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Active pharmaceutical ingredients (APIs) are vital to human health and welfare, but following therapeutic use, they may pose a potential ecological risk if discharged into the environment. Today's conventional municipal wastewater treatment plants are not designed to remove APIs specifically, and various techniques, preferably cost-effective and environmentally friendly, are being developed and evaluated. Microalgae-based treatment of wastewater is a sustainable and low-cost approach to remove nutrients and emerging contaminants. In this study, a North Sweden high-rate algal pond (HRAP) using municipal untreated wastewater as medium, was investigated in terms of API distribution and fate. Three six-day batches were prepared during 18 days and a total of 36 APIs were quantified within the HRAP of which 14 were removed from the aqueous phase above 50% and seven removed above 90% of their initial concentrations. Twelve APIs of a hydrophobic nature were mostly associated with the algal biomass that was harvested at the end of each batch. HRAPs treatment successfully removed 69% of studied APIs (25 of 36 studied) in six day time. The distribution of various APIs between the aqueous phase and biomass suggested that several removal mechanisms may occur, such as hydrophobicity driven removal, passive biosorption and active bioaccumulation.

Several micropollutants show low removal efficiencies in conventional sewage treatment plants, and therefore enter the aquatic environment. To reduce the levels of micropollutants in sewage effluent, and thereby the effects on biota, a number of extra treatment steps are currently being evaluated. Two such techniques are ozonation and adsorption onto activated carbon. In this study, we investigated the efficiency of Sweden's first full-scale ozonation treatment plant at removing a number of antibiotics, antimycotics and biocides. The effect of adding granular activated carbon (GAC) on a pilot scale and pilot-scale ozonation were also evaluated. The conventional treatment (13,000 PE) with the add-on of full-scale ozonation (0.55 g O3/g Total organic carbon (TOC)) was able to remove most of the studied compounds (>90%), except for benzotriazoles and fluconazole (<50%). Adsorption on GAC on a pilot scale showed a higher removal efficiency than ozonation (>80% for all studied compounds). Three types of GAC were evaluated and shown to have different removal efficiencies. In particular, the GAC with the smallest particle sizes exhibited the highest removal efficiency. The results demonstrate that it is important to select an appropriate type of carbon to achieve the removal goal for specific target compounds.

This study investigated the abatement of a number of antimicrobials frequently detected in municipal wastewater by conventional ozonation and a recently developed ozone-based advanced oxidation process, the electro-peroxone (E-peroxone) process. A synthetic water and a real secondary wastewater effluent were spiked with fourteen antimicrobials, including antibiotics and biocides, and then treated by the two processes. The results show that most of the antibiotics investigated (e.g., ofloxacin, trimethoprim, norfloxacin, and ciprofloxacin) readily react with ozone (O₃) and could therefore be efficiently eliminated from the water matrices by direct O₃ oxidation during both processes. In contrast, most of the biocides tested in this study (e.g., clotrimazole, pentamidine, bixafen, propiconazole, and fluconazole) were only moderately reactive, or non-reactive, with O₃. Therefore, these biocides were removed at considerably lower rate than the antibiotics during the two ozone-based processes, with hydroxyl radical (OH) oxidation playing an important role in their abatement mechanisms. When compared with conventional ozonation, the E-peroxone process is defined by the in situ electrogeneration of hydrogen peroxide, which considerably enhances the transformation of O₃ to OH. As a result, the E-peroxone process significantly accelerated the abatement of biocides and required a considerably shorter treatment time to eliminate all of the tested compounds from the water matrices than conventional ozonation. In addition, the E-peroxone process enhanced the contributions of OH fractions to the abatement of moderately ozone reactive benzotriazoles. These results demonstrate that the E-peroxone process holds promise as an effective tertiary treatment option for enhancing the abatement of ozone-resistant antimicrobials in wastewater.

Aqueous core-shell particles based on polystyrene, poly(methyl methacrylate) or polycaprolactone have been formulated using a facile double emulsion-based solvent evaporation method. The size distribution is narrow, and the morphology control is remarkable given the simple characteristics of the encapsulation method. The inner droplets are stabilized by oil-soluble poly(ethylene oxide)-based block copolymers which are anchored in the polymeric shell by using hydrophobic blocks of the same type as that of the shell-forming polymer. This facilitates the efficient encapsulation of dyes and hydrophilic biocides.

The World Health Organization (WHO) has identified antibiotic resistance as a major threat to human health. The environment has been suggested to play an important role in the emergence of antibiotic resistant bacteria. The external environment can act as a source of resistance genes that could potentially be transferred into human pathogens. It is also an important route for the dissemination of antibiotic resistance genes and bacteria. Sewage treatment plants (STPs) are among the most important routes by which antibiotics and antibiotic resistance genes enter the environment. It has been suggested that STPs are hotspots for the development of antibiotic resistance because they contain relatively high concentrations of antibiotics as well as both human and environmental bacteria. Further complicating matters, there is evidence that other substances with antimicrobial properties, such as biocides and metals, can cause antibiotic resistance due to co- and cross-resistance.

This thesis contributes new knowledge on the concentrations, mass flows, and removal efficiencies of antimicrobials in STPs and their connections to the emergence of antibiotic resistance. Paper I presents data on the levels of 40 different antimicrobials in the incoming wastewater, treated effluent, and digested sludge of eleven different STPs. Although not previously detected in STPs, chlorhexidine is shown to be ubiquitous in such plants. In Paper II, mass flows and removal efficiencies are calculated for eleven antimicrobials over various treatment steps in three STPs, showing that polar antimicrobials were inefficiently removed from the wastewater. In Paper III, the minimum selective concentration (MSC) for the antibiotic tetracycline was determined in a complex bacterial aquatic biofilm using both phenotypic and genotypic endpoints. It was found that 10 µg/L selected for phenotypic resistance, and 1 µg/L selected for certain resistance genes. Paper VI used metagenomics to determine whether there is selection for antibiotic-resistant bacteria in STPs and whether the extent of this selection can be correlated to the concentrations of antimicrobial compounds. No clear evidence for selection was identified. Paper V evaluates advanced wastewater treatment techniques for removing antimicrobial compounds using ozonation and granular activated carbon (GAC). The identity of the GAC material was found to strongly affect removal efficiency, and GAC was more efficient than ozonation for most compounds at the tested concentrations.

Antimicrobial compounds, such as biocides and antibiotics, are widely used in society with significant quantities of these chemicals ending up in sewage treatment plants (STPs). In this study, mass flows and removal efficiency in different treatment steps at three Swedish STPs were evaluated for eleven different biocides and antibiotics. Mass flows were calculated at eight different locations (incoming wastewater, water after the first sedimentation step, treated effluent, primary sludge, surplus sludge, digested sludge, dewatered digested sludge and reject water). Samples were collected for a total of nine days over three weeks. The STPs were able to remove 53-> 99% of the antimicrobial compounds and 0-64% were biodegraded on average in the three STPs. Quaternary ammonium compounds were removed from the wastewater N99%, partly through biodegradation, but 38-96% remained in the digested sludge. Chlorhexidine was not biodegraded but was efficiently removed from the wastewater to the sludge. The biological treatment step was the most important step for the degradation of the studied compounds, but also removed several compounds through the surplus sludge. Compounds that were inefficiently removed included benzotriazoles, trimethoprim and fluconazole. The study provides mass flows and removal efficiencies for several compounds that have been seldom studied. 

Abstract There is concern that heavy metals and biocides contribute to the development of antibiotic resistance via co-selection. Most antifouling paints contain high amounts of such substances, which risks turning painted ship hulls into highly mobile refuges and breeding grounds for antibiotic-resistant bacteria. The objectives of this study were to start investigate if heavy-metal based antifouling paints can pose a risk for co-selection of antibiotic-resistant bacteria and, if so, identify the underlying genetic basis. Plastic panels with one side painted with copper and zinc-containing antifouling paint were submerged in a Swedish marina and biofilms from both sides of the panels were harvested after 2.5–4 weeks. DNA was isolated from the biofilms and subjected to metagenomic sequencing. Biofilm bacteria were cultured on marine agar supplemented with tetracycline, gentamicin, copper sulfate or zinc sulfate. Biofilm communities from painted surfaces displayed lower taxonomic diversity and enrichment of Gammaproteobacteria. Bacteria from these communities showed increased resistance to both heavy metals and tetracycline but not to gentamicin. Significantly higher abundance of metal and biocide resistance genes was observed, whereas mobile antibiotic resistance genes were not enriched in these communities. In contrast, we found an enrichment of chromosomal RND efflux system genes, including such with documented ability to confer decreased susceptibility to both antibiotics and biocides/heavy metals. This was paralleled by increased abundances of integron-associated integrase and ISCR transposase genes. The results show that the heavy metal-based antifouling paint exerts a strong selection pressure on marine bacterial communities and can co-select for certain antibiotic-resistant bacteria, likely by favoring species and strains carrying genes that provide cross-resistance. Although this does not indicate an immediate risk for promotion of mobile antibiotic resistance, the clear increase of genes involved in mobilizing DNA provides a foundation for increased opportunities for gene transfer in such communities, which might also involve yet unknown resistance mechanisms.

Incoming sewage water, treated effluent and digested sludge were collected from 11 Swedish sewage treatment plants (STPs) on 3 different days. Analytical protocols were established for a large number of compounds (47) with antimicrobial properties and the collected samples were then screened for the presence of these selected substances. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) were used to analyse the samples. Thirty organic compounds and 10 metals were detected above their respective detection limit. Quaternary ammonium compounds were the most abundant substances in the particulate phases with levels up to 370 μg/g and benzotriazoles were the most common in the aqueous phases with levels up to 24 μg/L. Several compounds with no, or very limited, previously reported data were detected in this study, including chlorhexidine, hexadecylpyridinium chloride and 10-benzalkonium chloride. Some of these were both frequently detected (>60% detection frequency) and found in high levels (up to 19 μg/g d.w. sludge). This study gives a comprehensive overview of the presence in Swedish STPs of a number of antimicrobial substances, providing crucial information in designing relevant studies on potential microbial co- and cross resistance development between antibiotics, biocides, and metals in the sewage system.

Sewage treatment plants (STPs) have repeatedly been suggested as “hotspots” for the emergence and dissemination of antibiotic-resistant bacteria. A critical question still unanswered is if selection pressures within STPs, caused by residual antibiotics or other co-selective agents, are sufficient to specifically promote resistance. To address this, we employed shotgun metagenomic sequencing of samples from different steps of the treatment process in three Swedish STPs. In parallel, concentrations of selected antibiotics, biocides and metals were analyzed. We found that concentrations of tetracycline and ciprofloxacin in the influent were above predicted concentrations for resistance selection, however, there was no consistent enrichment of resistance genes to any particular class of antibiotics in the STPs, neither for biocide and metal resistance genes. The most substantial change of the bacterial communities compared to human feces occurred already in the sewage pipes, manifested by a strong shift from obligate to facultative anaerobes. Through the treatment process, resistance genes against antibiotics, biocides and metals were not reduced to the same extent as fecal bacteria. The OXA-48 gene was consistently enriched in surplus and digested sludge. We find this worrying as OXA-48, still rare in Swedish clinical isolates, provides resistance to carbapenems, one of our most critically important classes of antibiotics. Taken together, metagenomics analyses did not provide clear support for specific antibiotic resistance selection. However, stronger selective forces affecting gross taxonomic composition, and with that resistance gene abundances, limit interpretability. Comprehensive analyses of resistant/non-resistant strains within relevant species are therefore warranted.

Selection pressure generated by antibiotics released into the environment could enrich for antibiotic resistance genes and antibiotic resistant bacteria, thereby increasing the risk for transmission to humans and animals. Tetracyclines comprise an antibiotic class of great importance to both human and animal health. Accordingly, residues of tetracycline are commonly detected in aquatic environments. To assess if tetracycline pollution in aquatic environments promotes development of resistance, we determined minimal selective concentrations (MSCs) in biofilms of complex aquatic bacterial communities using both phenotypic and genotypic assays. Tetracycline significantly increased the relative abundance of resistant bacteria at 10 μg/L, while specific tet genes (tetA and tetG) increased significantly at the lowest concentration tested (1 μg/L). Taxonomic composition of the biofilm communities was altered with increasing tetracycline concentrations. Metagenomic analysis revealed a concurrent increase of several tet genes and a range of other genes providing resistance to different classes of antibiotics (e.g. cmlA, floR, sul1, and mphA), indicating potential for co-selection. Consequently, MSCs for the tet genes of ≤ 1 μg/L suggests that current exposure levels in e.g. sewage treatment plants could be sufficient to promote resistance. The methodology used here to assess MSCs could be applied in risk assessment of other antibiotics as well.