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Active ingredients (AIs), including pesticides and pharmaceuticals, are designed to have a specific mode-of-action (MoA) in target organisms. Despite this, there is substantial evidence of their occurrence in marine environments having the potential to impact non-target species with effects both as a result of, and independent from, their MoA. Furthermore, due to a large proportion of AIs being readily ionisable, there is growing evidence that their uptake and effects are sensitive to environmental parameters such as pH, which can be driven by changes in pCO2, both in natural cycles and as a consequence of anthropogenic activities. In this thesis I investigate the impacts of 5 common active ingredient contaminants: the selective serotonin reuptake inhibitor fluoxetine; the non-steroidal anti-inflammatory drugs ibuprofen and diclofenac; and the neonicotinoid pesticides imidacloprid and thiamethoxam; on marine invertebrates and investigate aspects such as bioconcentration, effects, and the sensitivity of these organisms to these compounds under changing pCO2-driven pH conditions. Firstly, fluoxetine was shown to elicit a pharmacological MoA effect in the marine worm Hediste diversicolor through an increase in serotonin concentration of approximately 10 % following exposure to 10 μg L-1 fluoxetine. Fluoxetine was also shown to induce weight loss (up to 2 % at 500 μg L-1 after 72 hours); decrease feeding rates (up to 68 % at 500 μg L-1); and alter multiple metabolic biomarkers. The uptake and effects of fluoxetine here were also shown to be sensitive to feeding mode, with filter feeding worms experiencing up to 130 times greater body burdens of fluoxetine, and consequently greater effects, than deposit feeding worms - a trait-based sensitivity. In a second series of experiments, an increase in pCO2 (1000 and 3000 μatm vs a 400 μatm control), and consequent reduction in pH from approximately pH 8.1 to pH 7.75 and 7.4 respectively, was shown to alter uptake and depuration profiles and bioconcentration of all 5 active ingredients in the marine worm Hediste diversicolor. Notably, the bioconcentration factor for fluoxetine decreased from 331 at pH 8.1 to 107 at pH 7.4, whereas the reverse trend occurred for ibuprofen, which increased from 31 at pH 8.1 to 121 at pH 7.4. Seawater pH was also shown to alter the MoA effects of some compounds, with fluoxetine only impacting its MoA endpoint, serotonin, at pH 8.1 and thiamethoxam showing a twofold greater decrease in its MoA, acetylcholinesterase activity, at pH 7.4 when compared to pH 7.75 or pH 8.1. Finally, the neonicotinoid pesticides imidacloprid and thiamethoxam were shown to alter sperm swimming parameters, and consequently fertilisation success, in the sea urchin Paracentrotus lividus, decreasing by 15 % at 0.32 μg L-1 thiamethoxam and 28 % at 2.38 μg L-1 imidacloprid. This was further shown to be sensitive to changes in pCO2 for thiamethoxam, with a twofold greater reduction in curvilinear velocity of sperm; threefold greater reduction in motile sperm; and 64 % greater reduction in fertilisation success at pH 7.75 compared to pH 8.1. Together these findings outline the importance of assessing the risk of active ingredients to non-target marine invertebrates and demonstrate the need for consideration of both MoA effects and independent toxicity assays. In addition, the potential for pCO2 and pH to significantly alter the uptake and effects of active ingredients here indicate that their consideration is vital for effective assessment of current and potential future risks of active ingredients in aquatic environments.

This documentation of my MFA thesis presents my process and progress through an investigation of the relationships between humor and graphic design. Notations in the side column provide resource information and refer to appendices located in the second section of the document.

The environment is increasingly recognised as a key player in the emergence and mobilisation of antibiotic resistance, which negatively impacts human health, healthcare systems, and farming practices worldwide. Recent work has demonstrated concentrations of antibiotics in the natural environment may select for resistance in situ, but a scarcity of meaningful data has prevented rigorous environmental risk assessment of antibiotics. Without such data, mitigation strategies, such as improved antibiotic stewardship or environmental discharge limits, cannot be effectively designed or implemented. This thesis designed and developed two methods for determining effect concentrations of antibiotics in complex microbial communities, thereby generating a significant amount of data to address this knowledge gap. Minimal selective concentrations (MSCs) were determined in long term selection experiments for four classes of antibiotic at concentrations as low as 0.4 μg/L, which is below many measured environmental concentrations. Lowest observed effect concentrations were determined using a short term, growth based assay which were highly predictive of MSCs. A novel finding was significant selection for cefotaxime resistance occurred at a wide range of antibiotic concentrations, from 125 μg/L - 64 mg/L, which has important clinical implications. Determination of MSC in single species assays was also shown to be a poor predictor of MSC in a complex microbial community. Co-selection for antimicrobial resistance was demonstrated in selection experiments and through improved understanding of class 1 integron evolution, assessing selective effects on resistance gene acquisition using a novel PCR method and next-generation sequencing. In the final study, a novel resistance determinant (UDP-galactose 4-epimerase) conferring cross-resistance to biocides and antibiotics was discovered, providing a target for further study. These findings indicate selection and co-selection for antimicrobial resistance is likely to occur in the environment, and provides the means to rapidly generate further data to aid in the development of appropriate mitigation strategies.

Antibiotics are designed to inhibit the growth of or kill bacteria. Of the many classes of antibiotics that have been synthesized two particular classes of antibiotic have been frequently detected in the natural environment, namely the tetracyclines and sulfonamides. Following use, these compounds can enter the environment via the application of animal manures or domestic seawage sludge to land as a fertiliser or from wastewater treatment plant effluents. Once in the environment the compounds can persist and, depending on their properties, are distributed around the different environmental media (i.e. surface waters, groundwaters, soils and sediments). Several studies have suggested that tetracyclines and sulfonamides may have significant impacts on microbial function in the natural environment. However most of these studies have used existing standardised test procedures and/or unrealistic exposure conditions. The aim of the current study therefore was to develop a more environmentally realistic test system for assessing the effects of antibiotics on aquatic microbial communities and to apply this to assess the potential impacts of a sulphonamide and a tetracycline antibiotic on aquatic microbial communities. The developed system, which was an aquatic microcosm composed of fresh river water, return line treated sewage and OECD synthetic sewage, was initially used to assess the effects of 3, 5-dichlorophenol, an OECD reference toxicant. Effects of DCP were seen within the concentration range that is considered valid by other standard microbial toxicity tests, such as OECD method 209 (3.2-32 mg/L). Since the developed system was able to quantify effects on aquatic microbial communities, it can be seen as being more environmentally relevant than existing standard tests, which rely on testing single microbial species or sludge communities. The test system was then used to test the effects of chlortetracycline (CTC) and sulfamethoxazole (SMX) on multisubstrate utilization. Following addition of the study antibiotics, effects were observed for both antibiotics on total substrate utilization and on the utilization of specific, ecologically relevant end-points (such as nutrient cycling and polymer degradation) also. Effects were also observed on the structure of microbial communities and on the functional diversity of substrate utilization (especially in the case of SMX exposure) and these effects generally persisted for up to 3 d after addition of the study compounds at a lower concentration of 0.1 mg/L for both compounds. The results indicate that both antibiotics were inhibiting various aspects of substrate utilization, most of which were related to ecologically relevant processes that occur in the aquatic environment. The results also suggest that SMX and CTC exposure result in changes in the community structure of ecologically relevant groups of microbes (such as bacteria involved in the nitrogen and carbon cycle bacteria). Microbial communities that were exposed to CTC and SMX showed a varying degree of recovery, although more long term effects were seen in SMX exposures; functional effects were observed for the duration of the exposure. It was hypothesized that the observed recovery may have been caused by the selection of antibiotic resistance in the exposed communities. Culture based resistance studies showed that there was a significant temporal rise in CTC resistance at all CTC concentrations, but not SMX resistance, across the dose range. In addition, a greater number of distinct microbial morphotypes could be isolated from combined microcosms on day 7 compared with day 1. An increase in CTC and SMX resistance in control exposures was also observed. In addition, distinct resistant morphotypes developed in both dosed and undosed microcosms on day 7 of the study. Furthermore, the results of a series of different culture based experiments suggested that multidrug resistance was present in morphotypes that were isolated from dosed and control microcosms. These data therefore suggest that multidrug resistance was present at the start of the exposure and may also have been co-selected by exposure to CTC and SMX. In general, CTC and SMX show signs that they may pose a risk to the environment and possibly the wider health of humans and animals.