Detection of Antibiotics in Drinking Water Treatment Plants in Baghdad City, Iraq

Mahmood, Ansam R.; Al-Haideri, Halah H.; Hassan, Fikrat M.

doi:https://doi.org/10.1155/2019/7851354

Advances in Public Health

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Abstract Introduction Methods Results and Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2019 | Article ID 7851354 | https://doi.org/10.1155/2019/7851354

Detection of Antibiotics in Drinking Water Treatment Plants in Baghdad City, Iraq

Ansam R. Mahmood,¹Halah H. Al-Haideri,²and Fikrat M. Hassan²

Academic Editor: Jagdish Khubchandani

Received02 Mar 2018

Revised30 Oct 2018

Accepted03 Dec 2018

Published01 Jan 2019

Abstract

Persistence of antibiotics in the aquatic environment has raised concerns regarding their potential influence on potable water quality and human health. This study analyzes the presence of antibiotics in potable water from two treatment plants in Baghdad City. The collected samples were separated using a solid-phase extraction method with hydrophilic-lipophilic balance (HLB) cartridge before being analyzed. The detected antibiotics in the raw and finished drinking water were analyzed and assessed using high-performance liquid chromatography (HPLC), with fluorometric detector and UV detector. The results confirmed that different antibiotics including fluoroquinolones and B-lactams were detected in the raw and finished water. The most frequently detected antibiotics were ciprofloxacin with highest concentration of 1.270 μg L⁻¹ in the raw water of Al-Wihda plant, whereas the highest concentration of levofloxacin was 0.177 μg L⁻¹, while amoxicillin was not detected in this plant. In contrast, ciprofloxacin was found in both raw water and finished water of Al-Rasheed plant and recorded highest concentration of 1.344 and 1.312 μg L⁻¹, respectively. Moreover, the residual amount of levofloxacin in the raw water was up to 0.414 μg L⁻¹, whereas amoxicillin was shown to be the most detectable drug in the raw water of Al-Rasheed plant, with a concentration of 1.50 μg L⁻¹. The results of this study revealed the existence of antibiotic drugs in raw and finished water and should be included in the Iraqi standard for drinking water quality assessment.

1. Introduction

Wide-spread distribution and persistence of resistant pathogenic bacteria and environmental pollution have led to the use of a wide spectrum of drug compounds for treatment. Active drug substances are chemical compounds that can damage the environment but have received little attention as potential environmental pollutants, particularly in the developing countries [1, 2]. Furthermore, their effects on the biotic environment can be serious. Antibiotics are produced in large quantities as they are widely used in veterinary therapy as well [3, 4]. The significant existence of antibiotics in the environment can be attributed to multiple factors including the release of unabsorbed antibiotics by animals and humans into the water stream. Moreover, most of the residual unused antibiotics from medicinal practices, laboratories, factories, residential, and commercial institutes are discarded into common waste water [5–7]. The detection of traces of drugs in the drinking water sources of various countries is due to the improper discarded of these drugs in our water supplies, which then affect directly and indirectly the environment and human [8]. The presence of these drugs at low levels in the environment is more than enough to have a harmful and deleterious impact; particularly, the long exposure time of pregnant women to the trace amount of these antibiotics may resulted in malformation of infants and children [9, 10]. Although antibiotics are considered as a harmful factor for human health, its existence in the environment is critical factor of contamination [11], so if these drugs are not disposed properly, they will lead to emergence of resistance bacteria in the environment. On the other hand, some unused antibiotics are usually thrown to the sewage system to be eliminated; however, if the drugs are not eliminated or degraded, they will directly move to the surface water, ground water, and drinking water [12]. Although there are different ways to eliminate the antibiotics from the sewage system, not all drugs were completely treated, then it may release into the natural waters. According to the high water solubility and weak degradability of antibiotics, it can easily pass across the membrane through filtration steps and reach to the drinking water [13]. The risk of existing of antibiotics in wastewater treatment has gained more attention, because it may lead to development of the antibiotic-resistant bacteria in nature, as well as in different environmental conditions [14].

Antibiotics have attracted significant attention due to the aggravation of resistant pathogens. Most notably, fluoroquinolones such as ciprofloxacin (CIP) and levofloxacin (LEV) and B-lactam amoxicillin (AMO) are wide-spectrum antibiotics that are effective against a broad range of infectious diseases. It was recorded that ciprofloxacin is able to inhibits the growth of multidrug-resistance microorganism, which exert resistant pattern to other antibiotics such as macrolide, beta-lactam and aminoglycosides [15]. Drugs like clofibric acid and diclofenac have been detected in different water sources including surface water, ground water, river, and sewage water in Berlin [16–18]. Drug contamination in drinking water units in Canada and United States has been confirmed [19, 20]. In addition, the presence of low levels of antibiotics has been associated with increased appearance of resistant pathogens that affect human health. Many studies have revealed the presence of antibiotic-resistant bacteria in drinking water supplies in the United States of America and Europe [21, 22]. In addition, similar composition of veterinary and human antibiotics can create cross-resistance [23, 24]. The antibiotic contamination of water also represents challenges for the water industry and water resource planning. Therefore, considerable attention has been paid to the presence of antibiotics in the environment and various aquatic media such as industrial wastewater, ground water, municipal wastewater, hospital wastewater, drinking water, and surface water [25–27]. As long as antibiotics are considered as environmental pollutants, they require robust preclinical and clinical approaches to assess their efficiency before commercialization. In UK, Australia, and US, the human risk assessments of antibiotics have been reviewed, where the concentration of antibiotics in drinking water is generally more than 1000-fold below the minimum therapeutic dose (MTD), which refers to the lowest active dose [28]. The extraction of antibiotics from different samples by solid-phase extraction techniques has been reported in many literatures [29, 30], as well as the identification and quantification methods using capillary electrophoresis, thin layer chromatography, and high-performance liquid chromatography [31–33]. The most recent advance in analytical techniques to investigate the antibiotics in different samples is HPLC technique [34]. Evaluating the presence of antibiotics in drinking water leads to realizing the critical development of antibiotic-resistant bacteria in the environment. The present study is conducted to investigate the presence of antibiotics in raw and finished drinking water in treatment plants in Iraq, which can serve as a reference to develop new strategies for drinking water treatment in the future and to determine the level of antibiotics at both beginning and end of water treatment.

2. Material and Methods

2.1. Study Area

Baghdad is a big city with a population exceeding seven million. Several factories and hospitals drain their wastewater illegally to the river. Many direct and indirect sources are responsible for the contamination of the river drainage area in Bagdad [26, 35]. In this study, two water treatment plants were selected for water sample collection: Al-Rasheed and Al-Wihda plants (Figure 1). Al-Rasheed plant is located in the south of Baghdad City within the site of multiple wastewater discharges and the waste of factories. It was firstly initiated in 1963, and the amount of production is estimated by 61.668 m³/day. This plant provides Al-Zufraniah area and Al-Rasheed district. Al-Wihda plant is located on the eastern bank of Tigris River and acts as a source to feed the surrounding area such as Al-Karrada area and the industrial era. The total capacity is 75.000 m³, where the available capacity is around 73.000 m³. Al-Wihda plant was firstly constructed in 1959.

2.2. Chemicals and Materials

Three antibiotic standards, including B-lactam antibiotic amoxicillin and fluoroquinolones (ciprofloxacin and levofloxacin) synthesized by Sigma-Aldrich (Germany), were utilized. LC grade methanol and deionised distil water (DD) were procured from the local market. Analytical grade reagents included sulfuric acid (purity 99%) from Fluke and sodium thiosulfate (Na₂S₂O₃) from Sigma-Aldrich. Formic acid, acetonitrile from Sigma-Aldrich hydrophilic-lipophilic balance (HLB) oasis cartridges (200 mg 6 mL⁻¹; Milford, MA, Waters, USA), was used. Furthermore, 0.45 μL cellulose acetate Millipore filters (Millifilter, Milford, USA) and Whatman filters (Sigma-Aldrich) were used.

2.3. Sample Collection

A total of 36 samples, including 18 raw water samples from the Al-Wihda (W1) and Al-Rasheed water treatment plants (R1), and 18 finished water samples from the Al-Wihda (W3) and Al-Rasheed (R3) water treatment plants were collected from May to July 2017. Samples of 1 liter were collected from the raw and finished water before its entry in the distribution system from water treatment facilities. Sodium azide was added to the raw sample to eliminate microbial activity. Sodium thiosulfate was added to quench the residual chlorine disinfectants in the finished water at the time of sample collection. After preparation, samples were collected in 1 L amber glass bottles, which were kept on ice during transportation, and stored at 4°C and extracted within 5 days of collection.

All samples were tested for their turbidity, temperature and pH prior to HPLC determination. The pH of samples was measured using pH meter, and the temperature of each sample was measured throughout the study period and taken into consideration the variability of temperatures within the season. Turbidity was measured to determine the cloudiness of samples and indicates the existing of suspended particles such as clay, organic particles, or microorganisms.

2.4. Sample Preparation: Solid-Phase Extraction (SPE)

The solid-phase extract was prepared according to the manufacturer’s protocol with some modifications. The Oasis HLB cartridge was preconditioned before use by adding 4 mL MeOH and 6 mL distil water (DW). The pH of the raw water samples and finished water samples was adjusted to 6 and 3, respectively. One liter of the sample was filtered using 0.45 μm Millipore filter to remove any impurities. The water sample was passed through the cartridge at flow rate of 5–8 mL /min using a vacuum extraction manifold. Next, 10 mL of ultra-pure water was used to wash the cartridge, which was subsequently air-dried for 5 min. Acidified methanol (10 mL of MeOH, 3 mL of 0.5 N HCL) was used to elute the analyte into a glass test tube. The extracts were reduced to a volume of 100 μL under a gentle flow of nitrogen, and the volume was increased to 250 μL using a mix of water/methanol (9:1). The extracts were filtered through 0.45 μm filters, transferred to auto sampler vials, stored at −15°C, and were analyzed within one week.

2.5. Analytical Methods

2.5.1. Devices

Devices used included the LC high gradient pump system (S1122, Sykam, Germany), auto sampler, degasser, heated oven, and fluorometric detector (RF) detector.

Chemical analysis of antibiotic content in the water samples was performed according to [36, 37]. Briefly, mobile phase A constituted of 0.1% formic acid and mobile phase B constituted of acetonitrile (v/v 30:70). Flow rate was 0.7 mL/min. RF detector with excitation wavelength of 278 nm; and emission wavelength of 450 nm for ciprofloxacin and levofloxacin, and UV detector of 230 nm for amoxicillin, was used. Chromatography was performed using 50 μL of the injected liquid at ambient temperature on a Pursuit Column C-18 (250 mm × 4.6 mm, 10 μm).

2.5.2. Standard Stock Solution

Stock solutions of 10 mg L⁻¹ were prepared by weighing the pure substances (ciprofloxacin, levofloxacin, or amoxicillin) and dissolving them in DD water [38, 39]. These solutions were used to prepare all working solutions and standards. All solutions were stored in amber glass vials at −20°C. The standards (0.05, 0.15, 0.25, and 0.5 μg L⁻¹) were prepared by diluting portions of the stock with DD water, and filtered using 0.45 μm filters. About 50 μL of the sample was injected into the HPLC column. The determination of antibiotics in the environmental samples was conducted by comparing the HPLC peaks with the corresponding standard solution peaks. The concentration was calculated based on the internal standards and retention times.

2.5.3. Quantification

Quantification procedure suggested by Hussain et al. [7] was employed. Analyte quantification was based on external calibration curves, which were plotted on the ratio of the peak area of the analytes signal for highest intensity and concentration.

2.6. LOD Analysis

In order to determine the detection limit of antibiotics in raw and finished water, signal-to-noise approach was performed according to [40]. Briefly, samples were measured and the signal with known concentrations of analyte was compared with the relative blank samples. The minimum concentration was established at which the analyte can be reliably detected. The signal-to-noise ratio 2:1 was used in this study, which is acceptable to estimate the detection limit. The LOD was expressed as 3X standard deviation according to the SD of the response and slop.

2.7. Statistical Analysis

The statistical analysis was performed using GraphPad Prism program and SAS-software system 2012 to analyze data of detected antibiotics in the study area by HPLC. The least significant difference (LSD) test (ANOVA) was used to determine the significant difference between detected drugs in raw and finished water plants at p< 0.05.

3. Results and Discussion

All the 36 collected samples (18 of Al-Wihda plant and 18 of Al-Rasheed plant) were subjected to HPLC to qualitative evaluation of remaining antibiotics in raw and finished water. The samples of raw and finished water of Al-Wihda and Al-Rasheed plants were coded as W1, W3, R1, and R3, respectively, and the turbidity, nature, Tm, and pH were determined and depicted in Table 1, which indicated that all samples were neutral in nature and temperature were ranged about 25-27°C. The results showed that all target analytes were detected in the drinking water treatments. Fluoroquinolones (ciprofloxacin and levofloxacin) were frequently detected in all water samples. Ciprofloxacin was detected in 11 out of 36 water samples, with maximum concentration at W1 (1.270 μg L⁻¹); nine samples confirmed the presence of levofloxacin, with a maximum concentration of 0.177 μg L⁻¹ at W1. Amoxicillin was found only in one sample in R1 at the concentration of 1.50 μg L⁻¹. Ciprofloxacin was the most frequently detected in both water treatment plants (Table 2 and Figure 2). Interestingly, the average BOD removal rate of Al-Wihda plant was zero, whereas in Al-Rasheed plant the average BOD removal rate was about 11.8509, thus indicating the efficient water treatment strategy in the former plant; this result was noticed by [41]. The occurrence of antibiotics in environments particularly in aquatic environment is under consideration due to the probable emergence of drug-resistant bacteria [40]. In such case, many studies reported that the development of drug-resistance bacteria is due not only to the presence of drugs in the aquatic environment, but also to the density of resistance bacteria, antibiotic exposure time, and nutrient enriched environment [14, 42]. Long exposure time to subtherapeutic dose of antibiotics potentially leads to creating suitable conditions for resistance gene transfer [42]. The presence of high concentrations of ciprofloxacin in the drinking water used for human consumption indicates the influence of wastewater discharge. Thus, the possibility and extent of pollution from animal sources should not be underestimated. For example, enrofloxacin is used only for animal treatments, but can be metabolized to ciprofloxacin under certain conditions [43, 44]. In this study, amoxicillin was detected in only one sample; this is because that B-lactam drug has chemically unstable rings that readily undergo hydrolysis and may not be detected easily in the finished water [10, 26]. Furthermore, many studies documented the prevalence of amoxicillin and ciprofloxacin resistant bacteria in river water, waste water, and drinking water [16, 45, 46].

Limit of detection either is defined as the characterization of the general chemical ingredients at very low concentration or recognizes only one chemical measurements process [40]. In the present study the LOD was calculated for detected drugs in all samples, combined with chemical formula and molecular weight, which are listed in Table 3.

Table 2 illustrates the concentration of antibiotics detected from all samples of raw and finished water of Al-Whida plant. Statistical analysis revealed significant differences (P<0.05) between antibiotics and the sites of Al-Wihda plant (W1, W3) for all antibiotics except amoxicillin.

Samples from the Al-Rasheed plant sites R1 and R3 showed higher levels of antibiotics than W1 and W3 of the Al-Wihda plant. Some antibiotics like ciprofloxacin, levofloxacin, and amoxicillin are not completely removed and can become a major health hazard if they persist in the finished water even at low levels (ng L⁻¹), due to the possible development of antibiotic-resistant bacteria in potable water (Figure 3) [29, 47]. The statistical analysis showed significant differences at (P<0.05) between antibiotics and sites of Al-Rasheed plant (R1, R3) for all antibiotics except ciprofloxacin, as seen in Table 4.

The study of [48] showed that nine commonly used antibiotics (like ciprofloxacin and amoxicillin) were tested for fate in wastewater and reclaimed water from two regions in US. They find that the concentration of detected antibiotics in influent samples is more than that of effluent samples. Moreover, the concentrations of these drugs did not change through passing from treatment plant to irrigation site, so antibiotics persist in treated municipal water at low concentration.

Unlike Al-Wihda plant, amoxicillin is detected in higher concentrations compared the levofloxacin and ciprofloxacin in raw and finished water in Al-Rasheed plants (Table 4). It was reported that the concentration of beta-lactam drugs in inlet pond is significantly higher than that of UV-treatment samples (p-value = 0.0006). This increase is might be due to the aeration of these ponds [48]. The degradability of antibiotics may be varied and relies on different treatment technologies as well as storage conditions, where some antibiotics like ciprofloxacin and ofloxacin are shown to be genotoxic [33].

The occurrence and existence of medically active drugs in raw and finished water in this study is consistent with other studies. Most of the active drugs are not fully degraded or consumed, may remain in drinking water in tiny amounts, and can lead to water contamination. In UK, it was reported that clofibric acid was indicated in the river and surface water [49], whereas it was detected in wastewater, ground, and drinking water at a maximum concentration of 70, 165, and 270 ng L⁻¹, respectively, in Germany [15–17]. Another study showed that macrolide antibiotics like tylosin were found in drinking water in UK, Germany, and Italy, at a variable concentration of 10, 6, and 1.7 ng L⁻¹ [15, 50, 51]. Furthermore, carbapenem was also detected in the sewage system in Italy at 3.5 ng L⁻¹ [50]. Moreover, the persistence of diclofenac and diazepam in wastewater, surface water, irrigation water, and drinking water has been recorded, where they were detected at a maximum level of 258 and 24 ng L⁻¹ in USA and Canada, respectively [18, 19].

HPLC is a widely selective separation method for the isolation of pure compounds from crude mixtures and was used successfully to detect antibiotics in low concentrations [52]. Twenty positive water samples were subjected to HPLC for quantitative analysis to investigate the presence of antibiotics in samples acquired from both plants. The chromatogram of raw water samples at the Al-Wihda plant (6 samples out of 18) confirmed the presence of ciprofloxacin and levofloxacin (Figure 4). Ciprofloxacin peaked at about 4.0 min retention time whereas levofloxacin exhibited peak at 5.6 min in comparison to the standard solution peak.

At the Al- Rasheed water treatment facility, the chromatogram showed that the raw water (12 samples out of 18) was contaminated by both ciprofloxacin and levofloxacin with sharp peak of ciprofloxacin at 3.9 min and peak of levofloxacin at 5.6 min (Figure 5). Notably, amoxicillin was found only in one raw water sample at the Al-Rasheed plant with peak at 4.5 min retention time (Figure 6).

Detection of ciprofloxacin by HPLC using spectroscopic detector resulted in high yield recovery of 75-84% within 13.8 and 15 min [53, 54]; however, ELISA and TLC techniques could be used to detect ciprofloxacin from different samples [55]. The majority of wastewater treatment systems worldwide are not fully equipped to eliminate antibiotics and disinfectants which are not biodegradable, though friendly environment, low cost, and efficient techniques should be assessed to remove antibiotics from wastewater and consequently drinking water [56].

This research signals the need for surveying multiple antibiotics in the raw and finished water of water treatment plants in Iraq. Future studies should concentrate on the presence and transportation of these compounds during the treatment of drinking water. Therefore, the persistence of a wide spectrum of antibiotics in some water sources suggests that the overall impact of antibiotics as a pollutant group should not be ignored.

4. Conclusion

The results of the present study revealed that ciprofloxacin, levofloxacin, and amoxicillin were detected in the raw water, while ciprofloxacin and levofloxacin were detected in the finished water as well. This has serious implications as it can contribute to the development of antibiotic-resistant bacteria. The possible persistence of ciprofloxacin and other antibiotics in potable water sources is a serious problem due to the health impacts of chronic exposure over a lifetime, even at low levels. Besides, the drugs emit unpleasant odors and can cause skin-disorders [57]. Antibiotic pollution of potable water has to be addressed with urgency as some diseases caused by pathogenic resistant bacteria cannot be treated by conventional antibiotics [58]. Thus, this study highlights the need for a comprehensive approach to the presence of antibiotic composites in rivers and potable water in Iraq.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Acknowledgments

The authors would like to acknowledge the Mayoralty of Baghdad and College of Science for Women/University of Baghdad. The authors also would like to thank Editage (www.editage.com) for English language editing.

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Copyright © 2019 Ansam R. Mahmood et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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