Removal of selected sulfonamides and sulfonamide resistance genes from wastewater in full-scale constructed wetlands.

Sulfonamides are high-consumption antibiotics that reach the aquatic environment. The threat related to their presence in wastewater and the environment is not only associated with their antibacterial properties, but also with risk of the spread of drug resistance in bacteria. Therefore, the aim of this work was to evaluate the occurrence of eight commonly used sulfonamides, sulfonamide resistance genes (sul1-3) and integrase genes intI1-3 in five full-scale constructed wetlands (CWs) differing in design (including hybrid systems) and in the source of wastewater (agricultural drainage, domestic sewage/surface runoff, and animal runs runoff in a zoo). The CWs were located in low-urbanized areas in Poland and in Czechia. No sulfonamides were detected in the CW treating agricultural tile drainage water. In the other four systems, four sulfonamide compounds were detected. Sulfamethoxazole exhibited the highest concentration in those four CWs and its highest was 12,603.23 ± 1000.66 ng/L in a CW treating a mixture of domestic sewage and surface runoff. Despite the high removal efficiencies of sulfamethoxazole in the tested CWs (86 %-99 %), it was still detected in the treated wastewater. The sul1 genes occurred in all samples of raw and treated wastewater and their abundance did not change significantly after the treatment process and it was, predominantly, at the level 105 gene copies numbers/mL. Noteworthy, sul2 genes were only found in the influents, and sul3 were not detected. The sulfonamides can be removed in CWs, but their elimination is not complete. However, hybrid CWs treating sewage were superior in decreasing the relative abundance of genes and the concentration of SMX. CWs may play a role in the dissemination of sulfonamide resistance genes of the sul1 type and other determinants of drug resistance, such as the intI1 gene, in the environment, however, the magnitude of this phenomenon is a matter of further research.

Application of UHPLC-MS/MS method to monitor the occurrence of sulfonamides and their transformation products in soil in Silesia, Poland.

Sulfonamides circulating in the environment lead to disturbances in food chains and local ecosystems, but most importantly contribute to development of resistance genes, which generate problems with multidrug-resistant bacterial infections treatment. In urban areas, sources of sulfonamide distribution in soils have received comparatively less attention in contrast to rural regions, where animal-derived manure, used as a natural fertilizer, is considered the main source. The aim of this study was to determine eight sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole, sulfapyridine, sulfathiazole, and sulfisoxazole) in environmental soil samples collected from urbanized regions in Silesian Voivodeship with increased animal activity. These soils were grouped according to the organic carbon content. It was necessary to develop versatile and efficient extraction and determination method to analyze selected sulfonamides in various soil types. The developed LC-MS/MS method for sulfonamides analyzing was validated. The obtained recoveries exceeded 45% for soil with medium organic carbon content and 88% for sample with a very low organic carbon content (arenaceous quartz). The obtained results show the high impact of organic matter on analytes adsorption in soil, which influences recovery. All eight sulfa drugs were determined in environmental samples in the concentration range 1.5-10.5 ng g-1. The transformation products of the analytes were also identified, and 29 transformation products were detected in 24 out of 27 extracts from soil samples.

Study of the Phytoextraction and Phytodegradation of Sulfamethoxazole and Trimethoprim from Water by Limnobium laevigatum.

Phytoremediation is an environmentally friendly and economical method for removing organic contaminants from water. The purpose of the present study was to use Limnobium laevigatum for the phytoremediation of water from sulfamethoxazole (SMX) and trimethoprim (TRI) residues. The experiment was conducted for 14 days, in which the loss of the pharmaceuticals in water and their concentration in plant tissues was monitored. Determination of SMX and TRI was conducted using liquid chromatography coupled with tandem mass spectrometry. The results revealed that various factors affected the removal of the contaminants from water, and their bioaccumulation coefficients were obtained. Additionally, the transformation products of SMX and TRI were identified. The observed decrease in SMX and TRI content after 14 days was 96.0% and 75.4% in water, respectively. SMX removal mainly involved photolysis and hydrolysis processes, whereas TRI was mostly absorbed by the plant. Bioaccumulation coefficients of the freeze-dried plant were in the range of 0.043-0.147 for SMX and 2.369-2.588 for TRI. Nine and six transformation products related to SMX and TRI, respectively, were identified in water and plant tissues. The detected transformation products stemmed from metabolic transformations and photolysis of the parent compounds.

Towards Sustainable Wastewater Treatment: Bioindication as a Technique for Supporting Treatment Efficiency Assessment.

Constructed wetlands (CWs) are a promising alternative for conventional methods of wastewater treatment. However, the biggest challenge in wastewater treatment is the improvement of the technology used so that it is possible to remove micropollutants without additional costs. The impact of wastewater treatment in CWs on toxicity towards Aliivibrio fischeri, Daphnia magna and Lemna minor was investigated. The effects of feeding regime (wastewater fed in five batches per week at a batch volume of 1 L, or twice per week at a batch volume of 2.5 L) and the presence of pharmaceuticals (diclofenac and sulfamethoxazole), as well as the presence of Miscantus giganteus plants in CW columns (twelve of the 24 columns that were planted) were analyzed. A reduction in toxicity was observed in all experimental setups. The effluents from constructed wetlands were classified as moderately toxic (average TU for A. fischeri, D. magna and L. minor was 0.9, 2.5 and 5.5, respectively). The feeding regime of 5 days of feeding/2 days of resting resulted in a positive impact on the ecotoxicological and chemical parameters of wastewater (removal of TOC, N-NH4 and pharmaceuticals). Extended exposure of Miscantus giganteus to the wastewater containing pharmaceuticals resulted in elevated activity of antioxidant enzymes (catalase and superoxide dismutase) in leaf material.

Determination of antimicrobial agents and their transformation products in an agricultural water-soil system modified with manure.

Manure fertilization is the primary source of veterinary antimicrobials in the water-soil system. The research gap is the fate of antimicrobials after their release into the environment. This study aimed to provide a detailed and multi-faceted examination of fertilized cultivated fields using two types of manure (poultry and bovine) enriched with selected antimicrobials. The research focused on assessing the mobility and stability of antimicrobials in the water-soil system. Additionally, transformation products of antimicrobials in the environment were identified. The extraction (solid-phase extraction and/or solid-liquid extraction) and LC-MS/MS analysis procedures were developed to determine 14 antimicrobials in the soil and pore water samples. Ten out of fourteen antimicrobials were detected in manure-amended soil and pore water samples. The highest concentration in the soil was 109.1 ng g-1 (doxycycline), while in pore water, it was 186.6 ng L-1 (ciprofloxacin). Sixteen transformation products of antimicrobials were identified in the soil and soil-related pore water. The same transformation products were detected in both soil and soil pore water extracts, with significantly higher signal intensities observed in soil extracts than in water. Transformation products were formed in oxidation, carbonylation, and ring-opening reactions.

Uptake of Pharmaceutical Pollutants and Their Metabolites from Soil Fertilized with Manure to Parsley Tissues.

Manure is a major source of soil and plant contamination with veterinary drugs residues. The aim of this study was to evaluate the uptake of 14 veterinary pharmaceuticals by parsley from soil fertilized with manure. Pharmaceutical content was determined in roots and leaves. Liquid chromatography coupled with tandem mass spectrometry was used for targeted analysis. Screening analysis was performed to identify transformation products in the parsley tissues. A solid-liquid extraction procedure was developed combined with solid-phase extraction, providing recoveries of 61.9-97.1% for leaves and 51.7-95.6% for roots. Four analytes were detected in parsley: enrofloxacin, tylosin, sulfamethoxazole, and doxycycline. Enrofloxacin was detected at the highest concentrations (13.4-26.3 ng g-1). Doxycycline accumulated mainly in the roots, tylosin in the leaves, and sulfamethoxazole was found in both tissues. 14 transformation products were identified and their distribution were determined. This study provides important data on the uptake and transformation of pharmaceuticals in plant tissues.

Solar-light driven photodegradation of antimicrobials, their transformation by-products and antibiotic resistance determinants in treated wastewater.

This study aimed to assess the possibility of using solar light-driven photolysis and TiO2-based photocatalysis to remove (1) antibiotic residues, (2) their transformation products (TPs), (3) antibiotic resistance determinants, and (4) genes identifying the indicator bacteria in a treated wastewater (secondary effluent). 16 antimicrobials belonging to the different classes and 45 their transformation by-products were selected for the study. The most susceptible to photochemical decomposition was tetracycline, which was completely removed in the photocatalysis process and in more than 80% in the solar light-driven photolysis. 83.8% removal (on average) was observed using photolysis and 89.9% using photocatalysis in the case of the tested genes, among which the genes sul1, uidA, and intI1 showed the highest degree of removal by both methods. The study revealed that applied methods promisingly remove the tested antibiotics, their TPs and genes even in such a complex matrix including treated wastewater and photocatalysis process had a higher removal efficiency of antibiotics, TPs and genes tested. Moreover, the high percentage removal of the intI1 gene (>93%) indicates the possibilities of use of the solar light-driven photolysis and TiO2-based photocatalysis in minimizing the antibiotic resistance genes transfer by mobile genetic elements.

Suspect screening of antimicrobial agents transformation products in environmental samples development of LC-QTrap method running in pseudo MRM transitions.

The aim of the work was to develop a new HPLC-MS/MS method that allows for the simultaneous detection of antimicrobials agents (targeted analysis) and their transformation products (non-targeted analysis), which enabled the elucidation of their transformation pathways in the environment. Targeted analysis was performed for 16 selected antimicrobials agents (AMs) in wastewater collected at different stages of the treatment process and river water from sections before and after wastewater discharge. The samples were collected in the Lyna sewage treatment plant (Olsztyn, Poland) in three measuring periods at different seasons. Analytes were selected from tetracyclines, fluoroquinolones, ß-lactams, macrolides, glycopeptides, lincosamides and synthetic antibiotics. As a part of the targeted analysis, 13 AMs were detected in wastewater samples, and 7 of them in river water samples. However, their presence and concentrations were closely related to the type of the sample and the season in which the sample was taken. The highest concentrations of AMs were detected in samples collected in September (max. 1643.7 ng L-1 TRI), while the lowest AMs concentrations were found in samples collected in June (max. 136.1 ng L-1 CLR). The total content of AMs in untreated wastewater was in the range of 1.42-1644 ng L-1, while in the river water was for upstream 1.22-48.73 ng L-1 and for downstream 2.24-149 ng L-1. In the non-target analysis, 33 degradation products of the selected AMs were identified, and the transformation pathways of their degradation were speculated. In the course of the research, it was found that as a result of the processes taking place in wastewater treatment plant, the parent substances are transformed into a number of stable transformation products. Transformation products resulted from hydroxylation, ring opening, oxidation, methylation or demethylation, carboxylation, or cleavage of the CN bond of the parent AMs.

Heterogeneous Photocatalysis of Metronidazole in Aquatic Samples.

Metronidazole (MET) is a commonly detected contaminant in the environment. The compound is classified as poorly biodegradable and highly soluble in water. Heterogeneous photocatalysis is the most promoted water purification method due to the possibility of using sunlight and small amounts of a catalyst needed for the process. The aim of this study was to select conditions for photocatalytic removal of metronidazole from aquatic samples. The effect of catalyst type, mass, and irradiance intensity on the efficiency of metronidazole removal was determined. For this purpose, TiO2, ZnO, ZrO2, WO3, PbS, and their mixtures in a mass ratio of 1:1 were used. In this study, the transformation products formed were identified, and the mineralization degree of compound was determined. The efficiency of metronidazole removal depending on the type of catalyst was in the range of 50-95%. The highest MET conversion (95%) combined with a high degree of mineralization (70.3%) was obtained by using a mixture of 12.5 g TiO2-P25 + PbS (1:1; v/v) and running the process for 60 min at an irradiance of 1000 W m-2. Four MET degradation products were identified by untargeted analysis, formed by the rearrangement of the metronidazole and the C-C bond breaking.

Application of UHPLC-MS/MS method to study occurrence and fate of sulfonamide antibiotics and their transformation products in surface water in highly urbanized areas.

Sulfonamide antibiotics (SAs) are used on a large scale in human and veterinary medicine. The main goal of this study was to develop a method for the detection of selected SAs (sulfamethoxazole, sulfadiazine, sulfamethazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethiazole, and sulfisoxazole) in aqueous samples (targeted analysis), and then conduct a non-targeted analysis to determine the transformation products to elucidate their degradation pathways. These analyses were performed using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. The procedure was used to detect selected antibiotics in water samples collected throughout a highly urbanized area. Among the studied compounds, sulfamethoxazole (max. 78.88 ng L-1) and sulfapyridine (max. 38.88 ng L -1) were the most common pollutants identified in surface waters. Trace amounts of sulfadiazine (below LOQ = 0.40 ng L-1) were also detected. Next, the samples were screened to detect the transformation products. Several sulfadiazine and sulfamethoxazole transformation products were detected and confirmed in the environmental samples.

Removal and transformation of sulfamethoxazole in acclimated biofilters with various operation modes - Implications for full-scale application.

The knowledge gaps regarding the degradation of sulfamethoxazole (SMX) in biofilters include the effect of aeration, constant feeding with readily biodegradable organic carbon and the presence of reactive media such as manganese oxides (MnOx). Thus, the goal of this study was to assess the removal of SMX in lab-scale biofilters with various operation variables: aeration, presence of MnOx as an amendment of filtering medium and the presence of readily biodegradable organic carbon (acetate). The sand used in the experiment as a filtering medium was previously exposed to the presence of SMX and acetate, which provided acclimation of the biomass. The removal of SMX was complete (>99%) with the exception of the unaerated columns fed with the influent containing acetate, due to apparent slower rate of SMX degradation. The obtained results suggest that bacteria were able to degrade SMX as a primary substrate and the degradation of this compound was subsequent to the depletion of acetate. The LC-MS/MS analysis of the effluents indicated several biotransformation reactions for SMX: (di)hydroxylation, acetylation, nitrosation, deamonification, S-N bond cleavage and isoxazole-ring cleavage. The relative abundance of transformation products was decreased in the presence of MnOx or acetate. Based on the Microtox assay, only the effluents from the unaerated columns filled with MnOx were classified as non-toxic. The results offer important implications for the design of biofilters for the elimination of SMX, namely that biofilters offer the greatest performance when fed with secondary wastewater and operated as non-aerated systems with a filtering medium containing MnOx.

Microbial and chemical pollutants on the manure-crops pathway in the perspective of "One Health" holistic approach.

This study determined the impact of poultry and bovine manure fertilization on the content of antibiotics, heavy metals (HMs), and the quantitative and qualitative composition of integrase and antibiotic resistance genes in soil, groundwater, and crops cultivated on manure-amended plots. Antibiotic concentration levels were analyzed using the HPLC-MS/MS, heavy metal concentration level were measured by HGAAS and ICP-OES, while the integrase genes and ARGs were quantified using Real-Time PCR (qPCR) method. Manure, soil, and crops samples contained the highest concentration of Zn (104-105 ng gdm-1) and Cu (103-105 ng gdm-1) of all HMs tested. Manure-supplemented soil was characterised by a high concentration of doxycycline and enrofloxacin. A high abundance of integrase genes was noted in samples of manure (109-1010 copies gdm-1) and soil (107-108 copies gdm-1). Among all the analyzed genes, sul1, sul2, blaTEM, and integrase genes were the most common. Results of the study demonstrate the selective character of ARGs transfer from poultry and bovine manure to plants. The only gene to occur in all studied environmental compartments was sul1 (from 102 - groundwater to 1011 - poultry manure). It was also found that animal manure may cause an increase in the HMs concentration in soil and their accumulation in crops, which may influence the health of humans and animals consuming crops grown on manure-amended soil. The high abundance of integrase genes and ARGs and their reciprocal correlations with HMs pose a serious risk of the rapid spread of antibiotic resistance in the environment. Moreover, unusual dependencies between integrase genes and selected ARGs indicate the possibility of changes in the mobility nature of genetic elements.

Wastewater treatment plants as a reservoir of integrase and antibiotic resistance genes - An epidemiological threat to workers and environment.

Conventional mechanical and biological wastewater treatment is unable to completely eliminate all pollutants, which can therefore enter surface water bodies together with treated wastewater. In addition, bioaerosols produced during wastewater treatment can pose a threat to the health of the wastewater treatment plant staff. In order to control the impact of a wastewater treatment plant (WWTP) on the surrounding environment, including its employees, samples of wastewater and water from a river which received treated wastewater were analysed in terms of their content of antibiotics and heavy metals, levels of selected physiochemical parameters, concentrations of antibiotic-resistance genes (ARGs) and genes of integrases. Furthermore, a quantitative analysis of ARGs in the metagenomic DNA from nasal and throat swabs collected from the WWPT employees was made. Both untreated and treated wastewater samples were dominated by genes of resistance to sulphonamides (sul1, sul2), MLS group of drugs (ermF, ermB) and beta-lactams (blaOXA). A significant increase in the quantities of ARGs and concentrations of antibiotics was observed in the river following the discharge of treated wastewater in comparison to their amounts in the river water upstream from the point of discharge. Moreover, a higher concentration of ARGs was detected in the DNA from swabs obtained from the wastewater treatment plant employees than from ones collected from the control group. Many statistically significant (p < 0.05) correlations between the concentration of the gene of resistance to heavy metals cnrA versus ARGs, and between the ARGs content and the concentrations of heavy metals in both wastewater and river water samples were observed. The study has demonstrated that the mechanical and biological methods of wastewater treatment are not efficient and may affect the transmission of hazardous pollutants to the aquatic environment and to the atmospheric air. It has been shown that an activated sludge bioreactor can be a potential source of the presence of multi-drug resistant microorganisms in the air, which is a health risk to persons working in WWTPs. It has also been found that an environment polluted with heavy metals is where co-selection of antibiotic resistance may occur, in the development of which integrase genes play an essential role.

The impact of antimicrobials on the efficiency of methane fermentation of sewage sludge, changes in microbial biodiversity and the spread of antibiotic resistance.

The study was designed to simultaneously evaluate the influence of high doses (512-1024 µg/g) the most commonly prescribed antimicrobials on the efficiency of anaerobic digestion of sewage sludge, qualitative and quantitative changes in microbial consortia responsible for the fermentation process, the presence of methanogenic microorganisms, and the fate of antibiotic resistance genes (ARGs). The efficiency of antibiotic degradation during anaerobic treatment was also determined. Metronidazole, amoxicillin and ciprofloxacin exerted the greatest effect on methane fermentation by decreasing its efficiency. Metronidazole, amoxicillin, cefuroxime and sulfamethoxazole were degraded in 100%, whereas ciprofloxacin and nalidixic acid were least susceptible to degradation. The most extensive changes in the structure of digestate microbiota were observed in sewage sludge exposed to metronidazole, where a decrease in the percentage of bacteria of the phylum Bacteroidetes led to an increase in the proportions of bacteria of the phyla Firmicutes and Proteobacteria. The results of the analysis examining changes in the concentration of the functional methanogen gene (mcrA) did not reflect the actual efficiency of methane fermentation. In sewage sludge exposed to antimicrobials, a significant increase was noted in the concentrations of ß-lactam, tetracycline and fluoroquinolone ARGs and integrase genes, but selective pressure was not specific to the corresponding ARGs.

Supramolecular Solvent-Based Microextraction of Selected Anticonvulsant and Nonsteroidal Anti-Inflammatory Drugs from Sediment Samples.

The increase in the production and consumption of pharmaceuticals increases their presence in the global environment, which may result in direct threats to living organisms. For this reason, there is a need for new methods to analyze drugs in environmental samples. Here, a new procedure for separating and determining selected drugs (diclofenac, ibuprofen, and carbamazepine) from bottom sediment and water samples was developed. Drugs were determined by ultra-high performance liquid chromatography coupled with an ultraviolet detector (UHPLC-UV). In this work, a universal and single-step sample treatment, based on supramolecular solvents (SUPRAS), was proposed to isolate selected anticonvulsants and nonsteroidal anti-inflammatory drugs (NSAIDs) from sediment samples. The following parameters were experimentally selected: composition of the supramolecular solvent (composition THF:H2O (v/v), amount of decanoic acid), volume of extractant, sample mass, extraction time, centrifugation time, and centrifugation speed. Finally, the developed procedure was validated. A Speedisk procedure was also developed to extract selected drugs from water samples. The recovery of analytes using the SUPRAS procedure was in the range of 88.8-115%, while the recoveries of the Speedisk solid-phase extraction procedure ranged from 81.0-106%. The effectiveness of the sorption of the tested drugs by sediment was also examined.

Is Biochar from the Torrefaction of Sewage Sludge Hazardous Waste?

Improved technologies are needed for sustainable management of sewage sludge (SS). The torrefaction (also known as biomass "roasting") is considered a pretreatment of SS before use in agriculture. However, it is not known whether the torrefaction has the potential to decrease heavy metals' (HMs) leachability and the SS toxicity. Thus, the aim of the study was to evaluate the influences of the SS torrefaction parameters (temperature and process time) on HM contents in biochar, HM leachability, and biochar toxicity, and compare them with raw SS. The experiments were designed in 18 combinations (six temperatures, 200, 220, 240, 260, 280, and 300 °C; and three process times-20, 40, 60 min). Standard tests were used to determine HMs content, leachability, and toxicity. Results indicated that the torrefaction did not increase (p < 0.05) the HM content in comparison to the raw SS. The leachability of Zn, Ni, Cu, Cr, and Mn from SS biochars was similar to raw SS. However, the degree of leachability varied significantly (p < 0.05) from as low as 0.1% for Cu to high as 16.7% for Cd. The leachability of Cd (<16.7%) and Pb (<11.9%) from biochars was higher than from raw SS (<6.1% and <2.4%, respectively). The leachability of Cd from SS biochar, in five torrefaction combinations, was higher than the threshold value for hazardous waste. It is recommended that site-specific decisions are made for torrefaction of SS with respect to its HM content, as the resulting biochar could be considered as hazardous waste, depending on the feedstock. Moreover, the biochar produced under the whole range of temperatures during 20 min retention time significantly (p < 0.05) increased the Daphnia magna Straus mobility inhibition by up to 100% in comparison to the biochar obtained during 40 and 60 min torrefaction. Taking into account the increased leachability of specific HMs and D. magna Straus mobility inhibition, biochar should be considered a potentially hazardous material. Future research should focus on biochar dosage as a fertilizer in relation to its toxicity. Additional research is warranted to focus on the optimization of SS torrefaction process parameters affecting the toxicity.

Development of a new SLE-SPE-HPLC-MS/MS method for the determination of selected antibiotics and their transformation products in anthropogenically altered solid environmental matrices.

The presence of antibiotic residues, their bioactive metabolites and other transformation products in the environment may adversely affect the organisms that live in the environment and may also contribute to increasing the antibiotic resistance of bacteria. It is particularly difficult to determine the types of contaminants in solid samples, in particular, those that are anthropogenically changed, e.g., as a result of controlled biochemical processes. Therefore, the aim of this research was to develop of a new method for the determination of twelve antibiotics belonging to different groups, such as penicillins, sulfonamides, tetracyclines, quinolones, imidazoles and cefalosporins, in digested manure and activated sludge samples, which were used as examples of anthropogenically altered environmental solid samples. The analyses were performed using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The solid-liquid extraction (SLE) method to isolate analytes from digested manure and activated sludge was developed and optimized, the same as clean-up procedure followed by solid phase extraction (SPE). The recovery ranged from 45 to 85%. Finally, the validated method was applied to the determination of the selected antibiotics in manure and activated sludge samples after an anaerobic digestion process.. An additional aim of the study was to verify whether the developed method allows simultaneous detection of transformation products of the studied antibiotics in solid samples. The study showed that by optimizing the analysis conditions, it is possible to simultaneously determine the selected antibiotics and their transformation products (including their epimeric forms), which can significantly improve the ability to control the efficiency of the biological processes used in this area. In practice, this means that the developed methodology may be particularly useful in the context of research and other works related to the anaerobic digestion of activated sludge, manure or other solid substrates of environmental origin.

Small-scale wastewater treatment plants as a source of the dissemination of antibiotic resistance genes in the aquatic environment.

Wastewater treatment plants (WWTPs) are significant source of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can spread further in the environment by reaching rivers together with effluents discharged from WWTPs. In this study untreated and treated wastewater (UWW, TWW), upstream and downstream river water (URW, DRW) were collected from 4 WWTPs, in the winter and autumn seasons. The occurrence of ARB resistant to beta-lactams and tetracyclines as well as the presence of antibiotics from these classes were analysed in water and wastewater samples. Additionally, the amounts of 12 ARGs, 2 genes of mobile genetic elements (MGEs), gene uidA identifying E. coli and 16S rRNA were also determined. Resistance to beta-lactams prevailed among ARB in water and wastewater samples (constituting 82-88% of total counts of bacteria). The dominant genes in water and wastewater samples were blaTEM, tetA, sul1. The gene blaOXA demonstrated high variability of its concentration in samples collected in both seasons. Despite the high per cent reduction of ARB and ARGs concentration observed during the wastewater treatment processes, their large quantities are still transmitted into the environment. The research focuses on WWTPs' role in the dissemination of ARGs and MGEs in the aquatic environment.

Evidence of mutations conferring resistance to clarithromycin in wastewater and activated sludge.

The occurrence of clarithromycin in wastewater samples and of the activated sludge bacteria possibly resistant to this pharmaceutical was the object of the study. Samples of wastewater or activated sludge were taken from a municipal wastewater treatment plant in summer and winter and characterised regarding their clarithromycin concentrations and the presence of nucleic acid fragments (Cla-sequences) known to be responsible for clarithromycin resistance in Helicobacter pylori. The concentrations of clarithromycin in raw wastewater were about 1086-2271 ng/L. Around 50-60% less of the pharmaceutical was found in treated wastewater. The concentrations were much higher in winter samples, as compared to summer samples. The clarithromycin resistance markers in H. pylori were detected by fluorescence in situ hybridisation in activated sludge bacterial cells. Cla-sequences were found in all the detected Proteobacteria, independently of the sampling season. Among nitrifying or phosphate or glycogen accumulating bacteria only Nitrosomonas spp. revealed presence of the clarithromycin sequences.

Antimicrobial pharmaceuticals in the aquatic environment - occurrence and environmental implications.

The environmental occurrence of antimicrobial pharmaceuticals and antibiotic resistant bacteria and antibiotic resistant genes has become a global phenomenon and a multifaceted threat. Integrated actions of many parties are needed to prevent further aggravation of the problem. Well-directed actions require clear understanding of the problem, which can be ensured by frequent revaluation of the existing knowledge and disseminating it among relevant audiences. The goal of this review paper is to discuss the occurrence and abundance of antimicrobial pharmaceuticals in the aquatic environment in context of adverse effects caused directly by these substances and the threat associated with the antibiotics resistance phenomenon. Several classes of antimicrobial pharmaceuticals (aminoglycosides, ß-lactams, glycopeptides, macrolides, fluoroquinolones, sulfonamides and trimethoprim, tetracyclines) have been selected to illustrate their sources, environmental abundance, degradation routes (transformation products) and environmental implications including their ecotoxic effect and the spread of antibiotic resistance within the compartments of the aquatic environment and wastewater treatment plants. Wastewater treatment plants are indeed the main source responsible for the prevalence of these factors in the aquatic environment, since predominantly the plants have not been designed to retain antimicrobial pharmaceuticals. In order to limit the prevalence of these impurities into the environment, better source control is recommended as well as the establishment of stricter environmental quality standards. Counteracting all the above-mentioned threats requires to undertake integrated activities based on cooperation of professionals and scientists from various fields of science or industry, such as environmental sciences, medicine, veterinary, pharmacology, chemical engineering and others.