An-Najah National University Faculty of Graduate Studies ASSESSING THE PREVALENCE OF PHARMACEUTICAL RESIDUES IN WADI ZOMAR CATCHMENT AREA IN PALESTINE: RISK ASSESSMENT FOR REUSE AND IMPACT ON HUMAN HEALTH By Ala'a Monther Hassan Jaddou Supervisors Dr. Souad Belkebir Prof. Saed Khayat This Thesis is Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Infectious Diseases Prevention and Control, Faculty of Graduate Studies, An-Najah National University, Nablus-Palestine. 2023 ii ASSESSING THE PREVALENCE OF PHARMACEUTICAL RESIDUES IN WADI ZOMAR CATCHMENT AREA IN PALESTINE: RISK ASSESSMENT FOR REUSE AND IMPACT ON HUMAN HEALTH By Ala'a Monther Hassan Jaddou This Thesis was Defended Successfully on 08/06/2023 and approved by Dr.Souad Belkebir Supervisor Signature Prof.Saed Khayat Co-Supervisor Signature Dr. Subhe Samhan External Examiner Signature Dr. Rasha Khayat Internal Examiner Signature iii Dedication In the name of Allah Almighty, the one to whom gratitude and thanks are acknowledged before anything. Allah has planted the first seed of science passion in me and guided me to the completion of this thesis. Second of all, I sincerely thank all those who believed in me, pushed me, and helped me through this journey; my parents and siblings, are the unknown soldiers. For all those who believe in science and human well-being. For all of them, I dedicate this and every work. iv Acknowledgment I’d like to extend my sincere thanks to my supervisors Dr. Suoad Belkebir, and Prof. Saed Khayat for their enormous support and guidance through this work. I’d like to thank the Royal Scientific Society personal especially Dr. Othman Almashaqbeh, manager of advanced materials & applications divisions, and scientist Layal Alsalhi for their assistance in the analytical process. Great thanks to Mr. Yousef Jaffal, chief director of West Nablus WWTP for his help and support in the sampling process and to Mr. Ahmad Abo-Alo’on, Anabtas’ Municipality hydrology engineer for his great assistance through this project. Many thanks to the Orange Knowledge Program-Institutional Collaboration Project, OKP-ICP-PAA-103455, funded by the Netherlands’ Ministry of Foreign Affairs and managed by Nuffic, the Netherlands for financial support. v Declaration I, the undersigned, declare that I submitted the thesis entitled: ASSESSING THE PREVALENCE OF PHARMACEUTICAL RESIDUES IN WADI ZOMAR CATCHMENT AREA IN PALESTINE: RISK ASSESSMENT FOR REUSE AND IMPACT ON HUMAN HEALTH I declare that the work provided in this thesis, unless otherwise referenced, is the researcher’s own work, and has not been submitted elsewhere for any other degree or qualification. vi Table of Contents Dedication ........................................................................................................................ iii Acknowledgment ............................................................................................................. iv Declaration ........................................................................................................................ v Table of Contents ............................................................................................................. vi List of Tables ................................................................................................................. viii List of Figures .................................................................................................................. ix List of Appendices ............................................................................................................ x Abstract ............................................................................................................................ xi Chapter one: Introduction and Literature Review ............................................................ 1 Chapter Two: Materials and Methods ............................................................................ 10 2.1 Study area ................................................................................................................. 10 2.1.1 West Nablus WWTP .............................................................................................. 10 2.1.2 Wadi Zomar ........................................................................................................... 11 2.2 Ethical approval ........................................................................................................ 13 2.3 Antibiotics dispensary data collection ...................................................................... 13 2.4 Sample collection, preparation, and extraction ......................................................... 14 2.4.1 Water Samples collection ...................................................................................... 14 2.4.2 Chemicals ............................................................................................................... 15 2.4.3 Sample preparation and extraction ........................................................................ 15 2.4.4 Analytical methods ................................................................................................ 16 2.5 Statistical Analysis .................................................................................................... 17 Chapter Three: Results .................................................................................................... 18 3.1 West Nablus WWTP performance ........................................................................... 18 3.2 Grab Samples and Removal Efficiency .................................................................... 18 3.3 POCIS Results .......................................................................................................... 23 3.4 Antibiotics dispensary data ....................................................................................... 26 vii 3.5 Promoting AMR development .................................................................................. 27 Chapter Four: Discussions and Conclusions .................................................................. 29 4.1 Occurrence of PhCs in wastewater samples and West Nablus WWTP removal efficiency ................................................................................................................. 29 4.2 Occurrence of PhCs in groundwater ......................................................................... 40 4.3 Potential Health effects of antibiotics measured environmental concentrations and the development of AMR ........................................................................................ 42 4.4 Strengths and Limitations ......................................................................................... 45 4.5 Conclusion and Recommendations ........................................................................... 46 4.6 Funding ..................................................................................................................... 46 List of Abbreviations ...................................................................................................... 47 References ....................................................................................................................... 50 Appendices ...................................................................................................................... 65 CDEFGب ............................................................................................................................... ا viii List of Tables Table 3.1: COD, BOD & TSS parameters of collected wastewater samples obtained from West Nablus WWTP ........................................................................... 18 Table 3.2: Summary of Grab Sampling Campaigns results ............................................ 24 Table 3.3: Physiochemical properties of detected pharmaceuticals ............................... 25 Table 3.4: Comparison between PNEC-MIC values provided by Bengtsson-Palme, Larsson et al. (52) and PNEC-ENV by Le page et al. (63) as endorsed by the AMR Industry Alliance and average concentrations of detected antibiotics in the WWTP effluents of the present study ................................................ 28 Table 4.1 General overview regarding the most frequently detected pharmaceutical compounds in our study modified after ....................................................... 32 ix List of Figures Figure 1.1: The introduction of antibiotics into the environment and the direct and in- direct human exposure to antibiotics ................................................................ 2 Figure 2.1: Location of (a) West- Nablus WWTP and sampling points with surrounding communities in respect to Wadi Zomar. (b) Palestine/ West Bank water supply map ..................................................................................................... 11 Figure 2.2: Vulnerability map of the studied area displaying the geological and hydrogeological structures ............................................................................. 12 Figure 2.3: The journey of the study, Wadi Zomar at Anabta sampling point, the grab samples of both campaigns, the deployment of the POCIS, the WWTP sampling points and shipment of the grab samples ........................................ 15 Figure 3.1: Summary of different PhCs detected (µg/L) in the first sampling campaign of April/2022 .................................................................................................. 19 Figure 3.2: Summary of different PhCs (µg/L) detected in second sampling campaign among wastewater samples ............................................................................ 21 Figure 3.3: West Nablus WWTP removal efficiency (%) in both sampling campaigns 22 Figure 3.4: PhCs detected in POCIS sampler (ng/POCIS) deployed at Anabta drinking groundwater reservoir in April/2022 for 28 day. ........................................... 23 Figure 3.5: Cumulative quantities of Antibiotics (in grams) administered for patients from included hospitals of the time period 1/1/2021-28/2/2022 .................... 26 Figure 4.1: Residual concentrations of different pharmaceutical in the influent, the effluent of the west Nablus WWTP and Anabta-Zomar in both sampling campaigns ....................................................................................................... 30 x List of Appendices Appendix A: IRB approval ............................................................................................. 65 Appendix B: Nablus municipality facilitating task letter ............................................... 66 Appendix C: Anabta municipality facilitating task letter ............................................... 67 Appendix D: Palestinian Ministry of Health facilitating task letter ............................... 68 Appendix E: Palestinian Ministry of Health permission for data collection from two governmental hospitals (Rafedia surgical hospital and Al-Watani hospital) ..................................................................................................... 69 Appendix F: Al-Itihad hospital facilitating task letter .................................................... 70 Appendix G: Najah National University Hospital facilitating task letter ....................... 71 Appendix H: List of tested pharmaceuticals in the collected water samples of this study .......................................................................................................... 72 xi ASSESSING THE PREVALENCE OF PHARMACEUTICAL RESIDUES IN WADI ZOMAR CATCHMENT AREA IN PALESTINE: RISK ASSESSMENT FOR REUSE AND IMPACT ON HUMAN HEALTH By Ala'a Monther Hassan Jaddou Supervisors Dr. Souad Belkebir Prof. Saed Khayat Abstract The occurrence of various classes of antibiotics and pharmaceuticals (PhCs) in the environment and their contribution to Antimicrobial resistance (AMR) development is questionable. AMR is recognized as a major health threat. Discharges from wastewater treatment plant (WWTP) is considered to be the major source contributing to the vast bulk of different pharmaceuticals in the environment. The researcher intends to investigate the prevalence of pharmaceutical residues in diverse aquatic matrices with more attention to the removal capacity of West Nablus WWTP with respect to the detected pharmaceuticals. Method: 2 raw wastewater, 2 treated wastewater, and 2 surface wastewater run-offs were collected in two grab sampling campaigns. An additional ground water sample was collected using a Passive Organic Chemical Integrative Sampler (POCIS). All samples were analyzed using LC-MS/MS. The Risk Quotient (RQ) was used for Measured Environmental Concentration (MEC) of detected antibiotics against Predicted No-Effect Concentration ((PNEC) to evaluate the risk for antibiotic resistance development according to the detected antibiotics residues. Sulfamethoxazole, Trimethoprim, Diclofenac, and Carbamazepine were the most frequently detected in all water samples. West Nablus WWTP delivered a significant removal efficiency in both campaigns. However, it was noticed that there was a significant spatial difference between the WWTP effluent discharge point and the Anabta-Zomar point of sampling directly after the rainy season. Ofloxacin residual concentration in immediate WWTP effluent discharges and surface run-offs along the sampling point is found to pose risk for AMR development in the environment. Groundwater is found to be polluted with Carbamazepine, Diclofenac, Ciprofloxacin, and Sulfamethoxazole. The local status indicates the need for further in-depth investigation regarding the risk of antibiotics to the environment and its role in the emergence of AMR concerning the detected antibiotics. Additional wastewater xii treatment methodologies are needed for better removal yield. Groundwater pollution requires urgent ecotoxicological studies for both human and animal health and environmental life forms. Keywords: Antibiotics; Antimicrobial resistance; Bacteria; Environment; Health; Pharmaceuticals; Wastewater; WWTP. . 1 Chapter one Introduction and Literature Review A healthy environment is as healthy as its smallest components can get to sustain its normal function. This is jeopardized as pharmaceutical (PhCs) pollutants of different sources are frequently found with different toxicities impacting various life forms(1). The use of different classes of therapeutics is indispensable for human and veterinary purposes, by which they are continuously being introduced into the environment(2–4). Antibiotics usage is intended to combat infectious conditions either as a treatment or as a prophylaxis, both in human and veterinary fields. Upon their administration, they undergo several metabolic processes by which they are broken down into active and inactive metabolites. None the less, not all administered dose is metabolized and a portion of the active parent drug remains intact. The excretion is limited to the urinary and/or biliary tracts (5). The veterinary field relies on the use of antibiotics extensively as 73% of the global production of antibiotics is intended for animals raised for food as Animal Growth Promoters (AGPs)(6). Even though the excretion of antibiotics in animals is species- dependent, their manure is found to harbor several classes of antibiotics and other pharmaceutical compounds (3,7). This is of concern for two reasons. The first reason is the implication of antibiotics use in animals and the emergence of antimicrobial resistance in zoonotic pathogens. The second reason is that manure is regarded as a natural fertilizer for crops and hence the dissemination of several pharmaceutical pollutants into the environment is a plausible possibility(8–10). The toxic effects PhCs exert on environmental life forms have been recognized after the near extinction of vultures in Pakistan in the time period of (2000-2003). The implicated PhC was found to be diclofenac which led to fatal renal disease. This PhC gained entry to the food chain by the administration to prey vultures feed (4). After this, the environment has been heavily investigated for the presence of different PhCs and possible access sources as follows. Pharmaceuticals can gain access to the environment (soil and water) in several anthropogenic r direct disposal of pharmaceutical wastewater into receiving environment (from manufacturing facilities or any health care setting), (b) the use of pharmaceuticals in aqua culture, (c) animal PhCs off with rain events, (d) treated municipal and industrial wastewater discharged into surface water, and (e) the use in agriculture Wastewater Treatment Plants (WWTP) have been recognized as the main source of antibiotics and other PhCs introduction into the aquatic environment WWTPs are typically designed to remove organic and some in the effluent is discharged into receiving body. Pharmaceuticals of any class aren’t removed in the process and their presence and effect in treated wastewater have been noticed. Where surface water is the end recipient for treated evident that trace levels (nanogram/Liter) of Endocrine Disturbing Substances (EDS) in the receiving aquatic mediums are sufficient for devastating biological alterations in fish (2,15). Figure 1.1 The introduction of antibiotics into the environment and the direct and in exposure to antibiotics 2 environment (soil and water) in several anthropogenic routes (figure direct disposal of pharmaceutical wastewater into receiving environment (from manufacturing facilities or any health care setting), (b) the use of pharmaceuticals in aqua culture, (c) animal PhCs-rich manure utilized for crops production and surface run off with rain events, (d) treated municipal and industrial wastewater discharged into surface water, and (e) the use in agriculture(3,7,11–13). ent Plants (WWTP) have been recognized as the main source of antibiotics and other PhCs introduction into the aquatic environment WWTPs are typically designed to remove organic and some in-organic pollutants before the effluent is discharged into receiving body. Pharmaceuticals of any class aren’t removed in the process and their presence and effect in treated wastewater have been noticed. Where surface water is the end recipient for treated wastewater effluents. It’s evident that trace levels (nanogram/Liter) of Endocrine Disturbing Substances (EDS) in the receiving aquatic mediums are sufficient for devastating biological alterations in fish uction of antibiotics into the environment and the direct and in outes (figure 1.1): (a) by the direct disposal of pharmaceutical wastewater into receiving environment (from manufacturing facilities or any health care setting), (b) the use of pharmaceuticals in s production and surface run- off with rain events, (d) treated municipal and industrial wastewater discharged into ent Plants (WWTP) have been recognized as the main source of antibiotics and other PhCs introduction into the aquatic environment (11,12,14). organic pollutants before the effluent is discharged into receiving body. Pharmaceuticals of any class aren’t removed in the process and their presence and effect in treated wastewater have been wastewater effluents. It’s evident that trace levels (nanogram/Liter) of Endocrine Disturbing Substances (EDS) in the receiving aquatic mediums are sufficient for devastating biological alterations in fish uction of antibiotics into the environment and the direct and in-direct human 3 Figure 1.1 displays how antibiotics have gained special attention after their detection in nature. Their delivery into the environment is via different anthropogenic routes. Hospital wastewater, animal manure, aqua culture and municipal wastewater. The cycle continues for human indirect exposure through contaminated water, crops and food- animals. Figure adopted from Serwecińska et al(16). Several studies reported antibiotics detection in aquatic compartments implying the in- ability of the WWTP to remove such PhCs before effluents are discharged. Tetracycline was detected at both the wastewater treatment plant (WWTP) influent and effluent in Spain with a residual concentration of 0.067 µg/L and 0.018 µg/L, respectively (17). Ciprofloxacin was detected in the raw influent of WWTP in Baghdad with a residual of 1.344 µg/L(18). The awareness of characteristics promoting the stability of PhCs in various environmental compartments aids in the quest to establish mitigation measures. Those characteristics helped PhCs to be considered recalcitrant and resist degradability in water and soil. They range from physiochemical properties (solubility and pH, net charge, long half-life, carbon content, etc.…), to resistance to biodegradation, resistant to solar degradation, and sorption and de-sorption ability. Sulfonamides, for instance, are an essential class of antibiotics used in both the human and animal medical fields. They were found to be labile for solar-degradation when exposed to direct sunlight. Furthermore, the speed of degradation is compound specific. Sulfamethoxazole was found to be, though to a lesser extent, sensitive for photo- degradation when irradiated with Ultra-Violet A/ Ultra-violet B (UVA/UVB) and direct sun exposure for 7 days (19). While J.K. Challis et al predicted the photodegradation of Sulfapyridine in water to be around 2.6 hours (20). Carbamazepine (CZB) physiochemical properties of hydrophobicity and net-charge of zero have made it to be resistant to solubility in aquatic mediums rendering it a mobile agent. Carbamazepine and Diclofenac have been recognized among the most frequently reported pharmaceuticals on a global scale (21). 4 Crops cultivated in soil- or irrigated with reclaimed water or contaminated surface water are found to uptake such PhCs from the soil. In a study investigating the occurrence of PhCs in Nakivubo wetlands in Kampala, Uganda, trimethoprim was found to have accumulated in the soil of wetlands and yam roots(22). Moreover, the ecotoxicity is concentration dependent. For example, CZB toxicity in inhibiting the growth rate of P. subcapitata is observed at a concentration of 10.4 mg/L while 6 mg/L of Diclofenac is sufficient for the same effect (23). The integration between factors favoring the accumulation of pharmaceutical pollutants in soil (i.e. chronic introduction into surface water with resistance to degradability and adsorption to soil particles) aids in the seep and detection of such PhCs in groundwater(24). Drinking water is also found polluted as well. In filtered tap water in China, the average total antibiotic concentration was found to be 0.182 µg/L(25). 0.096 µg/L of Sulfamethoxazole was detected in the effluent of a WWTP in Jordan(12). Dalahmeh et al. reported that Trimethoprim and Sulfamethoxazole were the most frequent antibiotics detected in various water samples collected from different sources in Kampala – Uganda(22). In addition to the environmental aspects(1,26), human health is in question. A study conducted in China estimating sources contributing to the un-intentional exposure of antibiotics through food and water, the main source was found to be food products derived from plants. In addition, preschool children had the highest daily intake rate of antibiotics from drinking water and food (310 ng/kg-bodyweight/day) (27). The frequent detection of antibiotics in water and soil mediums opens new arrays for research on its implication in the emergence of antimicrobial resistance (AMR). Water provides a rather supporting medium where resistance can get allocated easily. In addition, it supports its persistence and wide spread detection(28). It has been demonstrated that antibiotics detection in effluents of WWTPs is associated with the detection of resistant bacteria as well(29,30). Concerning the constantly nutrient-rich medium of wastewater with the presence of antibiotics and bacterial 5 populations as well, L. Rizzo et al. in his review, regarded urban WWTP as hot spots where resistance genes and resistance bacteria can easily disseminate to near-by environment. Resistance traits can be exchanged between bacterial colonies.(14). Antimicrobial resistance (AMR) is considered an urgent health crisis requiring prompt action to tackle. In 2019, the AMR bacteria-associated death toll reached 4.95 million on a global scale (31). This death toll is estimated to reach 10 million by the year 2050 unless an effective response is initiated. The economic impact is extravagated as well, with an expected 100 trillion dollars loss and a gradual decrease in global domestic production by 2 - 3.5% (32). The etiology underlying the development of AMR is rather ubiquitous. It can be as simple as a natural intrinsic phenomenon(33), or evolutionary response mechanisms bacteria develop due to selection pressure when co-existence with antibacterial levels (34,35) or it can be picked-up from the environment through horizontal gene transfer (36,37). Pereira et al in their systemic review regarding the toxicity and environmental risk assessment (ERA) of selected PhCs in different aquatic compartments, noted that antibiotics risk quotients alert for toxicity effects on the environment (1). To have a clearer picture regarding AMR development in any setting, one needs to understand both parties apart and combined – bacterial spp with/without AMR genes and antibiotics. Bacteria are diverse microorganisms and are considered integral normal inhabitants of any biosystem. They are resilient to function in rather complicated conditions naturally(38). Whereas antibiotics are merely chemical compounds with a variety of pharmacological and physiochemical characteristics that help them achieve their intended function: infectious disease treatment. Nevertheless, the same properties have aided in their persistence in many environmental compartments and their identification (39). If low levels of antibiotics and certain bacterial spp combine, then the susceptible bacteria will die while the resistant strain will dominate. This is the definition of selection pressure and the evolution of antimicrobial resistance. This level is referred to as Minimal Selective Concentration (MSC)(37,40). 6 Interestingly, it’s noticed that selection develops in concentrations well below the Minimally Inhibitory Concentration (MIC) thresholds proposed for clinically relevant bacteria by several folds (MIC: the lowest level of antimicrobial required to inhibit the growth of microorganisms in-vitro aided by the naked eye) (41,42). Gullberg et al reported that the MSC value for ciprofloxacin is 100 pg/L ≈1/10th MIC. In addition, AMR mutation is developed within susceptible bacteria in the presence of MSC despite the presence of already resistant bacteria (41). While the process may not be explained as intentional development rather than a survival and adaptation mechanism for microorganisms’ use, its consequences are prevalent in the detection of AMR genes in the environment and the emergence of “super-bugs”(36,43). On a higher level, environmental risk assessment adopted by the European Medicines Agency (EMA) is used to set-up threshold values for any drug residual in environmental compartments as follows: using the PNEC and Predicted Environmental Concentration (PEC: predicted level of antimicrobial to reach the environment); in order to evaluate their potential risk on the environment. A Risk Quotient (RQ) is calculated by obtaining the ratio of Measured Environmental Concentration (MEC) or PEC to PNEC for each pharmaceutical compound of question. If the RQ is > 1 then a potential impact is predicted on different trophic levels, and this calls for further studies determining what the effect shall be. Whereas if the RQ is < 1, then no risk is expected (44). In terms of the dual detection of residual concentrations of antibiotics and resistant bacteria in the environment, efforts are done to clarify the point where a certain concentration of an antibiotic paves the way for resistance development. Scientists Bengtsson-Palme and Larsson et al.developed Predicted No-Effect Concentrations (PNEC) derived from the MIC values established in the European Committee on Antibiotic Susceptibility Testing (EUCAST) for the majority of frequently detected antibiotics in the environment (PNEC –MIC: a level of which below no effect on the clinically relevant bacteria is observed – according to the MIC values). PNEC-MIC took into consideration the effects of antibiotics on clinically relevant bacteria (45). 7 On the other hand, the Environmental Predicted No-Effect Concentrations (PNEC- ENV) developed by Le Page et al(46) studied the effects of antibiotics on representative environmental bacteria (Cyanobacteria, V. fischeri and P. putida) (46). The residual concentration detected in the environment for antibiotics is then compared to the PNEC value to estimate the RQ value. If RQ is >1, then this antibiotic is expected to exert selection pressure against selected species and promote resistance evolution(45,46). The AMR Industry Alliance is a group of several pharmaceuticals, advance research, and diagnostic companies of the private sector collated to curb AMR. The alliance advocated the lowest value of PNEC-MIC and PNEC-ENV thresholds as antibiotics target markers for manufacturing companies to adhere to when considering pharmaceutical wastewater discharge into the environment. Those values are updated regularly. In addition, the alliance recommended a default threshold of 0.05 µg/L for antibiotics when no PNEC value is available(47). Several antibiotics are on the EU watch list of substances within the European Water Framework Directive on a union-wide monitoring system for high-quality data gathering. The watch list is established for substances whose residuals are potentially found to pose a significant risk to, or via, the aquatic environment and is updated every 2 years. Currently, of those substances the antibiotics: Trimethoprim, Sulfamethoxazole, Ofloxacin, and Clindamycin(48). Up to this point, ground water, surface water, wastewater, vegetables, and animal manure are all found to harbor residual concentrations of antimicrobials (18,49). This brings to mind several questions. First: what level of risk do those trace residues pose on human health? Second: the acute versus chronic exposure effects to the emerging new contaminant? Third: when should we include screening for antimicrobials as “pollutants” as part of regulating water use? And the clear need to implement advance wastewater management technologies. In the West Bank, the presence of several classes of antibiotics and herbicides was investigated in off-grid, household greywater reuse systems in farms where the 8 reclaimed water is intended to be used for crop irrigation. Several antibiotics were recovered from domestic sewage, filtered sewage, and pond water with varying concentrations and frequencies. Pipemidic acid, Azithromycin, and Oxolinic acid were the most frequent antimicrobials among the influents while only Oxolinic acid was the most frequent in the effluent samples(50). So far to the authors’ knowledge, published work regarding the detection of antibiotics in different aquatic compartments locally is absent. Palestinian hydrological sources are scarce. Their exploitation is rather restricted which imposes further pressure on the quality and proper utilization of accessible natural resources (51). This highlights the importance of aquatic management practices especially groundwater (52). Reclaimed wastewater is proposed as an alternative for agricultural purposes to elevate the demand for groundwater intended for agricultural consumption. AMR is regarded as a major public health issue and the local status indicates the presence of high resistance to antibiotics including last resort antibiotics: colistin, from both clinical human samples and animal samples (9,53–55). Given that there are no local studies regarding the role of aquatic environments in disseminating resistance and the urgent need to tackle AMR, we looked for the contribution of municipal and industrial sewage in occurrences of antibiotics in several water types (raw sewage, treated wastewater, treated surface wastewater and ground water). The West-Nablus WWTP is used here as a case study. Aim and Objectives We aim to investigate the occurrence of antibiotics and other pharmaceuticals (PhCs) in raw, treated, surface wastewater and ground water in west Nablus, North Palestine. Specific objectives: • To evaluate the removal efficiency of west Nablus WWTP for the detected PhCs if present. 9 • To test for the spatial difference in concentrations of detected compounds in collected samples. • To link between dispensary quantities of antibiotics from hospitals in the study area and the identification of antibiotics in wastewater. • To evaluate the potential health effect of measured environmental concentrations of antibiotics and the development of AMR according to the data in the literature. 10 Chapter Two Materials and Methods 2.1 Study area The study area covered the catchment area of west Nablus in the West Bank of Palestine. All the catchment area untreated sewerage is drained down to West Nablus WWTP. The WWTP inlet was taken as the first sampling location (raw wastewater) just before entering the WWTP. The finished treated wastewater (WW) was considered the second WW sampling site which is directly discharged into Wadi Zomar. Treated WW transports down Wadi Zomar through Anabta village where the third sample was collected at Anabta at a specific location for both sampling campaigns. 2.1.1 West Nablus WWTP Nablus city is located north of Palestine and 681 m above sea level. The WWTP is located west of Nablus and is 305 m above sea level. It receives sewage (domestic, industrial, and pharmaceutical) from west Nablus catchment area alongside 5 surrounding villages (Zawata, Beit Eba, Dair Sharaf, Beit Wazan, and Qusin), with approximate inhabitants of 120,00. The plant is in service since 2015 and operates as a secondary treatment facility. It uses activated sludge with mechanical treatment, biological treatment, and sludge treatment steps with gasutilization. West Nablus WWTP was designed to receive an estimated flow of raw sewage water of about 14,000 m3/day. In 2022, it received an average of 12,000 m3/day. According to West Nablus WWTP annual report of the year 2022, Biological Oxygen Demand (BOD) and Total Suspended Solids (TSS) average values in the inlet WW were 535 mg/L and 440 mg/L, respectively. WWTP performance capacity for the removal of BOD and TSS was 98% for both parameters (56). The treated wastewater is continuously being discharged into the environment of Wadi Zomar. This wadi passes through Anabta village, Tulkarem city (63 m above sea level), and finally into the Mediterranean Sea. 11 Figure 2.1 Location of (a) West- Nablus WWTP and sampling points with surrounding communities in respect to Wadi Zomar. (b) Palestine/ West Bank water supply map (a) (b) Figure 2.1 (a) displays the geographical locations of sampling points (red pin) at the inlet of west Nablus WWTP (purple circle) and outlet point and at Wadi Zomar-Anabta (red pin). Wastewater at Wadi Zomar has many tributaries (from Bet Imren, Bazaria and Kufr Rumman) along its natural path as displayed here. (b) Palestine/west bank water supply map adopted from Palestine Water Authority website. 2.1.2 Wadi Zomar The importance of Wadi Zomar originally comes from the fact that it’s the largest treated wastewater discharge in the northern part of the west bank. It’s 55 Km from the upper part of Alexander River in northern Palestine originating from the mountains of the West Bank and reaching the coastal plane. The investigated area of Wadi Zomar lies between West Nablus WWTP and Anabta village which compromise 5 Km. Where Wadi Zomar passes through the village and is surrounded by several groundwater wells. Most of these wells are municipal wells and some are agricultural wells. 12 Figure 2.2 Vulnerability map of the studied area displaying the geological and hydrogeological structures Figure 2.2 explains the geological structure around and underneath Wadi Zomar. It has diverse geological structures that permits the seep of different pollutants to layers below earth surface where wells are dugged normally. Vulnerability map Adopted from Saed Khayat et al.: Mechanisms of Groundwater Pollutants Transport in Tulkarm Area / Palestine, Resources and Environment 2012, 2(6): 281-290. The geological and hydrogeological structures of the studied area highly influence the infiltration of different pollutants into the sediment bed of Wadi Zomar. The main aquifer for Palestinians in the northern region of the west bank comes from the western and north-eastern water basins. Most of the groundwater wells are dugged in the shallow layer of the uppermost Jerusalem-Turonian aquifer which has characteristics that facilitate the movement of pollutants from the top layer (57–59). 13 2.2 Ethical approval This study was reviewed and approved by the An-Najah National University (ANNU), Institutional Review Board (IRB) (Appendix A). Designated facilities were informed of the scope and intent of the research before the initiation of samples collection. Approvals from hospitals, Nablus municipality and Anabta municipality were granted prior to antibiotics dispensary data collection (Annexes B-G). 2.3 Antibiotics dispensary data collection To have a baseline data regarding the expected human excretion of both quantity and class of antibiotics in the study area, we’ve requested dispensary data regarding antibiotic consumption from all hospitals located in West Nablus in the period of 1/1/2021 – 28/2/2022. Out of the 7 hospitals residing in west Nablus, 5 hospitals (2 governmental and 3 private hospitals) agreed to provide us with the required data. All hospitals' domestic sewerage is piped into the same system; with no previous treatment where all raw wastewater with PhCs residues drained directly to west Nablus WWTP. Medical waste (biomedical waste containing hazardous materials to human and environment) from hospitals are subjected to special waste disposal practices. Hospitals domestic sewerage isn’t included in such practices and the produced wastewater isn’t tested for pharmaceutical residues (Personal communication with Mr. Yousef Jaffal, West Nablus WWTP Chief Director and Eng. Suha Kharraz, Environmental Control Unit Chief Director at Nablus Municipality). Approvals from Nablus municipality, Anabta municipality, and all designated hospitals are supplemented in annexes (B-G). A plant for veterinary PhCs is located in west Nablus study area. Their industrial wastewater isn’t disposed down the sewerage systems as they developed an internal protocol that eliminate any disposal alongside west Nablus domestic sewerage (Personal communication with Nivin Ratrot, Dana Pharmaceuticals Quality department Chief Director). 14 2.4 Sample collection, preparation, and extraction 2.4.1 Water Samples collection Sampling campaigns were divided into 2 grab sample sets for wastewater and one Passive Organic Chemical Integrative Sampler (POCIS) (Environmental Sampling Technologies, EST Inc, St. Joseph, MO, USA) for groundwater. Three grab samples per set were collected from raw (WWTP inlet), treated (WWTP outlet), and surface wastewater (from Wadi Zomar at Anabta collection point) in early April/2022 and another set in September/2022 with a total of 6 grab samples. Grab samples were collected in 1L amber glass bottles that were previously rinsed with LC- grade Methanol (Sigma Aldrich, St. Louis, MO, USA), then washed with double deionized water (ChromasolvaTM Plus, for HPLC-gradient, obtained from Honeywell Riedel-de Haen, M, USA). Samples were immediately stored on ice bags and refrigerated at 4-8 ˚c until shipment to analyzing laboratory (Royal Scientific Society, Amman, Jordan) within 48hrs of collection. An additional 2 grab samples were collected from Wadi Zomar at the Anabta sampling point in each campaign, stored on ice bags, and tested for wastewater quality parameters at West Nablus WWTP Lab. Those parameters include Chemical Organic Demand (COD), Biological Oxygen Demand (BOD), Total Suspended Solids (TSS), Conductivity, and pH). The POCISs were deployed in early April/2022 for 28 days. Two POCIS with Hydrophilic- Lipophilic Balanced (HLB) sorbent membranes were used (Waters Corporation, Milford, MA). Two sets were used to avoid any misleading information regarding temporal variations of measured PhCs in groundwater samples and the measurements from both sets were averaged. The POCIS were constantly submerged at Anabta’s municipal groundwater reservoir after water is pumped from the groundwater wells. During installation, caution was taken to avoid heavy water movement (i.e. the pump) near the POCIS. On the 28th day, the POCIS were removed from the ground water tank, wrapped inaluminum foil, kept in an airtight, clean plastic bag, and preserved at 4◦c during transporting and shipping to analyzing laboratory (Royal Scientific Society, Amman, Jordan). 15 Figure 2.3 The journey of the study, Wadi Zomar at Anabta sampling point, the grab samples of both campaigns, the deployment of the POCIS, the WWTP sampling points and shipment of the grab samples 2.4.2 Chemicals All chemicals including all reference materials and labeled standards for antimicrobials and PhCs are obtained from Sigma–Aldrich (St. Louis, MO, USA). A list of tested antibiotics and other pharmaceuticals is detailed in Appendix I. Solvents used in sample preparation were of high-grade purity (OPTIMA, Fisher Scientific, St. Louis, MO, USA). 2.4.3 Sample preparation and extraction The extraction process was implemented according to the procedure provided by Water Sciences Laboratory at the University of Nebraska–Lincoln (WSL/UNL) in the United States (USA) and others (60,61) described as follows. Grab samples were first decanted to remove all suspended particles and then filtered through the vacuum filtration unit using a 0.45µl glass fiber filter. Then the filtered sample is passed through the cartridges of polymeric Hydrophilic-Lipophilic Balanced 16 (HLB) Oasis 6CC (200mg) using a vacuum manifold system. The Cartridges were first connected to a solid-phase extraction (SPE) manifold and vacuum pump and preconditioned by passing 6mL of acetone and then 6mL of methanol respectively, followed by passing 6mL of distilled deionized water (DDIH2O). The flow rate of samples through the SPE cartridge was set at 10mL/min or less. The cartridges were then rinsed with DDIH2O once the extraction process has completed. Drying was done using room air with continued suctioning for no less than 5min. All cartridges are then removed, labeled, and stored in a clean bag at (-20◦c)(61). POCIS were disassembled once arrived at the analyzing lab and the sorbent was transferred into glass gravity-flow chromatography columns. Chemical residues were recovered from the sorbent by organic solvent elution. Methanol was used to recover analytes from the pharmaceutical POCIS. The extracts were reduced in volume by rotary evaporation and under a gentle stream of nitrogen, then filtered through a glass- fiber filter, solvent exchange was used as necessary, and sealed in amber ampoules under nitrogen until further analysis(60). 2.4.4 Analytical methods The cartridges were eluted using 10mL of high-purity methanol into a disposable glass culture tube. The volume of the eluent then was reduced to 4mL under nitrogen at 40◦c. Subsequently, it was transferred to auto-sampler vials where the inserts were silane- treated(60,61). The separation of compounds was done using the AB-Sciex 5500 Qtrap (LC-MS-MS) equipped with Luna Omega polar C18 embedded column (100 mm*3.0 mm, particle size of 3µm). Gradient elution mode was considered in the following order: eluent A is 5mM ammonium formate and 0.1% formic acid, and eluent B is Acetonitrile and 0.1% formic acid. The initial mobile phase conditions started with 98:2 A/B for 1min then a linear gradient pattern was used to reach a ratio of 70:30 A/B at 3min, then the ratios were changed to reach 50:50 A/B at 6min. Before returning to the initial conditions, the last ratio was maintained for 10min. The flow rate was set at 0.6mL/min and the injection volume of 5 µL(60,61). Multiple reaction monitoring (MRM) was conducted with AB-Sciex 5500 Qtrap equipped with an electrospray ionization interface, using the positive-ion mode (Santa 17 Clara, CA, USA). Instrument control, data acquisition, and quantitation were run using analyst software. Setting the drying gas temperature at 350◦C, the capillary voltage of 4.0kV, drying gas flow of 12L/m, and the nebulizer pressure of 40 psi(60,61). Of the 60 PhCs tested, 14 compounds were detected in different samples. Their physiochemical properties (molecular weight g/mol, acid dissociation constant (pKa), Log of the octanol/water partition coefficient (Log Kow), water solubility, Chemical Abstracts Services (CAS) number, and chemical formula) are displayed in Table 3.3. 2.5 Statistical Analysis The statistical analysis was performed using Statistical Package for Social Sciences (SPSS version 22) to test for the significance of spatial differences between each point of sample collection with respect to seasonal differences as well. Descriptive statistics were reported in tables and figures. Wilcoxon Signed rank test was used to test the spatial differences between samples of the same campaign. The significance level (p- value) was set at 0.05. Note: The single detection of a compound among all collected samples in both campaigns is disregarded and excluded in data analysis where further validation is required. 18 Chapter Three Results 3.1 West Nablus WWTP performance According to wastewater quality parameters of West Nablus WWTP, during sampling, the plant showed high efficiency in the removal of BOD5, COD, and TSS at a rate of 97 %, 94.5 %, and 97% respectively (Table 3.1). All the parameters comply with the top requirements of the Palestinian mandatory standards for wastewater efficient treatment before effluent discharge into the environment (62). Table 3.1 also includes results of the same wastewater quality parameters of the 2 grab samples collected at the Anabta- Zomar point of sampling of both campaigns. Table 3.1 COD, BOD & TSS parameters of collected wastewater samples obtained from West Nablus WWTP Test Unit First sampling campaign 11 th April Second sampling campaign 19 th Sep. WWTP Inlet WWTP Outlet Anabta surface WW WWTP Inlet WWTP Outlet Anabta surface WW COD mg/l 687 38 78.4 1094 60 157 pH * 7.76 7.69 NA1 7.8 7.59 8.11 Conductivity µS/cm 1438 1360 NA 1730 1717 1724 TSS mg/l 118 0 16 504 24 50 BOD5 mg/l 543 12 11.4 461 12 22 1: not available 3.2 Grab Samples and Removal Efficiency In general, the results of the grab sampling campaigns showed consistency for the majority of detected PhCs and some heterogeneity in some others. In total, 14 PhCs were detected from different classes. Those can be described as follows. First grab sampling campaign results Figure 3.1 displays the findings of the first garb sampling campaign, which was undertaken in April 2022. A set of 10 PhCs were found in total for all sampling locations. Of which, 9 PhCs were detected in the WWTP inlet, 10 PhCs were found in the WWTP outlet, and both sampling locations shared 8 PhCs out of 10. 19 Figure 3.1 Summary of different PhCs detected (µg/L) in the first sampling campaign of April/2022 In comparison to the concentrations of the 10 PhCs detected in the WWTP outlet sample and 9 PhCs WWTP inlet sample, the removal efficiencies of West Nablus WWTP per PhC compound detected are presented in Figure 3.4. There was a significant difference between the inlet and outlet samples results of the first campaign (Wilcoxon signed rank test, p = 0.025), which in turn reflects the efficiency of west Nablus WWTP in average removal of detected PhCs. Whereas West Nablus WWTP displayed different efficiencies which can be summarized as follow. Complete removal was seen only for CIP 100%, followed by ~80% for Trimethoprim (TMP), 72 % for SXM, 62.4 % for OFX, 50 % for Sulfapyridine (SP), 36.2 % for Lincomycin (LCM), 31 % for CBZ, 5.6% for Pyrimethamine (PYR), and negative removal of -2.7 % for DIC. Interestingly, West Nablus WWTP treatment revealed the presence of trace amounts of Sulfaguanidine (SGD) and Sulfaquinoxaline (SQX) in the outlet sample where they 0 1 2 3 4 5 6 7 8 Ciprofloxacin Ofloxacin Sulfamethoxazole Diclofenac Carbamazepine Sulfapyridine Pyrimethamine Trimethoprim Lincomycin Erythromycin Sulfaguanidine Sulfaquinoxaline Concentration (µg/L) 20 were absent in the inlet sample. In addition, Diclofenac residual level increased slightly to reach (0.961 µg/L) in the outlet sample. A significant spatial difference was found between the concentrations of the PhCs in the WWTP effluent sample and the concentrations of detected PhCs in the wastewater sample in Wadi Zomar at the Anabta sampling location (Wilcoxon signed rank test, p = 0.016). It also detected the presence of Erythromycin (E) in trace concentration (0.022 µg/L) which was absent from the previous two grab samples. This in turn suggests the introduction of new raw wastewater prior to this sampling location. SGD wasn’t detected in this sample. Second sampling campaign results The results of the second grab sampling campaign done in September/2022 are presented in Figure 7. A total of 11 PhCs are detected in all autumn samples for all sampling locations. 11 PhCs are detected in the WWTP inlet sample, 8 PhCs are detected in the WWTP outlet sample and 9 PhCs were detected in the surface wastewater in Wadi Zomar at Anabta point of sampling. 7 PhCs out of 11 are in common between all samples. There was a significant difference in the concentrations of detected PhCs between the WWTP inlet and outlet autumn samples (Wilcoxon Signed rank test, p = 0.033). The removal efficiency of west Nablus WWTP regarding the detected PhCs in the outlet sample of the second campaign in comparison to the inlet results is in Figure 3.4. 21 Figure 3.2 Summary of different PhCs (µg/L) detected in second sampling campaign among wastewater samples The significant removal efficiency observed in the first campaign is again present in the second campaign. The WWTP showed complete removal of 4 PhCs; SQX, TMP, Flumequine (FLU), and Testosterone, and very high removal for CIP (94%). While it showed fairly good removal capacity for SXM (78 %), PYR (76%), and SP (74%). OFX and CBZ were removed at a rate of 66.5 % and 59%, respectively. Whereas DIC, interestingly, displayed a higher level of detection than what was present in the inlet sample; the concentration was doubled which resulted in negative removal efficiency of -105%. 0 1 2 3 4 5 6 7 8 Carbamazepine Ofloxacin Sulfamethoxazole Diclofenac Pyrimethamine Sulfapyridine Ciprofloxacin Testosterone Trimethoprim Sulfaquinoxaline Flumequine Lincomycin Concentration (µg/L) Treated surface WW (µg/L) WWTP outlet (µg/L) WWTP Inlet (µg/L) 22 Figure 3.3 West Nablus WWTP removal efficiency (%) in both sampling campaigns Figure 3.4 details the variable removal patterns by west Nablus WWTP observed in both campaigns with respect to detected PhCs. A better removal is noticed in the autumn campaign (brown line) than what’s noticed in the spring campaign (blue line). Negative removal is observed for DIC in both campaigns, however, it was higher during the autumn. Complete removal was noticed only for CIP in the spring sample and for TMP in the autumn. 9 PhCs are detected in the treated surface wastewater sample collected from wadi Zomar in Anabta in the autumn. The highest concentrations are in descending order: DIC (0.436 µg/L), CBZ (0.4 µg/L), SXM (0.257 µg/L), OFX (0.121 µg/L), and CIP (0.54 µg/L). FLU and TMP are detected in trace levels, 0.019 and 0.014 µg/L respectively. However, there was no significant spatial difference between the detected CBZ CIP DIC FLU LCM OFX PYR SXM SP SQX T TMP WWTP removal % 31.3 100.0 -2.7 0.0 36.2 62.4 5.6 72.0 50.0 0.0 0.0 79.3 WWTP removal % 58.5 93.9 -105. 100.0 0.0 66.5 75.8 77.6 74.2 100.0 100.0 100.0 Average 44.9 97.0 -53.8 100.0 36.2 64.5 40.7 74.8 62.1 100.0 100.0 89.7 CBZ CIP DIC FLU LCM OFX PYR SXM SP SQX T TMP -150.0 -100.0 -50.0 0.0 50.0 100.0 150.0 W es t N ab lu s W W T P R em o va l % WWTP removal % WWTP removal % Average 23 concentrations of PhCs in the WWTP outlet sample and treated surface wastewater in wadi Zomar at the Anabta sampling site of the autumn sample (Wilcoxon signed rank test, p = 0.646). 3.3 POCIS Results The POCISs revealed the presence of 4 PhCs which are displayed in Figure 10. CBZ had the highest concentration detected followed by DIC, SXM, and CIP in descending order respectively. The POCISs are merely done as screening tests for the presence or absence of any PhCs in groundwater at Anabta. Figure 3.4 PhCs detected in POCIS sampler (ng/POCIS) deployed at Anabta drinking groundwater reservoir in April/2022 for 28 day 188.3 5.8 7.2 6.7 Carbamazepine Diclofenac Sulfamethoxazole Ciprofloxacin 24 Table 3.2 Summary of Grab Sampling Campaigns results Inlet WWTP 1 (µg/L) Outlet WWTP (µg/L) Treated surface WW 2 (µg/L) WWTP average removal efficiency % PhCs F ir st sa m p li n g S ec o n d sa m p li n g F ir st sa m p li n g S ec o n d sa m p li n g F ir st sa m p li n g S ec o n d sa m p li n g Carbamazepine 0.806 1.044 0.554 0.433 0.529 0.4 44.9 Ciprofloxacin 8.043 0.312 0 0.019 0 0.054 97.0 Diclofenac 0.936 0.498 0.961 1.021 0.872 0.436 -53.8 Erythromycin 0 0 0 0 0.022 0 0.00 Flumequine 0 0.029 0 0 0 0.019 100.00 Lincomycin 0.058 0 0.037 0.01 0.015 0 36.2 Ofloxacin 2.67 0.824 1.002 0.276 0.584 0.121 64.5 Pyrimethamine 0.232 0.422 0.219 0.102 0.045 0.09 40.7 Sulfaguanidine 0 0 0.007 0 0 0 0.00 Sulfamethoxazole 0.95 0.818 0.266 0.183 0.143 0.257 74.8 Sulfapyridine 0.266 0.365 0.133 0.094 0.037 0.078 62.1 Sulfaquinoxaline 0 0.031 0.013 0 0.011 0 100 Testosterone 0 0.09 0 0 0 0 100.00 Trimethoprim 0.092 0.055 0.019 0 0.021 0.014 89.7 1 WWTP: Wastewater Treatment Plant, 2 WW: wastewater, 3 ND: Not-detected (below the detection limit of 0.005 µg/L) 25 Table 3.3 Physiochemical properties of detected pharmaceuticals Pharmaceutical Molecular Weight g/mol pKa 1 Log Kow 2 Water solubility (mg/L) CAS 3 Registry Number Chemical Formula Carbamazepine 4 236.3 13.9 2.45 18.0 298-46-4 C15H12N2O Diclofenac4 296.1 4.15 4.51 2.37 15307-86-5 C14H11Cl2NO2 Sulfamethoxazole4 253.3 pKa1: 1.6 pKa2: 5.7 0.48 610 723-46-6 C10H11N3O3S Ciprofloxacin4 331.34 acidic 6.09 basic 8.74 0.28 3 * 105 85721-33-1 C17H18FN3O3 Erythromycin4 733.9 8.9 3.06 F4.2 114-07-8 C37H67NO13 Lincomycin4 406.5 7.6 0.2 927 154-21-2 C18H34N2O6S Ofloxacin4 361.4 pKa1 5.97 pKa2 9.28 -0.39 1.08 * 104 82419-36-1 C18H20FN3O4 Pyrimethamine4 248.71 7.34 2.69 10 58-14-0 C12H13ClN4 Sulfaguanidine4 214.25 5pKa1: 2.21 pKa2: 11.97 -0.99 2.6 * 105 57-67-0 C7H10N4O2S Sulfapyridine4 249.3 8.43 0.35 268 144-83-2 C11H11N3O2S Sulfaquinoxaline4 300.34 5.1 1.68 7.5 59-40-5 C14H12N4O2S Trimethoprim4 290.3 7.12 0.91 400 738-70-5 C14H18N4O3 Flumequine4 261.3 6.5 1.6 2190 42835-25-6 C14H12FNO3 Testosterone4 288.4 19.09 3.32 23.4 58-22-0 C19H28O2 1pKa: acid dissociation constant, 2 Log Kow: Log of the octanol/water partition coefficient, 3CAS Chemical Abstracts Services, 4 Parameters are adopted from PubChem, and ChemSpider,5 as reported by Zrnˇci´c et al. 2015(63) 26 3.4 Antibiotics dispensary data The collected dispensary data from all participating hospitals are in figure 3.6 Data were collected for the period 1/1/2021 – 28/2/2022. Figure 3.5 Cumulative quantities of Antibiotics (in grams) administered for patients from included hospitals of the time period 1/1/2021-28/2/2022. The above listed antibiotics are the top 15 antibiotics prescribed in the participated hospitals in cumulative order and the next (Amikacin, Levofloxacin, Cephalexin, Amoxicilin, Gentamicin, Teigoplanin, Erythromycin tab, doxycycline tab, Ertapenem, Tigecycline, Chlorampenicol, Moxifloxacin and Nitrofurantoin tab) had a prescribed quantities in the range < 5000 gm in the same studied period. Cumulative quantities highlighted in red (18412 and 7403 gm) belong to the two classes of antibiotics which were detected in this study Ciprofloxacin and Trimethoprim- Sulfamethoxazole, respectively. [18412* [7402.98]* 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 P ip er ac il li n -T az o b ac ta m C o -a m o x y cl av C ef tr ia x o n e C ef u ro x im e V an co m y ci n M er o p en em C ef az o li n C ef ta zi d im e C o li st in e C ip ro fl o x ac in M et ro n id az o le A m p ic il li n T ri m et h o p ri m - … C li n d am y ci n A zi th ro m y ci n A m ik ac in L ev o fl o x ac in C ef o ta x im e C ep h al ex in A m o x ic il li n G en ta m ic in T ei g o p la n in I V E ry th ro m y ci n t ab D o x y cy cl in t ab E rt ap en em T ig ec y cl in e IV C h lo ra m p h en ic o l M o x if lo x ac in N it ro fu ra n to in t ab Q u an ti ti es in g m 27 3.5 Promoting AMR development We estimated the RQ (MEC/lowest PNEC value) according to the antibiotics class where threshold values were developed (Table 3.4). Not all detected antibiotics here in our study have a corresponding threshold value by which an RQ can be calculated. Ofloxacin residual concentrations in WWTP influent, effluent, and surface wastewater of both campaigns are found to pose risk for AMR development concerning the bacterial community of the WWTP and the environment (RQ>1). The residual concentrations of OFX exceed its respective PNEC of 0.5 µg/L. Ciprofloxacin concentration in the WWTP influent poses risk for resistance development for the bacterial community employed in the WWTP in both campaigns (RQ>1). As the detected residues in both influent samples are greater than the PNEC of 0.06 µg/L. For the remaining antibiotics with no corresponding PNEC value, a conservative threshold of 0.05 µg/L is used as guided by the AMR Industry Alliance. Sulfapyridine and Pyrimethamine are found to have a RQ > 1. The measured concentrations of the following samples: WWTP influent and effluent samples of both campaigns and the treated surface WW sample of the autumn campaign are found in concentrations greater than 0.05 µg/L. 28 Table 3.4 Comparison between PNEC-MIC values provided by Bengtsson-Palme, Larsson et al. (52) and PNEC-ENV by Le page et al. (63) as endorsed by the AMR Industry Alliance and average concentrations of detected antibiotics in the WWTP effluents of the present study Antibiotic PNEC- MIC (µg/L) PNEC- ENV (µg/L) Lowest value Inlet concentration Spring (µg/L) Autumn (µg/L) Outlet concentration Spring (µg/L) Autumn (µg/L) Ciprofloxacin 0.06 0.45 0.06 8.043 0.312 0 0.019 Flumequine NA 0.25 0.25 0 0.029 0 0 Lincomycin 2.0 0.81 0.81 0.058 0 0.037 0.01 Ofloxacin 0.5 10.0 0.50 2.67 0.824 1.002 0.276 Sulfamethoxazole 16.0 6.6 6.60 0.95 0.818 0.266 0.183 Trimethoprim 0.50 928.00 0.50 0.092 0.055 0.019 0 PNEC-MIC, ENV: Predicted no environmental concentration-minimum inhibitory concentration, environmental, respectively.µg/L: microgram per liter. 29 Chapter Four Discussions and Conclusions 4.1 Occurrence of PhCs in wastewater samples and West Nablus WWTP removal efficiency The investigation into the occurrence of antibiotics and other pharmaceuticals in sample locations from the study area showed the presence of several PhC residues with varied concentrations. Results also reflected a significant spatial difference between the three sampling locations in the spring season. The autumn campaign results showed only significant removal efficiency by the WWTP of detected PhCs residues and no significant spatial difference between the concentrations of detected PhCs residues detected in west Nablus WWTP effluent and Anabta-Zomar point of sampling. The results showed that the highest detection level for single PhC in wastewater samples was Ciprofloxacin (8.043 µg/L) which was detected in the influent of the first sampling campaign (spring), while Diclofenac of (1.021 µg/L) was detected in the effluent during the autumn season. In addition, Ofloxacin of (1.002 µg/L) was detected highly during the spring season in the effluent. Moreover, Diclofenac was found to be the highest residue of (0.872 µg/L) in surface-treated wastewater of the spring season sample. We also noticed that OFX residual concentrations detected may aid in the development of OFX resistance. Figure 4.1 Residual concentrations of different pharmaceutical in the influen west Nablus WWTP and Anabta The variation of detected PhCs and antibiotics ( quantitatively, can be accredited to: the variation in administration and disposal of detected PhCs concerning the study area, the different physiochemical properties of detected PhCs play a role in their persistence, west Nablus WWTP removal efficiency, abiotic- and biodegradation contributions, different tributaries along Wadi Zomar, agricultural and veterinary activities in the catchment area and the sporadic raw sewerage effluents along Wadi Zomar. The fate of each pharmaceutical compound is determined by a single or a combination of the aforementioned factors. All hospitals in Nablus send their untreated medical wastewater to the West Nablus WWTP, where it is assumed that the concentration of antibiotic residues will be higher than in any other locality. It somewhat reflects the local protocols used in various 0% 10% 20% CZP CIP DCL ERY FLU LIN OFL PYR SGD SXM SPD SQX TET TMP Spring 30 Residual concentrations of different pharmaceutical in the influent, the effluent of the west Nablus WWTP and Anabta-Zomar in both sampling campaigns The variation of detected PhCs and antibiotics (Figure 4.1), both qualitatively and quantitatively, can be accredited to: the variation in administration and disposal of detected PhCs concerning the study area, the different physiochemical properties of detected PhCs play a role in their persistence, west Nablus WWTP removal efficiency, and biodegradation contributions, different tributaries along Wadi Zomar, agricultural and veterinary activities in the catchment area and the sporadic raw sewerage effluents along Wadi Zomar. The fate of each pharmaceutical compound is determined by a single or a combination of the aforementioned factors. us send their untreated medical wastewater to the West Nablus WWTP, where it is assumed that the concentration of antibiotic residues will be higher than in any other locality. It somewhat reflects the local protocols used in various % 30% 40% 50% 60% 70% 80 Spring Spring Spring Summer Summer t, the effluent of the ), both qualitatively and quantitatively, can be accredited to: the variation in administration and disposal of the detected PhCs concerning the study area, the different physiochemical properties of detected PhCs play a role in their persistence, west Nablus WWTP removal efficiency, and biodegradation contributions, different tributaries along Wadi Zomar, agricultural and veterinary activities in the catchment area and the sporadic raw sewerage effluents along Wadi Zomar. The fate of each pharmaceutical compound is determined by a single or a combination of the aforementioned factors. us send their untreated medical wastewater to the West Nablus WWTP, where it is assumed that the concentration of antibiotic residues will be higher than in any other locality. It somewhat reflects the local protocols used in various 80% 90% 100% Summer 31 institutions as well as the trend. However, it is interesting to note that some classes of the found antibiotics here are not among the antibiotics that are typically recommended for human administration, according to data gathered from hospitals in the study area. This instead implies that additional sources, such as veterinary and industrial operations, are involved in this. Sadly, local laws governing the use of antibiotics in veterinary practice are not well-applied. (64). The majority of detected PhCs here share one property that favors their detection in raw wastewater; up to a certain degree, a given dose is excreted, species-dependent, unchanged in the urinary system or through the biliary system based on the route of administration(5) (Table 4.1). Adding to that, most PhCs noticed here are commonly used between both the human and veterinary field. Furthermore, the variation of PhCs residuals here can be attributed to seasonal variations of infections or medical conditions which require the administration of those specific PhCs. This is noticeable by the markedly high CIP residue in the WWTP influent of the spring sample in comparison to the autumn sample (26 times higher). The same is observed for DIC and OFX (2 and 3 times) higher than the autumn residues, respectively. Pitarch et al. reported the seasonal variation in the detection of different classes of PhCs in wastewater favoring the winter season(17). As this mainly could be attributed to more respiratory infections in the winter season requiring medication. In addition to less water consumption; together deliver a precipitation effect. 32 Table 4.1 General overview regarding the most frequently detected pharmaceutical compounds in our study modified after PhCs Acronym Class Indication Excretion Carbamazepine CBZ Anticonvulsant Epilepsy, Trigeminal Neuralgia, and Psychosis Metabolites in urine, small percentage as unchanged parent drug Diclofenac DIC Analgesic Pain relief and fever reducer In humans: 60-70% excreted in the urine. 30% in feces as metabolites and very little as the unchanged parent compound. In animals: species dependent. Ciprofloxacin CIP Antibiotics Fluoroquinolones Broad spectrum antibiotic. Respiratory tract infections Urinary tract infections. Orally: 40-50% in an unchanged active drug, 15% as metabolites in urine. Parenterally: up to 70% unchanged and 10% as metabolites within 24 hours. Ofloxacin OFX Antibiotics Fluoroquinolones Broad spectrum antibiotic. urinary tract infections and soft tissue infections colibacillosis in poultry Orally: 65-80% unchanged in the urine within 48 hours while biliary excretion accounts for 4-8% only. Flumequine FLU Antibiotics Fluoroquinolones In humans: uncomplicated urinary tract infection. Animals: enteric infections in food animals and treatment of bacterial infections in farmed fish Species dependent: in veal calves 3.2–6.5% excreted in the urine unchanged. In eels: long elimination time periods. Lincomycin LCM Antibiotics Lincosamide Gram-positive cocci and anaerobes, except Enterococcus spp. In animals: Bacterial pyoderma Arthritis and pedal osteomyelitis In Humans: no longer used. In animal: Variable elimination rate in dairy cattle, bovine, buffalo calves, pigs, cats, and chickens. Trimethoprim- Sulfamethoxazole TMP - SXM Antibiotics Sulfonamides Broad spectrum antibiotic used in combination with trimethoprim in synergy. Genitourinary, respiratory, and gastrointestinal tract infections. Prophylaxis (in HIV patients) and treatment of Pneumocystis carinii pneumonia. SXM: Up to 30% excreted in urine as unchanged parent drug. TMP: almost completely excreted unchanged in the urine. Sulfapyridine SP Antibiotics Sulfonamides As a moiety of Sulfasalazine for Rheumatoid Arthritis Anti-coccidiosis affecting chickens Mainly unchanged in feces Sulfaquinoxaline SQX Antibiotics Sulfonamides Anti-coccidiosis agent in poultry. Anticoagulant-based rodenticide. Mainly excreted in urine Pyrimethamine PYR Antiparasitic In human: chloroquine-resistant malaria and toxoplasma gondii. In animals: anti-coccidiosis. Mainly excreted through the biliary system in the feces. Note: (83,105,114–120,106–113) 33 In 2022, the world started to recover from the COVID-19 pandemic where the prescription of antibiotics in COVID-19 patients was high even though evidence for co- bacterial infection was low. Where FQs ranked first among all studied regions as the antibiotic class of choice prescribed for COVID-19 patients(65). The World Health Organization (WHO) categorized FQs among the watch list upholding the antibiotics with the highest potential for resistance development. The watch list should be used in specific infectious diagnoses only and not as an empirical therapy (66). The residual concentrations detected for CIP and OFX in this study and their association with AMR development are discussed later. In contrast, PYR, CBZ, and SP had a higher detection level in the autumn sample (0.422, 1.044, and 0.365 µg/L), respectively, relative to the spring sample (0.232, 0.806, and 0.266 µg/L), respectively. This could be related to the unregulated and injudicious use of antibiotics and PhCs detected here where a great deal of PhCs are considered OTC. However, according to the authors’ knowledge, the available scientific evidence indicating any seasonal variations in local infections which may indicate the administration of the most frequently detected PhCs here is absent. LCM detection was common among wastewater samples collected in the spring season with concentrations of (0.058, 0.037, and 0.015 µg/L) in WWTP influent, WWTP effluent, and surface wastewater in Wadi Zomar at Anabta sampling point, respectively. While LCM was detected in the WWTP effluent sample of 0.010 µg/L of the autumn campaign. LCM common detection in all samples of one campaign (spring) relative to the other campaign (autumn), can be attributed to the seasonality of dairy products manufactured by cattle and sheep in the spring (67), where Lincomycin is used to treat mastitis (68). LCM is excreted mainly unmetabolized in the urine, feces, and bile and detected in manure, which can be easily dispersed into the environment(69). The detection of LCM is mainly attributed to its free solubility in aquatic mediums of alkaline pH and thus its resistance to removal. In addition, this could be attributed to the negative competition effect of inorganic cationic molecules (present in Wadi Zomar) on the sorption ability of Lincomycin onto cationic sites in the soil. Furthermore, the poor 34 sorption ability is enhanced by the increase in pH values beyond the disassociation constant range (70). Another finding concerning the autumn campaign is the residual concentrations detected for Flumequine. It was detected in the WWTP inlet sample and surface wastewater with a residual concentration of 0.029 µg/L and 0.019 µg/L, respectively. Despite the fact it wasn’t detected in the WWTP outlet autumn sample, surface wastewater showed its presence reflecting an introductory source other than the treated wastewater discharged from the WWTP. Wadi Zomar has many scattered tributaries that deliver untreated wastewater that transports along the Wadi. Whereas the local administration of Flumequine is unknown, this FQ is mainly used for enteric infections in animals and in fish farming and also reported to have mutagenic side effects (71). The WWTP removal efficiency is variable for each compound here (Figure ). CIP removal in both sampling campaigns (> 90%), while OFX, SP, SXM, and TMP maintained a removal efficiency in both seasons of a rate of >50%. As opposed to DIC which showed increased concentrations in effluent samples of both campaigns. The main mechanisms by which PhCs are removed can be attributed to adsorption, biodegradation, and /or photolysis. Ciprofloxacin removal was 100% in the spring and 94% in the autumn. This removal efficiency by the WWTP can be the result of adsorption to sludge employed in the WWTP or the biodegradation by microorganisms used in the WWTP operations. Bisognin et al. reported the isolation of OFX and CIP from both the effluent and the sludge of a large WWTP in southern Brazil, with more than 83% of CIP and OFX removed (72). Carbamazepine was detected in all wastewater samples investigated here with the inability of the WWTP to remove it completely. Furthermore, the removal rate was poor in the first campaign (31%) and increased in the autumn campaign to (59%). Moderate removal can be reached by adsorption into particles of sludge and soil (73). This explains the relatively stable concentration of CBZ in both the WWTP effluent and Wadi Zomar surface wastewater samples of both sampling campaigns. West Nablus 35 WWTP removal efficiency for CBZ here may be considered a better removal yield in comparison to what Al-Mashaqbeh et al. reported for CBZ (22.5%) in Jordan (2018) (12), and even better removal yield than what was previously reported by Odeh et al. (2015) (< 20%) in the same study area (74). CBZ is a hydrophobic, non-polar compound with very poor solubility in aquatic mediums, hence the establishment of its persistence and stability in various ecosystems. CBZ stability in the environment is noticed as previous research in the same study area detected the occurrence of CBZ in concentrations three times higher than those detected in our study (average 3.046 µg/L) (74). In addition, a recent study in the investigated area concluded the detection of CBZ, SXM, and TMP in wastewater samples in concentrations higher than our study here (Samhan, S. PADUCO Conference; 2022; ANN). It’s concluded that the WWTP is solely responsible for CBZ removal here whereas the removal down Wadi Zomar is rather negligible (Figure 3.3). CBZ is poorly degraded in nature. It mainly depend on temperature. Tixier et al. estimated CBZ half-life in the environment by around 63 days in autumn days(75), while another study concluded that CBZ is persistent to abiotic photodegradation and predicted its half-life time of around 100 days in winter (76). Taking CBZ as an example in terms of water solubility and the impact it enforces on the fate of a chemical compound in the aquatic mediums and its resistance to biodegradation, OFX removal in both campaigns is rather stable. With a removal of 62 % in the spring season and 67% in the autumn season. This can be attributed to the fact that the solubility of OFX in aqueous mediums is pH dependent (2.8 – 5.5) (77), and the operating conditions of the WWTP lie in the alkaline spectrum (pH >7)(56), indicating OFX resistance to solubility in such alkaline mediums and the partial average removal efficiency is due to adsorption to particles either the sludge or suspended solids in the WW or biodegradation by microorganisms in the WWTP (72). SP was detected in all wastewater samples with concentrations ranging from 0.037 µg/L in the spring surface wastewater to 0.365 µg/L in the autumn WWTP influent sample. 36 The WWTP delivered variable removal capacity in both campaigns (50% in the spring vs. 74 % in the autumn). The stability of SP in WW samples and resistance to degradation can be attributed to its moderate water solubility (268 mg/L) and resistance to biodegradation by bacteria at the WWTP. Indeed, the removal ability is mainly related to the adsorptive ability of SP onto an organic matter which is influenced by the pH, type of organic content of the medium they are detected at (78). The photolytic properties of sulfonamides additionally enhance the removal due to sun exposure (estimated half-life 2.6 hours)(20) as its removal improves in the autumn sample. However, for SP and PYR, the removal down Wadi Zomar in the autumn sampling is marginal at 17%, and 11% in comparison to the removal observed in the spring sample at 72% and 79%, respectively. This variation due to dilutional effect delivered by the rainy season in the spring campaign which led to false higher removal percentages in the spring campaign. Or due to the sporadic sewerage discharges containing investigated PhCs along Wadi Zomar past the WWTP effluent point; as the wastewater quality parameters at the Anabta sampling location displayed increased levels in the autumn (Table (4.1). TMP, SXM, CIP, and FLU are found to have higher concentrations in the surface wastewater autumn sample than what was measured in the WWTP effluent autumn sample. This reflects the introduction of additional residues of PhCs from different tributaries along Wadi Zomar directly after the discharge from West Nablus WWTP. Where one may argue that a concentrated effect due to the high autumn temperatures is the causative reason for such difference, which is probably unlikely given the fact that SXM is photo-labile. Due to sun exposure, SXM residual concentration in the surface wastewater autumn sample is expected to be less than what it is detected in the WWTP effluent autumn sample, yet it was 37% more in the surface wastewater autumn sample than what it was detected in the WWTP autumn effluent sample (18). Another photo-labile compound here is DIC, and its removal down Wadi Zomar is seen to be best in the autumn season (57%) whereas the spring season delivered a rather poor removal down Wadi Zomar (9%). This can be attributed to the rapid photodegradation 37 of DIC in the autumn times as it’s estimated to be less than an hour with more sunlight exposure (Wadi Zomar is 100% sun-exposed). While in winter, it’s estimated that DIC undergoes photodegradation in around 5 days (76). It is noted that West Nablus WWTP displayed not effective removal about (36 %) for LCM during the first sampling campaign. The biodegradability of LCM (with an estimated half-life of 30 hours) is thought to be the driving force for its removal as long as the initial concentration didn’t exceed the MIC of the present bacteria(80). Wastewater treatment relies on microorganisms’ ability to break down various organic compounds present in the wastewater to reach a specific threshold before effluent wastewater is discharged into an environmental body. The WWTP is required to uphold certain threshold values as standards. As noted in this study, the WWTP has variable removal efficiencies to the measured PhCs. On the other hand, some PhCs may exert a negative pressure on wastewater purification processes. Carucci et al. stated the significant inhibitory effect of Lincomycin present in wastewater on the nitrification treatment employed in the WWTP, which negatively reflects on the WWTP's overall performance(81). The nitrification treatment is thought to be the main mechanism by which TMP is removed (82), a process by which if the present LCM level had any inhibitory effect, the removal of TMP is further rendered. This requires additional studies regarding the matrix effect of wastewater as a stimulant/inhibitory for various PhCs (81). Sulfamethoxazole and trimethoprim are found to be detected in all wastewater samples (only TMP is absent from the WWTP autumn effluent sample). The residual concentration of SXM is higher than TMP in all measured samples. In the medical field, the administration of this combination is fixed at a ratio of 5 SXM: 1 TMP to combat antimicrobial resistance (83). Nonetheless, the detected ratio in wastewater samples exceeds the aforementioned ratio. It’s detected in 10:1 in the WWTP spring influent sample and 14:1 in its respective autumn sample. Furthermore, their measured residual levels are higher in the spring WWTP influent sample than in the autumn WWTP influent sample. This reflects the 38 higher administration of SXM/TMP in the spring season in comparison to the autumn season. In our opinion, we conclude that the scientific evidence regarding the local infectious status requiring the prescription of this antimicrobial is absent. Moreover, the higher ratios detected favoring SXM that can be attributed to the sole administration of SXM alone and not in the form of combination therapy. The removal efficiency delivered by West Nablus WWTP is high for both SXM and TMP and in both campaigns. This is explained due to their biodegradability by microorganisms employed in the WWTP. As other sources of carbon are depleted in wastewater treatment, pharmaceuticals here are utilized as an energy source instead (82). Interestingly though, antimicrobial resistance to TMP/SXM is exploited by some bacterial spp as a vital source for growth, as it’s the case with TMP/SXM-dependent Pseudomonas. This antibiotic-dependent strain is isolated from a cystic fibrosis patient. The patient was put on this regimen as prophylaxis, along which resistance has evolved to become the reason for this strain to survive and cause harm to the patient(84). This could be found in the bacteria-rich working environment of the WWTP with chronic exposure to different antibiotics. This theory can’t be verified unless microbiological studies with susceptibility profiles are well conducted. SXM-TMP combination therapy is commonly administered concurrently, and together with their stability and persistence in wastewater, the detection of this combination in wastewater can be exploited as a pollution marker (85). Sustained surveillance systems would permit the allocation of advanced wastewater treatment processes where most needed according to the major source contributing to their introduction in the wastewater. A proposed approach for the removal of PhCs from wastewater prior to discharge into the environment is the use of laboratory-engineered, bacterial spp. capable of degrading a selection of antimicrobials as needed(82). As if using AMR backward for better overall health using an environmentally friendlybacterial spp. However, a delicate 39 balance should be considered when such a solution is implemented as spreading this resistant bacterium has additional side effects we are hardly able to control. The detected residues in the WWTP effluent have demonstrated a decreased level for all PhCs than what was detected in the WWTP influent in both campaigns except for Diclofenac. The residual level of DIC in the WWTP influent of the spring sample (0.936 µg/L) has increased to (0.961 µg/L) in the WWTP effluent, a marginal increment for the spring effluent sample yet it was further noticed in the autumn campaign. DIC has increased from (0.498 µg/L) in the WWTP autumn influent to (1.021 µg/L) in the WWTP autumn effluent sample. This can be attributed to several factors of which the increase in effluent concentrations of some PhCs is assigned to the reverse conversion of the drug metabolites into its parent compound through the different enzymatic processes and microbial activity in the plant rather than the introduction of new DIC residues (86) According to research results here, this effect ranged from almost negligible to doubling the initial concentration detected. Adding to that, the complex matrix present in the wastewater influences the stability and removal of existing chemical compounds (87). The operating conditions of the WWTP highly influence the removal of DIC from wastewater. Which requires acidic pH and prolonged contact time with sludge (87). Whereas the working conditions of the WWTP are in the range of neutral to slightly alkaline. In addition, DIC is resistant to biodegradation by bacteria when other sources of carbon are present(88), as it’s the case with high COD levels present in influents of the WWTP. Another finding regarding the detection of PhCs in the effluent samples which were not detected in the influent samples is LCM of (0.010 µg/L) in the autumn campaign and Sulfaguanidine and Sulfaquinoxaline of (0.007 and 0.013 µg/L), respectively, in the spring effluent sample. Whereas the discrepancy seen in both campaigns could be attributed to either the nature of the momentary effect in grab sampling or it can be attributed to the reversal of the glucuronic conjugate antibiotic metabolites to its parent drug during treatment(89). The PhCs SGD and SQX are veterinary sulfonamides used 40 as anticoccidials in poultry and cattle. SGD and SQX physiochemical properties indicated by studies on sorption into different types of soil are governed by the increase in organic content and decreased with increased pH and ionic strength (90,91). Overall, we noticed that the removal efficiency by the WWTP in the autumn campaign was higher than the spring campaign. This could be related to the higher temperature of the autumn season which acted as a more accelerating factor for better removal efficiency as well (79). As the physiochemical structures of any chemical compound determine its fate in the fluid it’s being detected at, the fluid movement plays a role as well in the adsorption and desorption process at sediment materials covering both flanks of Wadi Zomar or suspended particles in the WWTP. With water movement, the adsorbed PhC can be liberated from the adsorbate material and get re-introduced into Wadi Zomar flow rather than adding new residues of the PhCs. The findings of our study are consistence with what is reported elsewhere in the literature (92). 4.2 Occurrence of PhCs in groundwater The PhCs investigated in this study were also screened for in groundwater as we considered it the vertical end point that residues can reach. Four classes are detected that comply with the most frequently detected PhCs in the wastewater samples. Those are CBZ, DIC, CIP, and SXM (188.3, 5.8, 6.7, and 7.2 ng/POCIS, respectively). The groundwater and tap water, on a global scale, are found to harbor 16 different PhCs in several countries as well. Where Carbamazepine, Diclofenac, Sulfamethoxazole, Ibuprofen, and Naproxen are most frequently detected worldwide and in different aquatic and soil compartments (21). The WWTP effluent in both campaigns had residual concentrations of the aforementioned PhCs (except CIP in the spring effluent), their residues are further noticed in the surface wastewater sample at the Anabta-Zomar sampling location indicating the sorption of PhCs into the soil of Wadi Zomar and along it’s both banks. Even though the soil hasn’t been screened for PhCs here, the residuals detected in groundwater imply no other alternative. It’s found that soil polluted with high loads of 41 PhCs in a certain area is accompanied by contaminated groundwater in the same area as well (24). The location of the treated surface wastewater sample in Anabta-Zomar is between two groundwater wells utilized as regular groundwater (Figure ). Water recharge is crucial in maintaining the groundwater sources in this area as it’s the sole source of viable groundwater. In addition, Wadi Zomar natural path is situated in close proximity to the aquifers of the studied location, combining that with the hydrological structure of the area, the discharged-WWTP effluent, the sporadic discharge of untreated sewage prior and at Anabta village and the agricultural activity of the area; the leach of different contaminants in groundwater sample is observed. For example, in India, Ciprofloxacin was detected (among others) in high alarming levels in water wells (>1 µg/L) in villages near the effluent of a WWTP for 90 bulk drug manufacturers(93). Emphasizing the importance of location in risk assessment. The significant removal detected in the spring campaign between the WWTP effluent and Anabta-Zomar point of sampling could be attributed to rain events with dilutional effects in addition to sorption and degradation along the Wadi. Rainfall events aided with the natural hydrological structure of Wadi Zomar in the studied area (57,59),and the chronic endurance of such PhCs making groundwater pollution a question of time; Piston-effect. It’s reasonable to deduce the presence of a tight link between the occurrence of PhCs in treated wastewater and surface water. The leach of different classes of PhCs here is assigned to the adsorption potential of each compound and is resistant to biodegradation (94). SXM, CBZ, and DIC share low water solubility (Table 3.3). The ingestion of water contaminated with different classes of antibiotics here raises concerns regarding the potential health effects for humans and animals. Concerns are at a point here regarding the risk associated with the wide usage of groundwater by all study area population age groups. The very young and very old are considered vulnerable sub-populations and more sensitive to any change in the gut microbiome. 42 This change is associated with daily intake of pharmaceutically polluted groundwater (27). The detection of different classes of PhCs in treated surface wastewater and groundwater is mainly attributed to wastewater effluents in the receiving environment. The literature review points towards the application of further treatment of wastewater for removal of different pharmaceutical residuals including antibiotics (76,95–99) which requires the application of advance infrastructure for the WWTP (Oxidation &/ Ozonation coupled with UV application), membrane bioreactors or simply solar systems (100). However, this is governed by the status of the local area regarding which PhCs are in great need of removal. As groundwater is contaminated with the most frequently detected PhCs here, urgent mitigation protocols are required. 4.3 Potential Health effects of antibiotics measured environmental concentrations and the development of AMR The main critical health effect of high residual concentrations of antibiotics in the environment is the development of antimicrobial resistance. Table 3.4 presents the detected residuals of antibiotics in wastewater influent and effluent samples of our study here for comparison against the threshold values of PNEC – MIC developed by Bengtsson-Palme, Larsson et al.(45) and PNEC – ENV developed by Le page et a.l(46). The lowest threshold value of both PNECs is considered the critical value to determine AMR risk on conservative grounds. We estimated the RQ (MEC/lowest PNEC value) according to the antibiotics class where threshold values were developed. Not all detected antibiotics here in our study have a corresponding threshold value by which an RQ can be calculated (45,46). According to the concentrations measured in the WWTP influent samples, we noticed a potential for AMR development in the bacterial community involved in the WWTP conventional operations employed for CIP and OFX (RQ > 1). The remaining antibiotics with a corresponding PNEC values were found not to constitute any risk for AMR according to the residuals detected (RQ <1). Nonetheless, the chronic effect due to prolonged exposure times is yet needed. 43 CIP of 8.043 µg/L and OFX of 2.67 µg/L in the WWTP spring influent sample and CIP of 0.312 µg/L and OFX of 0.824 µg/L in the WWTP autumn influent sample are all found in concentrations exceeding the lowest PNEC threshold value in both seasons. This alerts for studying the association between the detection of highly alarming residual concentrations of antibiotics and the presence and extent of FQ’s resistant genes/bacteria in effluents and the sludge. As the discharged effluent is being disinfected before releasing it into Wadi Zomar, evading disinfection is still a possibility (40). Water provides a rather supporting medium where resistance can get allocated easily. Furthermore, it supports its persistence and widespread detection(28). The wastewater medium is considered a nutrient-rich habitat for both sensitive and resistant bacteria to proliferate. Horizontal gene transfer can easily happen and mobile genetic elements (plasmids and transposons) can be exchanged in the gene pool. For instance, the high abundance of antibiotics residues (e.g. Macrolides with a range of 0.1-10 ng/L, and FQs range of 0.10- 1000 µg/L) in the influent of a WWTP was found to be significantly positively associated with the genetic material responsible for their resistance(30). Additionally, it’s found that only OFX residual concentration in the WWTP spring effluent sample poses risk for AMR development (RQ >1) against clinical bacteria present in the environment but not a risk for the environmental bacteria itself. The detected spring residual concentration of OFX (1.002 µg/L) exceeds the lowest threshold values for the PNEC-MIC and PNEC-ENV which is the PNEC-MIC value (0.5 µg/L). This is in agreement with what Booth et al. concluded in their retrospective study which indicated that OFX was among the most frequently detected antibiotic in concentrations exceeding PNEC-MIC in surface water while CIP ranked first in percentages of analyses exceeding the PNEC-MIC on a global scale (101). This is of importance as Wadi Zomar naturally passes through populated and active agricultural lands on both of its flanks. This is a worrisome point here considering the potential threat of effluents in disseminating resistance to the environment impacting human and animal health and agricultural life forms. A previous study yielded the detection and enhanced survival of clinically relevant bacterial spp. (E. coli, 44 Pseudomonas, Klebsiella and Enterococcus) in sediments of Wadi Zomar which precludes the possibility of detection of resistant strains as well(59). Moreover, resistant gram-negative bacteria were detected in in-house, greywater samples collected from farms in the West Bank, Palestine, which Craddock et al. had investigated. In their study, resistance was reported mainly for ampicillin followed by TMP-SXT, tetracycline, and cefazolin in decreasing order. Additionally, an alarming finding is the detection of multi-drug resistance isolates (~8%) as well. The effluent which was found to harbor resistant isolates is used traditionally for crop irrigation to cover the water scarcity (29). The RQ of OFX is more than 1 in spring surface wastewater when compared to the residual concentration.. The residual concentration of (0.584 µg/L) in the surface wastewater sample at the Zomar-Anabta point of sampling reflects a possibility for resistance development and dissemination. In comparison to the autumn sample, the RQ < 1. The slightly increased removal capacity delivered by the WWTP in the autumn (66%) has aided in decreasing the residual concentration in the WWTP effluent prior to discharge to the concentration of (0.276 µg/L). This level, if not increased, to our belief and based on the literature threshold values, holds no risk for AMR in the bacterial community of Wadi Zomar in the autumn. On the other hand, the removal effect for OFX down Wadi Zomar in the autumn additionally lowered OFX residue by a 56%. Rendering the ability for AMR emergence. Combining the detection of residual antibiotics here in levels promoting selection pressure in WWTP effluent samples and the physiochemical properties that permits antibiotics to sustain their persistence in the wastewater samples collected with enough exposure time, then the possibility for AMR genes/bacteria is high. Furthermore, in Hanna et al. study, they found that the ESBL-coding gene and plasmid-mediated quinolone-resistance gene are the most frequently detected in river water and sediment over a three-year study(102). Guo et al. reported the vast abundance of ARGs in dewatered sludge, where ARGs copies were detected in the final effluent with varying concentrations in comparison to the influent. ARGs are influenced by both the bacterial community and the residual concentration of antibiotics present (30). 45 In this study, the microbiological profile of the wastewater samples collected wasn’t investigated yet the detection of antibiotics itself might be indirectly an indicator for the presence of AMR bacteria and AMR genes have-given their common excretion source (3,7,11). The RQ for SP and PYR is found to be > 1 in the WWTP influent and effluent samples of both campaigns. This was noticed when comparing the MEC of SP and PYR in the influent and effluent samples with a default threshold value of (0.05 µg/L) proposed by the AMR Industry Alliance (47). However, their role in promoting AMR is unknown; health problems arising from the present trace residuals are lagging, and the development of target threshold values is still ongoing. In our opinion, this suggests a more strict and protective approach for adhering to a target threshold of zero µg/L before any effluent is discharged into the environment to avoid any harm. Even though there is a theoretically propo