were done biweekly. Macrophyte harvesting for dry weight estimation was done at the end of the experiment. Throughout the experiment water level decreased 0.41 &plusmn / 0.06 m in each enclosures. Water level was so critical for macrophyte development that no significant macrophyte growth was observed in enclosures located at 1.6 m (HW). However, fish predation did not prevent the growth of macrophyte in enclosures located at 0.8 m (LW) but it was important in HW enclosure for affecting water clarity. Fish predation affected chlorophyll a, zooplankton and nutrient concentrations and the effect was mostly pronounced at LW enclosures. They had high chlorophyll a and nutrient concentrations but it did not repress macrophyte growth as in temperate lakes. Despite high water clarity in HW fishless enclosures, very low macrophyte biomass may be attributed to enhanced periphyton development. Zooplankton community shifted to small sized ones under fish predation while fishless enclosures had higher zooplankton /phytoplankton ratio for each depth. Hence, regarding these results it can be stated that decrease in water level can compensate the negative effects of fish predation on macrophyte growth in warm Mediterranean lakes.

A mesocosm experiment was conducted in Lake Eymir between June &ndash / September 2009 in order to elucidate the effects of water level changes and fish predation on periphyton growth and macroinvertebrates in semi-arid shallow lakes. Twenty four cylindrical enclosures, each with 1.2 m diameter, open to lake bottom and atmosphere, were placed at three different depths, i.e. 0.8 m (low water level, LW), 1.6 m (high water level, HW) and 2.3 m (however, data regarding the enclosures at 2.3 m were excluded in this study due to complications after fifth sampling) to simulate water level fluctuations. At each water level, four replicates were stocked with omnivorous&ndash / planktivorous fish (Tinca tinca and Alburnus escherichii) and the other four replicates were left fishless to observe the effect of fish predation. Ten shoots of submerged macrophytes (Potamogeton pectinatus) were planted and six polyethylene strips were hung in the water column in each enclosure to monitor macrophyte and periphyton growth. The mesocosms were sampled for physical, chemical and biological parameters weekly in the first month and fortnightly thereafter. Benthic macroinvertebrate samples were taken before the start, in the middle and at the end of the experiment with Kajak corer. Macrophytes were harvested after the last sampling for determination of dry weight, epiphyton, and the associated macroinvertebrates. All macroinvertebrate samples were sieved through 212 &mu / m mesh size before identification and counting. Over the course of the experiment, an average of 0.46 &plusmn / 0.03 m water level decrease in the mesocosms triggered submerged macrophyte growth in all LW enclosures, overriding the negative effects of fish predation. The results indicate that while fish predation pressure had negative influences on macroinvertebrate communities in terms of both abundance and richness, structural complexity created by dense vegetation in the LW mesocosms weakened the top-down effect of fish on macroinvertebrates by acting as a refuge in this semi-arid shallow lake.

Numerous causes have been implicated in contributing to amphibian population declines since the 1980's, with habitat modification, ultraviolet radiation (UV-B) and environmental contaminants (such as glyphosate-based herbicide) being among the most common. This study identifies the effects of a generic glyphosate-based herbicide (GLY- 4 Plus) on mortality, immune function, body condition, and morphological plasticity of larvae of the spotted salamander (Ambystoma maculatum) under conditions that reflect open and closed canopy light regimes. Larval salamander responses to glyphosate-based herbicide varied depending on UV-B conditions. In general, it appears that an open canopy (i.e. greater UV-B exposure) may confer fitness benefits. In the presence of herbicide, survival was higher in an open canopy UV-B regime and pooled open canopy survival was higher than that of closed canopy treatments. In the absence of herbicide, body condition and immune function were positively related with amount of UV-B. Finally, herbicide presence appeared to affect morphology under low UV-B conditions. UV-induced breakdown of surfactant or a complex interaction between temperature stratification and trophic relations is potentially responsible for the observed patterns in survival and body condition. However, the mechanistic underpinnings of improved immune function and morphological differences are less clear. As deforestation is likely to continue, amphibians may find themselves in ponds with increasingly open canopies. Combined with the knowledge that some amphibians can become locally adapted to UV exposure and develop pesticide tolerance, the probability of surviving exposure to this herbicide may be elevated in open canopy ponds. These results emphasize the complexity of natural systems and the importance of including multiple factors in experiments.

This MCs thesis is part of the Grant Agency of Czech Republic GACR P504/11/1151 The role of plants in bilance of greenhouse gases in Carex fens. The results of this project will serve as a basis for another analyses of greenhouse gases in natural wetland biotops. Growth and morphological changes of Carex acuta were monitored in mesocosm experiment durig the growing seson. Carex acuta has already been the subjekt of numerous studies. The aim of this study is to compare the growth rate of plants C. acuta grown under controlled conditions, simulating different regimes of flooding habitat. Number of shoots and maximum plant height were observed during the growing season. Destructive method has been used to fall aboveground and belowground biomass in autumn. Elevated water levels in the middle of the growing season caused a slowdown in growth in height and the creation of new shoots. Reduced water level of the mid-growing season seemed to faster growth of plants in height. Underground biomass significantly negatively affected by increased water level in the middle of the growing season.

This thesis is an extension of my Bc. thesis. Based upon previously conducted research in a wet grassland, Mokré Louky, near Třeboň, Czech Republic, a mesocosm experiment was designed for investigating the effect of two nutrient regimes (non fertilized / fertilized) and three flooding regimes (saturated / spring flood / flooded) on Carex acuta and Phalaris arundinacea plants to determine the effect of management type for restoring a more diverse wet meadows system.

Grunda, näringsrika sjöar förekommer i två stadier: antingen ett med grumligt vatten och hög biomassa av växtplankton, eller ett med klart vatten och riklig undervattensvegetation. Det klara stadiet är eftersträvansvärt eftersom det skapar goda förutsättningar för en rad ekosystemtjänster, inklusive vattenkvalitet, rekreation och förutsättningar för biodiversitet. Vid eutrofiering, d v s ökat tillskott av näringsämnen, kvarstår det klara stadiet tills näringsbelastningen blir så hög att sjön övergår till ett grumligt stadium. Sjön Tåkern, i Östergötland, har under de senaste åren blivit alltmer eutrofierad och kalkning av vattnet har diskuterats som en möjlig åtgärd för att bevara det klara stadiet som sjön numera befinner sig i. Undersökningar om kalkning av eutrofierade sjöar har gjorts tidigare, men det har varit få studier av effekterna av kalkning på undervattensvegetation, substratlevande djur och påväxtalger. För att undersöka detta, gjordes ett fält- och ett laboratorieexperiment inriktade på effekter på överlevnad av vattengråsugga, Asellus aquaticus, fysiologisk status (halten klorofyll a) i kransalgen Chara globularis och tillväxt av nyetablerade påväxtalger i sjön Tåkern. Resultaten visar att A. aquaticus hade lägre överlevnad efter kalkning till pH 10 i laboratorieexperiment, men i fältförsöket hade kalkning till detta pH inte någon inverkan. C. globularis hade en högre halt klorofyll a efter kalkning till pH 10 i laboratorieexperimentet, men i fältexperimentet hade kalkning ingen effekt på halten klorofyll a. Påväxtalger hade en lägre tillväxthastighet efter kalkning i fältexperimentet. Slutsatsen är att kalkning kan vara en metod för att motverka eutrofiering av sjön Tåkern. Några inkonsekvenser mellan mesokosm- och laborationsresultaten tyder på att fler studier behövs göras för att undersöka hur kalkning till pH 10 påverkar bentiska organismer. / Shallow, nutrient-rich lakes can occur in two alternative states: either a turbid water state with high biomass of phytoplankton, or a clear water state with abundant submerged vegetation. The clear water state is worth striving for because it creates good conditions for a range of ecosystem services and biodiversity. Lake Tåkern, Östergötland, has in recent years become increasingly eutrophic and liming has been discussed as a method to reduce the phosphorus level and maintain, or trigger a shift to, the clear water state. Investigations regarding the effects of liming of eutrophic lakes on phosphorus level and plankton have been performed in the past, but few studies have examined the effects on submerged vegetation, benthic invertebrates and periphyton. To study this, a field and a laboratory experiment were performed to examine how liming to pH 10 affects survival of the benthic invertebrate Asellus aquaticus, the chlorophyll a content in the stonewort Chara globularis and growth of periphyton. Survival of A. aquaticus was not affected in a field mesocosm experiment in Lake Tåkern, but decreased after liming in a laboratory experiment. The chlorophyll a content of C. globularis was not affected in the field experiment but higher than in the controls in the laboratory experiment. Growth of periphyton was reduced in the field experiment. Based on the field experiment, effects on A. aquaticus and C. globularis were negligible, but the laboratory experiment indicate that liming to pH 10 may under certain conditions affect benthic organisms. The conclusion is, however, that liming can be a suitable method to counteract the eutrophication of Lake Tåkern. Some inconsistencies between the field and laboratory results suggest that more studies needs to be made to see how liming to pH 10 affect benthic organisms.

Global records of temperature show a warming trend both in the atmosphere and in the oceans. Current climate change scenarios indicate that global temperature will continue to increase in the future. The effects will however be very different in different geographic regions. In northern Europe precipitation is projected to increase along with temperature. Increased precipitation will lead to higher river discharge to the Baltic Sea, which will be accompanied by higher inflow of allochthonous organic matter (ADOM) from the terrestrial system. Both changes in temperature and ADOM may affect community composition, altering the ratio between heterotrophic and autotrophic organisms. Climate changes may thus have severe and complex effects in the Baltic Sea, which has low species diversity and is highly vulnerable to environmental change. The aim of my thesis was to acquire a conceptual understanding of aquatic food web responses to increased temperature and inputs of ADOM. These factors were chosen to reflect plausible climate change scenarios. I performed microcosm and mesocosm experiments as well as a theoretical modeling study. My studies had a holistic approach as they covered entire food webs, from bacteria and phytoplankton to planktivorous fish. The results indicate a strong positive effect of increased temperature and ADOM input on the bacterial community and the microbial food web. However, at the prevailing naturally low nutrient concentrations in the Baltic Sea, the effect of increased temperature may be hampered by nutrient deficiency. In general my results show that inputs of ADOM will cause an increase of the bacterial production. This in turn can negatively affect the production at higher trophic levels, due to establishment of an intermediate trophic level, consisting of protozoa. However, the described effects can be counteracted by a number of factors, as for example the relatively high temperature optimum of fish, which will lead to a more efficient exploitation of the system. Furthermore, the length of the food web was observed to be a strong regulating factor for food web responses and ecosystem functioning. Hence, the effect of environmental changes may differ quite drastically depending on the number of trophic levels and community composition of the system. The results of my thesis are of importance as they predict possible ecological consequences of climate change, and as they also demonstrate that variables cannot be examined separately. / <p>This thesis was supported by grants from the Swedish Research Council FORMAS to AA and SL (217-2006-674), the Centre for Environmental Research in Umeå (CMF) to UB, AA and SL, and by the Swedish strategic research program ECOCHANGE to Umeå University.</p>