The Effect of Disinfection on the Behaviour of Two Reserve Antibiotics in Hospital Wastewater

The Effect of Disinfection on the Behaviour of Two Reserve Antibiotics in Hospital Wastewater E. Felis*, S. Bajkacz**, J. Wilk**, P. Sowik*, M. Harnisz*** and E. Korzeniewska*** * Department of Environmental Biotechnology, Silesian University of Technology, Akademicka 2 Str., 44-100, Gliwice, Poland (E-mail: [email protected]; pauli[email protected]) ** Department of Inorganic Chemistry, Analytical Chemistry, and Electrochemistry, Silesian University of Technology, Krzywoustego 6 Str., 44-100, Gliwice, Poland (E-mail: sylwia.baj[email protected]; joanna.w[email protected]l) *** Department of Engineering of Water Protection and Environmental Microbiology, University of Warmia and Mazury in Olsztyn, Prawocheńskiego 1 Str., 10-720, Olsztyn, Poland (E-mail: [email protected]; [email protected]) Abstract The study shows the effect of the disinfection process of real hospital wastewater on the behaviour of two reserve drugs, i.e. Linezolid and Aztreonam, and other physicochemical parameters of these wastewater, such as dissolved organic carbon, dissolved total nitrogen, etc. The studies were conducted in hospital wastewater, in which full-scale in-situ disinfection process was carried out, before it was introduced into the municipal sewage system. The samples were taken in winter and summer period. The results showed that the maximum concentration of Linezolid in hospital wastewater was equal to 2535.93 ± 95.44 ng/L and it was observed in the summer period. Each time the chlorine disinfection process caused the concentration of this drug in filtered wastewater after the process to be higher than before the disinfection process. This phenomenon requires further research, but a hypothesis was put forward that Linezolid in hospital wastewater occurs in a dissolved form and in conjunction with the wastewater suspension solids, and the disinfection process causes the disintegration of the suspension, which contributes to the increase in the concentration of dissolved Linezolid. It coincides with the increase in the concentration of dissolved organic carbon in the wastewater samples after the process. Keywords Chlorination process, dissolved organic carbon, hospital effluents, Linezolid and Aztreonam INTRODUCTION Antibiotics are identified in the environment worldwide, in concentrations from a few ng/L to a few dozen μg/L [Felis et al., 2020]. Their so-called "hot spots" include effluents from municipal wastewater treatment plants, where domestic, industrial, and hospital wastewater are treated. Although legislative changes in European Union countries resulting from the implementation of the so-called revised Urban Wastewater Treatment Directive significantly tighten the requirements for the quality of treated urban wastewater [Directive EU, 2024], the requirements related to the quality of individual components of this sewage directed to municipal sewage treatment plants, e.g. hospital sewage, are not fully regulated, and often such regulations are of a local nature. For example, in Poland, there is no obligation to pre-treat hospital wastewater before it is introduced into the municipal sewage system, and only wastewater from so-called "infectious diseases wards" are legally obliged to disinfect wastewater. However, this obligation applies to effluents from specific wards, not hospital wastewater considered as a whole. Only a small percentage of hospitals in Poland disinfect the entire stream of generated wastewater before discharging it to the sewage system. The presence of antibiotics in the environment, as well as their excessive use, is associated with the problem of multi-antibiotic resistance in bacteria, which is becoming a global problem. In 2017, the WHO Expert Committee on Selection and Use of Essential Medicines proposed the AWaRe antibiotic classification as a tool supporting the monitoring of antibiotic use at local, national and international levels. The AWaRe classification is updated every 2 years and currently includes 258 antibiotics, dividing them into 3 groups, i.e. antibiotics from the access group (Access), antibiotics from the observation group (Watch) and reserve antibiotics (Reserve) [WHO, 2023]. Reserve antibiotics can only be used in hospital treatment. They are commonly called "last chance antibiotics". Therefore, the aim of this study is to assess how full-scale disinfection of hospital wastewater affects the presence of two reserve antibiotics, i.e. Linezolid and Aztreonam in the matrices, as well as dissolved organic carbon (DOC) and total dissolved nitrogen (TDN). The hospital wastewater samples were collected in the summer and winter period, from hospitals located in Poland, where full disinfection of hospital wastewater is carried out before it is discharged into the municipal sewage system. The tests were performed in wastewater before and after the full-scale chlorine disinfection process. MATERIALS AND METHODS The hospitals selected for the study differed in terms of treatment profile, number and type of wards, number of hospital beds and patients served, antibiotic policy and location in Poland, their common feature was disinfection of the entire stream of generated sewage before its introduction into the municipal sewage system. Due to the fact that at present we do not have full consents to use data concerning hospitals, we are obliged to use data anonymization, including the location of hospitals, therefore in this study the following nomenclature is used: H1, H2, H3, H4, H5, H6, H7 referring to subsequent 7 hospitals from which the wastewater was collected for the study, without providing details facilitating the identification of a given medical facility. The hospital wastewater samples were collected in the winter and summer of 2024. The study focused on two reserve antibiotics, i.e. Linezolid and Aztreonam. Antibiotics analyses were performed, after solid-phase extraction, using a high-performance liquid chromatograph coupled to a hybrid triple quadrupole-linear ion trap QTRAP 4000 tandem mass spectrometer obtained from AB Sciex LLC (Framingham, MA, USA), operating in multiple reaction monitoring (MRM) mode, dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were determined using a Shimadzu carbon and nitrogen analyzer, while analyses of selected anions were carried out using a Thermo Scientific Dionex Aquion high-performance ion chromatograph equipped with a selfregenerating anion suppressor ADRS 600. All wastewater samples were filtered through a 0.45 μm filter prior to instrumental analyses. RESULTS AND DISCUSSION The studies have shown that the effect of disinfection process on both tested antibiotics is various. In wastewater before the disinfection process, the concentration of Linezolid, depending on the wastewater sample, varied from LOD to 248.95 ± 8.25 ng/L in the winter season and from LOD to 2535.93 ± 95.44 ng/L in the summer season. An interesting observation was that higher concentrations of this drug were observed in the summer season. Generally, this antibiotic is used for treating infections caused by multidrug-resistant strains of bacteria including Streptococci and Staphylococcus aureus. In combination with other drugs, linezolid is also used in the treatment of (multi-drug-resistant) tuberculosis. An important observation was that in the case of Linezolid, its dissolved concentration in hospital wastewater increased after the disinfection process (Table 1) and this was observed almost every time, regardless of the source and the parameters of the hospital wastewater. This phenomenon was associated with observations concerning the concentrations of dissolved organic carbon (DOC) (Table 2) - also in selected cases the concentration of this parameter was also higher after the disinfection process than before. Table 1. Average concentrations of Linezolid and Aztreonam in hospital wastewater before and after disinfection process in different seasons Linezolid, ng/L Aztreonam, ng/L Winter Summer Winter Summer Hospital wastewater N1 D2 N D N D N D H1 4.71 29.15 9.49 544.72 25.27 4.13 8.57 16.39 H2 <LOD3 49.12 1.11 <LOD <LOD 39.24 <LOD <LOQ H3 <LOD <LOD <LOD <LOD <LOD 14.26 <LOD <LOQ H4 248.95 366.19 2535.93 4072.02 5.75 4.36 11.17 3.25 H5 <LOD 28.38 <LOD <LOQ <LOD <LOD <LOD 5.92 H6 1.91 7.35 <LOQ4 <LOQ 1.62 <LOQ 5.09 <LOQ H7 1.14 70.60 21.77 1086.64 <LOD 6.67 17.92 4.82 1before the disinfection process (not disinfected); 2after the disinfection process (disinfected), 3LOD - limit of detection; 4LOQ - limit of quantification. Table 2. Average concentrations of dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) in hospital wastewater before and after disinfection process in different seasons DOC, mg/L TDN, mg/L Winter Summer Winter Summer Hospital wastewater N D N D N D N D H1 133.25 156.3 153.80 180.70 26.90 24.1 22.11 53.58 H2 185.25 327.15 175.90 80.40 28.80 14.80 50.35 29.17 H3 337.90 203.25 103.75 52.10 20.81 19.41 14.51 19.85 H4 99.05 85.95 530.50 108.05 8.12 7.83 17.27 16.68 H5 3.81 146.35 90.05 137.60 10.03 10.14 23.85 19.35 H6 25.48 32.01 115.60 146.45 4.87 6.32 19.20 15.82 H7 350.30 253.75 881.50 263.20 27.34 21.62 47.77 27.55 Therefore, it was hypothesized that linezolid may occur in hospital wastewater in a dissolved form, but it cannot be ruled out that it may also occur in a form partially bound to organic suspension, i.e. suspended solids naturally occurring in hospital wastewater. The disinfection process could cause partial disintegration of the suspension into a more dissolved form, which was in some cases observed by increasing the concentration of DOC and linezolid in the wastewater after filtration step in that all instrumental analyses were performed. In the case of the second reserve antibiotic, i.e. Aztreonam, this phenomenon was not as pronounced (Table 1). Most likely, the final effect was influenced by the composition of the matrix, but this phenomenon will be investigated further, e.g. by conducting analyses of intermediate products of the tested drugs and correlating the concentrations of these compounds with other parameters of the tested wastewater. CONCLUSIONS The effect of chlorine disinfection on the behaviour of the tested drugs cannot be determined unequivocally and the justification of this phenomenon requires further research. However, it has been hypothesized that Linezolid may be present in hospital wastewater in a dissolved form and in a form associated with wastewater suspended solids or in the form of other conjugates. As a result of chlorine disinfection, disintegration of the sewage suspension and/or conjugates may occur, which causes an increase in the concentration of dissolved organic carbon and this drug in the liquid phase, which may indirectly contribute to an increase in the mobility of this drug in the aquatic environment. Acknowledgments This research was financial supported by the National Science Centre Poland, under the project “OPUS 23” No. UMO-2022/45/B/NZ7/00793. REFERENCES Felis, E., Kalka, J., Sochacki, A, Kowalska, K., Bajkacz, S., Harnisz, M., Korzeniewska, E. 2020. Antimicrobial pharmaceuticals in the aquatic environment - occurrence and environmental implications. European Journal of Pharmacology 886, 172813. Directive (EU) 2024/3019 of the European Parliament and of the Council of 27 November 2024 concerning urban wastewater treatment. WHO, 2023. Reference Number: WHO/MHP/HPS/EML/2023.04