Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Gabriela Tkáčová Supervisor: RNDr. Jitka Vytlačilová, Ph.D Title of diploma thesis: The occurence of pharmaceuticals in the environment I. Drugs are biologically active substances used for treatment and prophylaxis of human and animal illnesses. Some drugs, however, remain biologically active even after they are excreted from the organism. By various means these substances can appear in the environment and can be detected in wastewater, surface water, seawater, groundwater, drinking water, sediments and soil. Chronic presence of low concentrations of drugs in the environment can negatively affect non-target organisms. Aim of this diploma thesis is to study presence of SSRI, statins and hypoglycemic drugs in the environment. Drugs from these farmacodynamic groups were detected in the environment in concentrations ranging from ng/l to μg/l. Selected drugs from SSRI group were fluoxetine, citalopram, fluvoxamine, sertraline, paroxetine and escitalopram. In water samples they were detected in concentration ranging from tens to hundreds ng/l, they were also detected in soils and sediments. Toxicity for water biota was also proven. Fluoxetine caused changes in secondary sexual characteristics of fish...

The main objective of this study was to assess the influence of sample storage on analysis, the seasonal changes in concentrations and removal efficiencies (REs) in a wastewater treatment plant (WWTP), and the photostability of pharmaceuticals under different sources of light were studied. Data obtained from stability studies showed that storage conditions had a significant impact on the stability of samples, and thus were very important for reliable determination of target compounds. The season can have an important influence on the variability of concentration and elimination of pharmaceuticals and personal care products (PPCPs) in WWTPs throughout the year. The target analytes were 10 antibiotics, 4 antidepressants, 3 psychotropics, 2 antihistamines, 2 lipid regulators, 4 UV filters, 3 analgesics/anti-inflammatories, and 9 anti-hypertensive/cardiovascular drugs. Wastewater samples (136 influents and 136 effluents) were collected from March 2011 to February 2012 and analyzed using two-dimensional liquid chromatography coupled with tandem mass spectrometry. The concentration of most pharmaceuticals was higher in winter. In most cases, elimination of PPCPs was insufficient, and removal rates varied strongly from 38% to 100%. There were significant seasonal trends in the observed REs, which decreased in winter. Photodegradation of 88 pharmaceuticals was investigated in buffered purified water. These experiments were performed both in sunlight and in the laboratory under artificial UV light. We found that photostability of PPCPs depends on the light sources, and that the rate of photolysis was slower under artificial UV light in most pharmaceuticals.

Main objective of the diploma thesis was to observe the concentrations of selected types of drugs in the environment around the area of the village Horní Beřkovice. This goal was achieved by monitoring of the drugs concentration in surface and ground waters at Horní Beřkovice site. Monitoring system allowed to detect the passage of pollution from the source of the contamination, through sewage treatment plant, the unsaturated zone and aquifer to a potential user of contaminated groundwater. The diploma thesis is based on the field data from TACR project. I have analyzed data from the six samplings at the area of the village Horní Beřkovice with regard to the observed drugs or their residues in surface and ground waters. I have compared the results with data from the sewage treatment plants in the river basin of Želivka. Keywords: ground water, surface water, sewage treatment plant, drugs, contamination

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany and Ecology Candidate: Tereza Olivová Supervisor: RNDr. Jitka Vytlačilová, Ph.D Title of diploma thesis: The occurrence of pharmaceuticals in the environment II. Pharmaceuticals are biologically active substances used for treatment or prevention of human and animal diseases. Some of these substances remain active even after being excreted from an organism and they can enter the environment. The residues of pharmaceuticals are present not only in rivers, but also in sea, groundwater and in soil, into which the contaminated water and manure are applied. The concentration of pharmaceuticals in water and in soil differ from place to place (it is higher e. g. in close proximity of sewage treatment plants) and from season to season (it depends on levels of precipitations and sunshine). Nowadays, thanks to sensitive analytical methods it is not a problem to detect these xenobiotics even in very small amounts, in which they usually occur in nature (usually in order of units or tens of ng/l or ng/g). However, even these low concentrations might have a negative impact on the life of non-target organisms (e. g. reproduction disorders) and therefore, the calculations, which measure the lever of risk of particular...

Present methods for diagnosis of neurodegenerative diseases are rather limited and in clinical practise are missing. This thesis is focused on utilization of the positron emission tomography using isotope 64 Cu(II). For the project, two contrast agents were proposed containing ligand for Cu (II) coordination and thioflavine T derivative as fluorescent dye, also as a targeting compound. Structure of the thioflavine T derivative was defined by X-ray structural analysis. The selected fluorescent dye has a high affinity for incipient amyloids and, when bound to their structure, has enhanced fluorescent properties. The proposed ligands are tetraazacyclic. The first one is a diamide, where the thioflavin T derivative is part of a macrocyclic ring. The second selected ligand is linked to the thioflavin T derivative via a linker. Key words: complexes; pharmaceuticals; transition metals

Pharmaceuticals are micropollutants, that enter the environment mainly through Wastewater Treatment Plants (WWTPs). In this work microbial community has been studied in sediments of a stream, which is located near a WWTP. This sediment is contaminated with pharmaceuticals. The subject of this thesis was to determine the presence of pharmaceuticals and microbial community in this study site and to point out possible relationships between these factors. Twelve pharmaceuticals were identified at concentrations reaching levels of ng/g. The concentrations of the compounds form a gradient that decreases with the distance from WWTP. Microbial biomass was estimated using the analysis of phospholipid fatty acids and microbial community was described using next-generation DNA sequencing. The analysis of phospholipid fatty acids pointed out, that with the increasing distance from WWTP the amount of microbial biomass decreases. DNA sequencing revealed large microbial diversity in the studied sediment. For evaluation of the relationship between the microbial community and pharmaceuticals in the stream sediment, Principal Component Analysis (PCA) was used. The result of PCA showed, that in the stream sediment (depth 10-30 cm), Betaproteobacteria negatively correlated with triclosan and Clostridia negatively...

Volume of consumed antibiotics in developed countries have increased eminently in past few decades. These substances are excreted from human body in various abundance and end up in the sewage. Sewage water flow to sewage treatment plants to be partly cleaned and finaly discharged to surface waters. Pharmaceutical residuals leaking to water environment of recipients affect local biota. Impact of pharmaceutical residuals present in biological pond Čežárka was assessed in this study. Čežárka biological pond (2,6 ha) is a recipient of discharged waters of sewage treatment plant in Vodnany, the town with 7 000 inhabitants. Biological ponds are good solution for final treatment of wastewater discharged from sewage treatment plant efluents. The nutrients from wastewater may enter the food chain of pond ecosystem. In total 62 pharmaceuticals were identified in water of sewage pond. Fish exposed in the pond for 30, 90, 180, 320 and 360 days were compared with fish of control group. Growth, condition of fish and biomarkers including level of vitellogenin in blood plasma, lipid peroxidation rate, antioxidative activity of catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase and superoxide dismutase were compared. The study proved the environmental impact of STP dischanrge containing pharmaceutical residuals in fish. Significant adaptation of fish to environment of biological pond was observed after 90 days of exposure.

The diploma thesis deals with the issue of health literacy of seniors in the field of pharmacology. The aim of the work was to find out, which skills and abilities seniors have, to understand, search and use information, services, and tools related to pharmacology. Thanks to these social and cognitive skills, seniors can have greater control over their health, they can make good decisions about their health condition, and they can maintain and support their health in general. The second aim of this thesis was to find out what problems the seniors encounter in the daily use of medicaments and what aspects make them the most difficult. With today's overusing drugs, seniors face more than one problem and it is affecting their quality of life and whole drug therapy. To achieve my goals, I have used quantitative research, using questionnaires. The examined sample was male and female from the age of 65 and the total number of respondents was 120. I set 2 goals with eight related hypotheses. Based on the gained results, out of the eight hypotheses, two of them were accepted and six of them were rejected. Based on the gained information from the research, in collaboration with graphic designer Dominika Pýchová, I created a stylized illustration (Annex 2), which could be introduced in the packaging to simplify pharmacy's using by seniors and simplify the information from information flyers. The reason, why seniors don't read the information flyers is the fact, that printed letters are too small and not readable. I was present while filling out the questionnaires and that is why I found out, that seniors would welcome better and uniform drug list which would make them easier to use. That's why I redrew the American version of the drug list, which is listed in (Annex 3). Overall research shows that half of the seniors are using more than 5 drugs, they have bigger problem of knowing the effects of drugs than knowing their names. Almost nobody knows the side effects of the drugs, as well as a clinical pharmacist who would facilitate pharmacotherapy in many cases. Practitioners do not check their patients' medication, and in many cases, when hospitalized, practitioners find out that the patient is taking unnecessary medication or even those that have not been prescribed for a long time. As long as the seniors do not improve their knowledge with help from practitioner and nurse, or by introducing of drug list's regular checks, seniors will be in alarming danger and their health literacy will not improve but it will be further reduced.

In this thesis occurrence of emerging pollutants in different compartments of aquatic environment was studied. Different environmental matrices were covered: wastewater, surface water and fish tissues with the focus on pharmaceuticals, illicit drugs and perfluorinated compounds. Although numerous pharmaceuticals have been already detected in aquatic environment all over the world, still there is need for new reliable methods for their detection and quantification. The output of the study is three multi-residue methods for the analysis of pharmaceuticals and illicit drugs in water and fish samples. Their main advantage is that wide range of compounds from different classes is covered, which enables obtaining maximum information with minimum analytical effort. All the methods were validated including such key parameters as limits of quantification, selectivity, recovery and repeatability, and showed excellent performance allowing determination of target compounds at trace levels. Consequently, the methods were applied for the monitoring of POCs in different environmental samples. Wide occurrence of pharmaceuticals in Swedish WWTP effluents was shown. Rather high concentrations for the representatives of anti-hypertension drugs, some antibiotics and anti-depressants were detected. This fact stresses the importance of WWTP´ effluent monitoring as it is the main source of PPCPs in the aquatic environment. Passive sampling approach was tested for the monitoring of POCs in wastewater. Both standard configurations of POCIS were calibrated under the field conditions for a broad range of contaminants. Sampling rate values were calculated for the case of wastewater effluent. This is a significant contribution to the potential widespread application of POCIS for environmental monitoring programs. Analysis of biota samples is an essential part in the assessment of pollutants? fate in aquatic environment. Therefore, the screening of antibiotics in fish available at the Czech market was carried out. Thirty-two antibiotics from different classes were analyzed. The fish and shrimp samples were bought from the three biggest supermarkets in České Budějovice, which belong to a network of shops covering the whole Czech Republic. In total 97 samples were obtained. Found concentrations did not exceed MRL values established in the European Union. No antibiotics were detected in the fish of Czech origin. Still, presence of antibiotic residues in edible fish tissues could be a matter of concern and the sign of improper use of antibiotics in aquaculture industry, what consequently can cause adverse effects for the environment and human health.

In this thesis, the impact of atenolol, verapamil, dexamethasone, clotrimazole, and PBSA on the physiological condition and health of rainbow trout (Oncorhynchus mykiss) were studied in vivo and in vitro. The sub-chronic test of atenolol, including at the environmentally relevant concentration, demonstrated that only the fish exposed to atenolol at a concentration of 1000 ?g?L-1 may be at a higher risk of oxidative stress. The second pharmaceutical compound studied was an L-type calcium channel blocker, verapamil. Despite the relatively high concentration of verapamil included in the experiments, the rates of seven hepatic CYP450-mediated reactions (EROD, MROD, PROD, BFCOD, COH, and PNPH) were unchanged in the exposed rainbow trout. This indicates that the activities of the selected CYP450 enzymes were not affected. Exposure to the highest concentration of PBSA (1000 ?g?L-1) led to significantly inhibited glutathione reductase activity. The activities of the EROD, MROD, and PROD enzymes gradually increased, beginning at the environmentally relevant concentrations. We suggest that long-term exposure to PBSA could cause oxidative stress in fish, as indicated by the decreased activity of GR. Moreover, PBSA could interact with intracellular receptors due to the elevated activities of CYP1A (EROD and MROD) and CYP2B (PROD). Rainbow trout showed multiple responses after exposure to clotrimazole. Changes in the haematological parameters in the exposed rainbow trout suggested that the immune system and haemoglobin synthesis were affected. The antioxidant defence enzymes that responded to the clotrimazole exposure influenced the oxidative stress status of the fish. Sub-chronic exposure to clotrimazole did not lead to oxidative stress in any of the fish tissues studied, as indicated by the unchanged TBARS levels. Hepatic energetic pathways were activated in the tested groups after 42 days of exposure to clotrimazole. High levels of glutathione S-transferase were seen in the liver and gill tissues, possibly associated with the detoxification of clotrimazole. The interaction of clotrimazole with intracellular receptors can be expected due to the changes in the catalytic activities of CYP1A1 and CYP3A. The BCF levels in the muscle tissue suggest a moderate bioaccumulation of CLO. The relatively long elimination period for CLO in the fish kidney (half-life = 29 days) may imply a greater potential for adverse effects in this organ. This finding is significant in view of the structural disturbances that appeared in the kidney and testis. The changes observed in the kidney were most prominent in the tubular epithelial cells. Changes in the gonads were only visible in the male specimens, and the pathological changes in the testis may indicate a disruption of steroidogenesis. The potential of clotrimazole and dexamethasone to inhibit CYP450 enzyme activity in rainbow trout hepatic microsomes was investigated in vitro. Clotrimazole non-competitively inhibited EROD activity in the hepatic microsomes, while BFCOD activity was competitively inhibited. PNPH activity was only slightly reduced in the presence of clotrimazole, indicating that it does not significantly interact with CYP2E1. The presence of dexamethasone in the incubations did not affect any of the CYP450 enzymes investigated. Everything considered, the results of the study confirmed that the presence of clotrimazole in the environment is of concern with respect to its impact on the health status of fish. In this thesis, the data of the actual effects of several PPCPs on fish were discussed. Based on the response of selected molecular endpoints, the cardiovascular and anti-inflammatory drugs studied do not present a significant risk for fish, while the antifungal drug and UV-screening agent that were tested may disrupt several physiological processes in fish.