Environmental Health Risks of Antibiotic Residue in Sewage Discharged from Mosul City: An ELISA-Based Investigation

This study aimed to showed that all sixteen samples collected from different areas of the city had detectable levels of antibiotic residues, with a mean concentration of 4.1 ng/ml and a standard deviation of 0.656 ng/ml. These findings indicate that antibiotics are being overused or not disposed of properly in the area, posing a potential risk to environmental health. The ELISA technique was effective in creating a standard curve for determining the antibiotic concentration in unknown samples. The standard curve showed a linear relationship between the concentration of antibiotics and the absorbance values obtained from the ELISA assay. By comparing the mean concentrations of antibiotic residues between different areas of the city, significant differences were found. For example, the left side of the city had a mean concentration of 3.97 ng/ml, while the right side of the city had a mean concentration of 4.19 ng/ml. The difference in the mean concentrations between the two areas was statistically significant (p<0.05). The high mean concentration of antibiotic residues detected in the samples suggests that antibiotics are being overused or not properly disposed of in the area. In conclusion, this study shows that there are detectable levels of antibiotic residues in the discharged water of Mosul City, indicating a potential threat to environmental health. Implementing measures to reduce the use and ensure proper disposal of antibiotics can help mitigate the potential risks associated with the development of antibiotic-resistant bacteria, protecting public health and environmental well-being.


Introduction
The overuse and misuse of antibiotics in human and animal health has resulted in a significant increase in antibiotic-resistant bacteria, which poses a major public health threat [1,2].The World Health Organization has identified antibiotic resistance as one of the biggest threats to global health, food security, and development [3].Antibiotic residues from human and animal waste discharged into the environment contribute to the spread of antibiotic resistance, making infections difficult to treat, and leading to increased healthcare costs [4,5].Mosul, a major city in Iraq, is experiencing rapid population growth, urbanization, and industrialization, which has resulted in a significant increase in the discharge of untreated sewage into the Tigris River [6].This untreated sewage contains high concentrations of antibiotics, which can contaminate the environment and pose a potential health hazard to humans and wildlife [7].This study aims to investigate the environmental health risks associated with antibiotic residues in the sewage water discharged from Mosul City using the Enzyme-Linked Immunosorbent Assay (ELISA) analysis to detect and quantify the presence of antibiotics.ELISA is a sensitive and reliable method for detecting antibiotic residues in environmental samples.The results of this study will help to shed light on the extent of antibiotic contamination in the environment and the potential health risks associated with it.This information will be useful to policymakers, researchers, and public health officials working to reduce the risk of antibiotic resistance in the environment.It will also contribute to the development of effective strategies for managing antibiotic residues in sewage water to protect public health and the environment.In summary, this study is critical in addressing the environmental health risks associated with antibiotic residues in the sewage water discharged from Mosul City.It will provide valuable insights into the extent of antibiotic contamination in the environment and the potential health risks associated with it, ultimately contributing to the global effort to combat antibiotic resistance.

Sample Collection and Preparation
Sixteen samples, eight from the left bank and eight from the right bank water discharged sewage main pipes in Mosul City, Iraq, were collected in sterile containers and transported to the laboratory for analysis.The samples were prepared for ELISA analysis according to the manufacturer's instructions.Solid particles were removed by centrifuging the samples at 10,000 rpm for 10 minutes, and the supernatant was diluted 1:10 with phosphate-buffered saline (PBS) solution.The diluted samples were then used for ELISA analysis [6] .

ELISA Analysis
A commercially available kit from Melsin manufacturer designed for the detection of antibiotic residues in water samples was used to perform the ELISA analysis, following the manufacturer's instructions (figure 1).Briefly, the diluted samples were added to the wells of a microtiter plate that was pre-coated with antibodies specific to the target antibiotic.The plate was incubated at room temperature for 30 minutes.After washing the wells, an enzyme-labeled antibody was added to the plate, followed by incubation at room temperature for 30 minutes [8].Next, a substrate solution was added to the plate, and the plate was incubated at room temperature for 15 minutes.The reaction was stopped by adding a stop solution, and the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader [8,9].

Data Analysis
The ELISA analysis's absorbance values determined the antibiotic residues' presence and concentration in the samples.Using the standard solutions provided in the kit, a standard curve was generated, and the concentration of antibiotic residues in the samples was calculated based on the absorbance values and the standard curve.

Results
The analysis conducted on the 16 samples of antibiotics residues by ELISA technique revealed that all the samples showed detectable levels of antibiotic residues (figure 2A).Furthermore, the results indicated that the concentrations of antibiotic residues were relatively high, with a mean concentration of 4.1 ng/ml and a standard deviation of 0.656 ng/ml.The high levels of antibiotic residues in the samples indicate that the antibiotics were being overused or not properly disposed of in the area.
The ELISA technique was utilized to create a standard curve for determining the antibiotic concentration in unknown samples.The standard curve was constructed using various known concentrations of antibiotics, ranging from 40 ng/ml down to 0 ng/ml, with concentrations of 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, and 0 ng/ml included.The standard curve depicts a linear relationship between the concentration of antibiotics and the absorbance values that were obtained from the ELISA assay (figure 2B).Upon comparing the mean concentrations of antibiotic residues between different areas, it was found that some areas had significantly higher levels of antibiotic residues than others.For instance, the left side of the city had a mean concentration of 3.97 ng/ml, while the right side of the city had a mean concentration of 4.19 ng/ml (table 1).The difference in the mean concentrations between the two areas was statistically significant p<0.05 (figure 3).The results of the analysis emphasize the importance of effectively managing the use and disposal of antibiotics in the examined area.Overuse and inadequate disposal of antibiotics can contribute to the emergence of antibiotic-resistant bacteria, which poses a significant risk to public health.Therefore, it is essential to establish and enforce measures that can effectively reduce the usage of antibiotics and ensure their proper disposal.These measures can help minimize the amount of antibiotic residues present in the environment and mitigate the potential risks associated with the development of antibiotic-resistant bacteria.Table1.The concentration of antibiotic residues present in 16 samples of discharged water was determined by plotting the samples onto a standard curve.

Discussion
The findings of this study are consistent with previous research that has identified overuse and improper disposal of antibiotics as significant contributors to the presence of antibiotic residues in the environment and the emergence of antibiotic-resistant bacteria [10].For example, a study conducted in India found detectable levels of antibiotic residues in samples of wastewater and suggested that the lack of effective wastewater treatment and inadequate regulatory measures were major factors contributing to the presence of antibiotic residues in the environment [11].Similarly, a study conducted in China identified high levels of antibiotic residues in samples of surface water and sediment, indicating potential risks to environmental health and the development of antibiotic resistance [12,13].Furthermore, another study conducted in Iraq examined the levels of antibiotic residues in chicken meat and found detectable levels of residues, with some samples exceeding the maximum residue limits set by the European Union [6,7].This highlights the need for effective management of antibiotic use not only in human medicine but also in veterinary medicine.Taking together, the findings of this project add to the body of evidence supporting the importance of proper management of antibiotic use and disposal to reduce the presence of antibiotic residues in the environment and mitigate the risks associated with the development of antibiotic resistance [14,15].Effective regulatory measures, wastewater treatment, and responsible use of antibiotics in both human and veterinary medicine are essential for protecting public health and environmental wellbeing [16,17].

Conclusion
The outcomes of the project suggest the presence of detectable levels of antibiotic residues in the discharged water of Mosul City, indicating a possible threat to environmental health.The high mean concentration of antibiotic residues detected in the samples implies that antibiotics are likely being overused or not disposed of properly in the area.The ELISA technique demonstrated its effectiveness in determining the concentration of antibiotics in the samples.The significant differences found in the mean concentrations of antibiotic residues between various areas of the city emphasize the need for appropriate management of antibiotic use and disposal in the region.These findings are in line with previous studies that have reported the overuse and improper disposal of antibiotics as major contributors to the emergence of antibiotic-resistant bacteria.Therefore, it is critical to develop and implement measures to reduce the use of antibiotics and ensure their proper disposal to decrease the levels of antibiotic residues in the environment.Such measures can mitigate the potential risks associated with the development of antibiotic-resistant bacteria and safeguard public health.

Figure 1 .
Figure 1.An ELISA kit designed by the Melsin manufacturer was used to detect the presence of antibiotic residues in water samples.

Figure 2 .
A. Microplates ELISA stages technique for determining the concentration of antibiotics in water discharged samples.B. Standard curve for antibiotic concentration (0, 2.5, 5, 10, 20 and 40 ng/ml) determination using ELISA technique.Notably, the optical density of ELISA reader was 450 nm.

Figure 3 .
Figure 3. Distribution of antibiotic residue concentrations in 16 samples of discharged water from Mosul city.The samples were collected from 8 locations on the left 8 locations on the right side of the city.The histogram displays the frequency of samples at different concentration ranges of antibiotic residues.