Assessing biosolids quality at the Mu'ta-Mazar wastewater treatment plant for the years 2016–2019

Sludge is generated during stages of the wastewater treatment process as a collection of a variety of compounds. Part of these compounds has agricultural value (organic matter, nitrogen, phosphorus and potassium, calcium, sulphur, and magnesium). While other parts are considered harmful to the environment such as heavy metals, organic pollutants, and pathogens. The quality of sludge depends on the original composition of the treated water, and also on the type of treatments carried out on the wastewater during the whole stages. The aim of treating the sludge before disposal or recycling is to reduce its water content, to decrease the possibility of the harmful fermentation process in the environment, and to eliminate the pathogens. There are several treatment processes such as thickening, dewatering, stabilization and disinfection, and thermal drying. The sludge may undergo several types of treatment depending on its characteristics and the sought reuse and/or disposal options (Andersen 2001; Aubain et al. 2002).

There is an exacerbated global problem with disposing of the huge amount of generated sludge by the wastewater treatment plants ‘WWTPs’. Sustainable management options, such as recycling, is very crucial at this time to utilize from the produced biosolids in beneficial applications, and to decrease the cost of the disposal process and the related environmental impact of the accumulated sludge. Jordan has been transitioning from stabilization ponds to mechanized wastewater treatment plants. The majority of these plants are secondary wastewater treatment plants, achieving nutrient and pathogen reduction by activated waste sludge processes, which produce treated solid residuals that are of Class B microbial quality (US-EPA 1994). Currently, there are no specific plans or firm regulations governing the beneficial use of generated biosolids in Jordan. Therefore, treated sludge/biosolids accumulated at domestic wastewater treatments plants (DWWTPs) are uncontrolled and inconsistently processed, which may have negative impacts on human health and the surrounding environment. Key stakeholders in Jordan are seeking sustainable methods for safe and economic recycling of biosolids for beneficial reuse.

Worldwide, EU and USA have developed advanced analyses of the risks and benefits of the different use and disposal options. Many other countries have built their understanding and policies from this foundation of knowledge and experience that integrated their local needs and conditions into the policies, laws, and regulations. In general, the USA has adopted the concept of risk assessment in its environmental regulations. The federal wastewater sludge regulations (40 CFR Part 503) are based on an extensive risk assessment completed in the early 1990s (Part and Provisions).

Regarding Pharmaceuticals and Personal Care Products (PPCPs), no regulation exists in the United States for PPCPs contained in sludge, and a need for more information on the occurrence of and risk from these compounds has been noted by the National Research Council of the National Academies of the United States (National Academies of Sciences, Engineering, and Medicine 2002). In contrast, the EU has adopted a precautionary approach or a no-net-degradation approach in some of its environmental policies and applies. Because of this, for example, the EU is well ahead of the USA in researching and phasing out chemicals of concern in personal care and commercial products, such as certain PDBEs (flame retardants), ingredients of personal care products, pharmaceuticals, some industrial chemicals, etc. These actions are partly driven by the interest in protecting the quality of treated sludge recycled to soils.

Despite these broader environmental policy differences, the USA and EU have created similar wastewater sludge regulations. The EU's central initiative on wastewater sludge management is the 1986 Directive for the Use of Sewage Sludge in Agriculture (which has seen additional development over the past twenty years). The EU directive and the US federal regulations both address pathogen reduction, the potential for accumulation of persistent pollutants in soils (heavy metals and persistent chemicals), and the application of appropriate amounts of nutrients. One notable difference is that the EU directive generally limits rates of applications of treated sludge to lower levels than those allowed in the US regulations.

In Jordan, standards for reuse of biosolids are established by the Jordan Standards and Metrology Organization (JSMO). Until recently, Jordanian Standard (JS): 1145/2016 has been the standard for recycling of biosolids in Jordan and was adopted from the 1993 version of the CFR 503 standards developed by the United States Environmental Protection Agency (USEPA 1993). The JS for the reuse and disposal of treated sludge No. (1145/2016) established numeric limits, management practices, and operational standards to protect public health and the environment. Concentrations of 10 heavy metals (As, Cr, Cd, Cu, Pb, Hg, Mo, Ni, Se, and Zn) are typically monitored in sewage sludge according to the JS (1145/2016), which classifies treated sludge according to some microbiological and physical aspects into three types (as shown in Table 1); Type I and Type II can be used as a soil amendment and Type III is to be disposed of in sanitary landfills. Hence, the JS: 1145/2016 standard is more rigid today than the 1999 modified CFR 503 standard since the latter has 12 alternative treatment processes that can be utilized in the treatment process to generate Class A-designated biosolids, while the Jordanian standards only have four. The goal of these treatment processes is to reduce pathogen densities below specified detection limits for three types of organisms: Salmonella sp. <3 (MPN/4 g total solids), enteric viruses <1 (PFU ‘plaque-forming unit’/4 g total solids), and helminths <1 (viable organism/4 g total solids).

In municipal wastewater, there is the potential for residual contaminants to remain in the sludge following wastewater treatment. In the past, analytical methods were limited, especially for trace analyses of complex environmental samples. In 2007, the release of US EPA method 1694 (6) for the analysis of PPCPs in various matrices afforded the opportunity to analyze sludge samples using a standardized protocol. Following, many surveys and studies have confirmed the presence of pharmaceutical compounds in municipal wastewater and effluents (US-EPA 1989). Therefore, there is a need to review JS (1145/2016) to include the requirement for the analysis of pharmaceutical compounds and any additional toxic pollutants.

In this study, a complete assessment of the sludge qualities in terms of physical, chemical, pharmaceutical, and microbial parameters for three types of sludge samples (thickener, drying bed, and treated organic matter (TOM)) collected during the 2016–2019 period from the Mu'ta-Mazar WWTP was conducted. This study includes an overview of sludge quality and discusses the major findings of the results of the analysis.

A total of 19 sludge samples were collected from the Mu'ta-Mazar WWTP to be analyzed in the laboratories of the Royal Scientific Society of Jordan ‘RSS’ (Amman-Jordan) between June 2016 and October 2019. Nine dewatered sludge composite samples were collected from the drying bed where the sampling was performed in accordance with US EPA guidelines (EPA 1989; US-EPA 1989). In addition, five TOM samples generated from a prototype digestion system (at Mu'ta University) that uses a mixed feed consisting of food wastes and sewage sludge were collected during 2017–2019, while five thickener samples were collected only during 2016 and 2017. The types of collected sludge samples and time points of the collection are summarized in Table 2. The samples were packed on ice and transported to the RSS laboratories for physical, chemical, pharmaceutical, and microbial analysis.

The samples were analyzed for physical and chemical properties following the ‘Standard Methods for the Examination of Water & Wastewater’, online 2011. Previously for the samples of 2016 and 2017, the analysis of pharmaceutical compounds was according to Martín et al. (2010). The pharmaceutical compounds monitored were five nonsteroidal anti-inflammatory drugs (diclofenac, ibuprofen, ketoprofen, naproxen and salicylic acid), two antibiotics (sulfamethoxazole and trimethoprim), a ß-blocker (propranolol), two lipid regulators (clofibric acid and gemfibrozil), an anti-epileptic drug (carbamazepine), four estrogens (17α-ethinylestradiol, 17ß-estradiol, estriol and estrone), and a nervous stimulant (caffeine). Salicylic acid and clofibric acid are metabolites of pharmaceuticals (acetilsalicylic and clofibrate, respectively) extensively metabolized. Subsequently, and after developing the method at RSS laboratories, analysis of pharmaceutical compounds was conducted based on the ultrasonic-assisted extraction, clean up by Solid-Phase Extraction (SPE) and analytical determination by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) for samples collected in 2018 and 2019 years. Sixty-one pharmaceutical compounds were analyzed. The Limit of Quantification (LOQ) for the tested compounds is shown in Table 3.

Samples were assayed for fecal coliforms using the Most Probable Number (MPN) method as recommended in US-EPA (2010): ‘Fecal Coliforms in Sewage Sludge (Biosolids) by Multiple-Tube Fermentation Using Lauryl Tryptose Broth (LTB) and EC Medium’ (Agency 2006a), and for Salmonella using a modified version of US-EPA (2006), ‘Salmonella in Sewage Sludge (Biosolids) by Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium’ (Agency 2006b). Assays for helminths ova were conducted using a series of sedimentation and flotation steps to isolate viable ova (US-EPA 2003).

Conventional cell culture methods used to detect human enteric viruses are costly and time consuming. The advent of recombinant DNA technology and related fields have made it possible to detect these pathogens in a matter of hours using the polymerase chain reaction (PCR). Therefore, this technique was adopted for the analysis of enteric viruses in sludge samples (the recovery of viruses from wastewater sludge (ASTM Standard D 4994-89 1993); detection of enteroviruses by Reverse-Transcriptase PCR) (Hot et al. 2003; Shaban & Malkawi 2007). The full list of other tested parameters is shown in Table 4.

Together with the traditional and analytical methods, the molecular detection of the antibiotics-resistant genes (ARGs) by PCR assays, it is possible to estimate: the presence of resistance genes, the influence of antibiotic presence in wastewater to gaining resistance against them and the direction of microbial community changes linked with bacterial changeability (Ziembińska-Buczyńska et al. 2015).

Some bacterial species are difficult to be cultivated in the laboratory; in addition to this, some genetic materials are carried by mobile elements inside the sludge culture (e.g. plasmid). Hence, a direct extraction of the bacterial DNA was carried out (Singka et al. 2012). Then, the ARGs were directly detected by PCR method. The selected common resistant genes, the corresponding antibiotic families, and the referenced methodologies are shown in Table 5.

RSS Environmental Laboratories are ISO 9001:2008 and ISO/IEC 17025:2017 certified, and are nationally accredited by the Jordanian Accreditation System (JAS). Moreover, many of the tests (for water and wastewater samples) conducted at the Environmental Laboratories are internationally accredited by the United Kingdom Accreditation Services (UKAS).

For the analysis of sludge samples, lab blanks were analyzed before each sample analysis. A duplicate sample analysis was performed by the laboratory staff for each batch between 7 and 20 samples. In addition to these standard procedures, a blind duplicate was included in the sample set to evaluate analysis precision.

To determine the concentration of heavy metals, validation tools for sludge were analyzed. The validation tools included: accuracy calculations, precision, matrix spike and recovery, linearity studies for each element, Initial Demonstration Capability (IDC), analysis of Certified Reference Materials (CRMs) and previous Proficiency Testing (PT) samples, detection limits, and uncertainty calculations. The appropriate linear or non-linear correlation coefficient for standard concentration to instrument response was ≥0.995. Duplicate samples were used to measure the precision of the analytical process; a minimum of one duplicate with each set of 20 or fewer samples. Relative Percent Difference (%RPD) was less than 10%. Percentage recoveries for fortified samples were between 80 and 120%. The control chart provides a better indication of system performance and was used to assess calibration verification, check standards, precision, and duplicates.

A total of nine composite dewatered sludge samples were collected during 2016–2019. Sludge drying beds, the most widely used method of sludge dewatering, rely on natural evaporation and percolation to dewater the solids (Page et al. 1988). Water removal from the sludge improves the efficiency of subsequent treatment processes, reduces storage volume, and decreases transportation costs. The average solids contents in the sludge sample from the drying bed were 91.6% (Supplementary material).

Volatile Solids (VS) provide an estimate of the readily decomposable organic matter in sludge and are usually expressed as a percentage of total solids. VS are an important determinant of potential odor problems at land application sites. The average Total Volatile Solids (TVS) in the tested dried sludge was 63.2%. The average ratio (TVS/TS) is 0.69; this value was more than the recorded acceptable limit for stabilized sludge (at most 0.6) according to USEPA regulation. A number of treatment processes, including anaerobic digestion, aerobic digestion, alkaline stabilization, and composting, can be used to reduce VS content; and, thus, the potential for odor.

The application of dewatered sludge on land is one of the most effective and attractive methods because it has a relatively high content of nutritive elements such as Ca, Mg, P, N, and organic carbon. The usual amount of phosphorous and nitrogen, which determines the fertilizer's value of sludge for phosphorous and nitrogen are 0.3–5.5% and 0.1–3.5% of sludge, respectively Farzadkia & Taherkhani 2005. According to the results obtained, the average amount of phosphorous and nitrogen were 1.768 and 6.606%, respectively; where the amount of nitrogen was more than the standard and it was considered as a restrictor factor for using the sludge on farm. The average potassium content was 0.613% for the collected dry samples. Some reported results proved that the potassium level in sludge is usually low and can range from 0.02 to 2.645%, but is enough for plant uptake and is still sufficient for crop requirements Sommers 2000.

However, there is a risk that toxic constituents in sludge, such as trace metals and chlorinated hydrocarbons, may accumulate in soil and contaminate ground water, the crops, and enter the food chains (Dacre 1980). Trace elements are found in low concentrations in sludge. The trace elements of interest in sludge are those commonly referred to as ‘heavy metals’. Some of these trace elements (e.g. copper, molybdenum, and zinc) are nutrients needed for plant growth in low concentrations, but all of these elements can be toxic to humans, animals, or plants at high concentrations. Possible hazards associated with a buildup of trace elements in the soil include their potential to cause phytotoxicity (i.e. injury to plants) or to increase the concentration of potentially hazardous substances in the food chain. The JS for the Reuse and Disposal of Treated Sludge No. (JS: 1145/2016) has established standards for the following ten trace elements: arsenic (As), cadmium (Cd), copper (Cu), chromium (Cr), lead (Pb), mercury (Hg), molybdenum (Mo), nickel (Ni), selenium (Se), and zinc (Zn) (Table 1). The levels of the heavy metals examined in the sludge samples from the drying bed were much lower than the limits mentioned in the JS: 1145/2016 (Supplementary material). Therefore, the use of sludge in terms of these elements for mentioned application is permitted. However, these elements have the accumulative property; and therefore, paying attention to the use level of this sludge in particular soils each time during a year and over a year is important. Moreover, other parameters such as soil condition and characteristics should be considered in designing the sludge application rate.

The ARGs in the sludge samples were not detected in most of the drying bed sludge samples except for limited number of genes in May 2017 sample (qnrA3, qnrB1, qnrB4, qnr, mef, and tet B) and July 2019 sample (mef gene); this might be due to the low number of pathogens inside such an ecosystem. The decrease in moisture content after dryness (the average water content in the tested sludge samples was 8.5%) affects the viability of most microorganisms in the sludge sample, as well as denaturing the genetic elements.

Regarding Contaminants of Emerging Concern (CECs), 16 pharmaceutical compounds including nonsteroidal anti-inflammatory drugs, antibiotics, an anti-epileptic drug, a ß-blocker, a nervous stimulant, estrogens and lipid regulators were analyzed according to Martín et al. (2010) for samples collected in 2017 and 2018. The concentration of all tested pharmaceutical compounds was <10 μg/kg in the samples from the drying beds. Regarding samples collected in 2018 and 2019, 61 pharmaceutical compounds were analyzed (Table 3). Although the ARGs were not found in all dry samples, some pharmaceutical compounds were found in different concentrations in the sludge samples from the drying bed of the Mu'ta-Mazar WWTP. Mainly, there were high concentrations of Diclofenac and Carbamazepine in the two sludge samples collected from the drying beds in July and October 2019.

A total of five ‘TOM’ samples were collected and tested from the Mu'ta-Mazar WWTP during 2017–2019. The results of physical, chemical and microbial characteristics of the tested co-digest samples are demonstrated in Supplementary material. The average content of organic matter in the tested samples was (35.62%), and the average VS for the TOM samples was (65.88%), which can be easily converted to methane using the anaerobic process. The TOM was mostly from the semi-continuous reactor where fresh biomass is added on a daily basis. The experiments were continued until gas rate production is constant, but this does not mean that the VS is fully digested (because it is a semi-continuous reactor, not a batch). The elemental analysis of the co-digested samples showed that the heavy metals concentrations were way below the JS (1145/2016)-Type I ceiling limits.

Thousands of compounds with a wide range of chemical structures are used in pharmaceutical products (Halling-Sorensen et al. 1998). Testing ARGs showed that four genes (qnrA3, qnrB4, qnr, and mef) were detected in the TOM sample collected in September 2017, three genes (mef, qnrA3, and qnr) were detected in the TOM sample collected in August 2018, and four genes (qnrA3, qnrB1, qnrB4 and qnr) were detected in the TOM sample collected in October 2019. The detected ARGs are correlated with the resistance mechanisms in two different antimicrobial families: Fluoroquinolones and Macrolide. Besides, the concentration of all (60) tested pharmaceutical compounds was <20 ng/kg in the TOM samples collected in 2018. But, seven pharmaceutical compounds (Ciprofloxacin, Carbamazepine, Diclofenac, Ofloxacin, Pyrimethamine, Sulfapyridine, and Progesterone) were detected in the TOM sample collected in October 2019.

Regarding the microbial characteristics of the TOM samples, the counts of Fecal Coliforms were below 1,000 MPN/g in all samples, except for the sample collected in October 2019, the count was 5.4 × 10³MPN/g. Salmonella species were detected in three samples (September 2017, June and August 2018) with an average amount of 52.54 MPN/4 g. IPN eggs were <1 egg/4 g for all samples. Enteric viruses were not detected in any of the samples. Based on the JS No. (1145/2016) criteria for well-stabilized sludge, the maximum amount of fecal coliform density is 1,000 and 2 × 10⁶ MPN/g for Type I and Type II, respectively, while the count of Salmonella spp. must be <3 MPN/4 g. Consequently, the co-digested sludge samples (except the sample of July 2017) can be classified as Type II according to the JS (1145/2016).

A total of five sludge samples from the thickener of the Mu'ta-Mazar WWTP were collected and tested during only 2016–2017. The results of physical, chemical and microbial characteristics of the tested sludge samples from the thickener are demonstrated in Supplementary material. The tested samples of sewage sludge had a high percentage of organic matter (41.14%) and the average VS for the sludge sample was 71.24%, which can be easily converted to methane using the anaerobic process.

According to the results obtained (Supplementary material), the average amount of phosphorous and nitrogen in the two sludge samples were 1.98 and 12.91%, respectively; where the amount of nitrogen was more than the limit set in the Standard and it is considered as a restrictor factor for using the sludge in the farm. The average potassium content was 1.86% for the samples. The levels of the heavy metals examined in the sludge samples from the thickener were much lower than the limits mentioned in the JS: 1145/2016-Type I.

However, the average fecal coliform density in dried solid (g) was more than the Standard maximum level of 1,000 MPN based on the JS: 1145/2016-Type I. The count of Salmonella spp. exceeded the standard limit (<3 MPN/4 g) in samples (June 2016 and March 2017). Hence, the sludge generated from the thickener at the Mu'ta-Mazar WWTP could be classified as Type II according to the JS (1145/2016).

Furthermore, 16 pharmaceutical compounds including nonsteroidal anti-inflammatory drugs, antibiotics, an anti-epileptic drug, a ß-blocker, a nervous stimulant, estrogens and lipid regulators were analyzed for collected sludge samples from Thickener in 2016 and 2017. The concentration of all tested pharmaceutical compounds was <10 μg/kg in all tested samples from the thickener (analyzed according to Martín et al. 2010).

For the sludge sample collected in July 2017, five ARGs were detected; qnrB1, qnr, erm, mef, and tet B. These five ARGs are correlated with the resistance mechanisms in four different antimicrobial families: Fluoroquinolones, Macrolide, Lincosamide, and Tetracycline, respectively. The ARG test was not carried out on 2016 samples.

In the last few years, major construction of modern activated sludge treatment plants has taken place in Jordan. This has resulted in the production of large quantities of biosolids, which must be either disposed or recycled through land application. Jordan is an arid country with soils of low fertility in many areas and land application of biosolids would provide a major benefit to the agricultural community. However, it is important that biosolids be used in a safe manner and meet certain standards to protect the environment and human health. In this regard, the characterization (contaminants and pathogens composition) for the biosolids collected from the drying beds in the Mu'ta-Mazar WWTP have been done during 4 consecutive years (2016–2019).

The two components in sludge that are technically and economically feasible to recycle are nutrients (primarily nitrogen (N) and phosphorus (P)) and energy (carbon) Tyagi & Lo 2013. As sludge contains organic matter, energy can be recovered while treating it. In the tested sludge samples from the drying beds, there was a considerable amount of nutrients, especially P and N. However, P is fast becoming the most significant nutrient due to depleting sources.

The content of heavy metals was proved to be not of real concern at this WWTP. It is known that the presence of high amounts of heavy metals in sludge is one of the most significant reasons that restricted its application in agricultural lands. The tested sludge samples in this investigation were within the existing limits set for concentrations of heavy metals in treated sludge according to JS No. (1145/2016) for restricted land applications in Jordan. However, prior to sludge application onto land, a composite sludge sample should be analyzed for the metals and the level of pathogenic organisms as specified by JS No. (1145/2016). Additionally, composite soil samples should also be collected from the land and analyzed for heavy metals. Thus, the quantity of sludge applied onto the land and the application rate can be calculated.

On the other hand, trace elements are important for activating and maintaining the enzyme activities of anaerobic microorganisms. The availability of certain trace elements has been shown to strongly impact the biogas production. Thus, although organic food waste has high-energy potential it usually has a lower concentration of trace elements than sludge, which are required for anaerobic digestion (Kubaska et al. 2010). In this regard, wastewater sludge might be advantageous when co-digested with food waste by overcoming trace element limitations (Vlachopoulou 2010).

The microbiological characteristics were varying between the collected samples for the three collected types of sludge. The average fecal coliform density in dried solid (g) was higher than the standard maximum level of 1,000 MPN based on the JS: 1145/2016-Type I, in one sample of each TOM and drying bed collected sludge samples. The Salmonella spp. was detected in some tested samples as well. The presence of Salmonella species was confirmed using molecular techniques.

Previously, it was demonstrated that the risks of infection from Salmonella found in wastewater residuals are essentially dependent on the Salmonella concentrations within the biosolids, and the subsequent exposure of individuals (Gerba et al. 2008). For Class B (Type II) biosolids (Supplementary material includes a description of Classes A and B), Salmonella concentrations determined over a 5-year period were found to be low with a mean of 10⁵ MPN/g. The risk of infection from direct contact with Salmonella within Class B (Type II) biosolids were close to the United States Environmental Protection Agency's (US EPA) recommended upper limits and therefore caution should be applied. However, based on the conservative assumptions made in this risk characterization, US EPA's regulations for land application of Class B (Type II) biosolids appear to be appropriate. Risks from potentially aerosolized organisms from Class B (Type II) biosolids were low. In contrast, concentrations of Salmonella within Class A (Type I) biosolids following regrowth approached 10⁶/g. In this case, risks of infection from direct contact or aerosolized organisms were highly significant. Since regrowth in Class A (Type I) biosolids has been shown to occur under saturated anaerobic conditions, great care must be taken when Class A (Type I) biosolids are stored prior to land application. Specifically, such biosolids should be covered to prevent saturated anaerobic conditions that could occur following rainfall events. Without such regrowth events, risks of infection from biosolid-borne Salmonella are low.

Based on the study of Gerba et al. (2008), we conclude that it is highly unlikely that Salmonella infections will occur from land applied Type I or II treated sludge (biosolids). However, risks become significant if Type I biosolids are stored anaerobically, i.e. saturated, prior to land application.

IPN eggs were <1 egg/4 g of dry sludge. In addition, the enteric viruses were not detected in either the thickener, TOM or the sludge samples from the drying beds.

On the other hand, the key outcome of this study is a robust and validated analytical method for the analysis of a selection of pharmaceutical compounds in sludge samples. Previously, the method of analysis for samples 2016 and 2017 was according to Martín et al. (2010) and tested only 16 compounds with a Limit of Detection (LOD) of <10 μg/kg. While the method of analysis for samples collected in 2018 and 2019 was based on ultrasonic-assisted extraction, clean up by SPE and analytical determination by LC-MS/MS. The method provided an overall recovery of between 70 and 110% and a LOD of <20 ng/kg, and tested around 60 compounds. The newly adopted method helped in the detection of some pharmaceutical compounds in 2019 samples such as Ciprofloxacin, Carbamazepine, Diclofenac, Ofloxacin, Pyrimethamine, Sulfapyridine, and Progesterone, which were detected in the TOM and drying bed samples collected in 2019.

The results of this study have shown that metal concentrations in the biosolids at the activated sludge plant easily met the current JS (1145/2016)-Type I in most drying bed sludge samples. Besides, the results of the microbiological analyses have shown that drying beds in the Mu'ta-Mazar WWTP are effective biosolids stabilization methods.

Biosolids drying beds are physical treatments that can be considered an effective way of biosolids treatment. However, the performance of these beds depends on the physical condition of the bed, the type of biosolids, temperature, drying period, and meteorological conditions.

Great thanks and appreciation to the Water & Environment Centre and Testing Laboratories Sector at Royal Scientific Society (Amman-Jordan) for their dedicated work in collecting and analyzing samples, respectively. We would like to extend our gratitude to the Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH for their ongoing support of our programs, as well as the German Federal Ministry for Economic Cooperation and Development (BMZ) for funding this research.

The study was implemented by the Royal Scientific Society (RSS) of Jordan with financial support from the Deutsche Gesellschaft fuer Internationale Zusammenarbeit (GIZ) GmbH in the context of Decentralized Integrated Sludge Management-DISM project (14.2483.7–001.00). The contents of this manuscript are the sole responsibility of RSS and does not necessarily reflect the views of the GIZ.

N.D.A.-H. designed the study, supervised the activities of the study, managed and supervised the technical and financial aspects of the study, interpreted the results and wrote the manuscript. M.M.A. coordinated the sampling events, conducted the molecular tests (PCR and ARG), analyzed the data, interpreted the results, and wrote the manuscript. B.O.H. contributed in designing the study, editing the manuscript, and interpretation of the results.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.