Analysis of four human antibiotics and antibiotic resistant bacteria in water and soils impacted by wastewater treatment plant effluent.
Open Access
- Author:
- Franklin, Alison Mary
- Graduate Program:
- Soil Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- March 23, 2015
- Committee Members:
- John Earl Watson, Thesis Advisor/Co-Advisor
- Keywords:
- Antibiotics
Antibiotic Resistance
Soil
Water
Sulfamethoxazole
Trimethoprim
Ofloxacin
Lincomycin
Sorption
Living Filter - Abstract:
- Since the late 1990’s, low levels of pharmaceuticals and personal care products (PPCPs) have been detected in waterways and soil systems throughout the United States. One of the main pathways that PPCPs reach the environment is by discharge of wastewater treatment plant (WWTP) effluent directly into natural waterways. Although concern of overt toxicity is low, pharmaceuticals are designed to elicit highly specific physiological responses. Release into the environment may result in unexpected biological and ecological responses in target and non-target organisms that permanently alter the dynamics within an ecological system. Antibiotics, in particular, are an area of rising concern, especially for human health, due to selection and development of drug-resistant bacteria that limit the drugs available to fight infections. As cases of resistance increase, the integrity and efficacy of antibiotics comes into question, especially with the development of human pathogens with multidrug resistance. Although, historically, environmental and clinical antibiotic resistance genes (ARGs) have been viewed as discrete entities, recently, environmental resistance genes have been linked to human pathogens. Scenarios for shared resistance between human pathogens and the environment are becoming more feasible given the influxes of anthropogenic antibiotics into the environment creating selective pressures for environmental bacteria to evolve cross-resistance to commonly prescribed antibiotics. Currently, sewage systems are not equipped to completely remove antibiotics. A main concern about this uncontrolled release of anthropogenic antibiotics into waterways is the conduit created for intimate interactions of environmental bacteria with synthetic antibiotics that may alter microbial communities and negatively impact ecological systems. Due to the nature of wastewater reuse, the presence of anthropogenic antibiotics in effluent and subsequent aquatic environments may allow antibiotics and antibiotic resistance to cycle through ecosystems and eventually impact ecological and human health. One possible approach to mediate the release of anthropogenic antibiotics into waterways is tertiary treatment of effluent in soil, which will not only potentially remove contaminants, but also conserve water resources. Recent research has shown that anthropogenic antibiotics released into the environment have variable impacts on antibiotic resistance in bacteria depending on the system: water, soil or sediment. Of note, the level of antibiotic resistance in effluent impacted soil systems appears to be significantly lower than waterways and sediments. Therefore, tertiary treatment of effluent may effectively remove antibiotics and limit the development of antibiotic resistance throughout ecosystems. The Pennsylvania State University has a unique wastewater renovation system known as the Living Filter where WWTP effluent is spray irrigated on cropped, grassed and forested lands. This site has been in full operation since the 1980’s, is continuously spray irrigated year-round and receives on average 2” of effluent per week. Given the continuous application of WWTP effluent, this site offers a unique opportunity to analyze the potential for soil to tertiary treat the effluent and remove antibiotics as well as analyze the potential impact of low concentrations of antibiotics within the soil and water environment. The purpose of this thesis was to (i) develop and optimize chemical analytical methodology to isolate and analyze concentrations of four antibiotics in soil and water systems, (ii) examine the behavior of these four antibiotics in different soil types, (iii) monitor the presence of these antibiotics in influent, effluent and groundwater at the Living Filter, and (iv) perform preliminary analysis of antibiotic resistance in impacted soil systems at the Living Filter. The antibiotics that were analyzed were sulfamethoxazole (SMX), trimethoprim (TMP), ofloxacin (OFL), and lincomycin (LIN). These antibiotics were selected based on presence in local and national waterways, average annual prescription rates (nationally and locally), presence in the WWTP effluent, potential environmental toxicity, as well as overall public health impact if resistance to these particular antibiotics increased. For the development of protocols for the simultaneous extraction of SMX, TMP, OFL, and LIN from water and soil matrices in Chapter 2, one procedure for either type of extraction did not result in ideal extraction efficiencies for all antibiotics. Therefore, the methods selected were based on the best overall recoveries that could be obtained with one procedure. For water extraction, the Oasis HLB Plus cartridge without a pH adjustment of the aqueous sample was the best method and resulted in recoveries of 118±5.2%, 86±4.0%, 83±5.4%, and 31±1.5% for SMX, TMP, LIN, and OFL, respectively. For appreciable recoveries of OFL (75±1.3%), a pH adjustment was necessary using the HLB Plus cartridge. The soil extraction procedure selected for the simultaneous recovery of antibiotics was the Accelerated Solvent Extraction (ASE) method with an extraction solution of acetonitrile/water (v/v, 50:50, pH 2.8). Although this method resulted in recoveries of 92±5.5%, 35±7%, and 31±5% for SMX, TMP, and LIN, respectively, OFL was not recoverable during preliminary methodology development with any method. When soils were ground before extraction, the recoveries of TMP, LIN and OFL increased compared to not grinding the soil; however, for SMX recoveries decreased. When ground soils were freeze-dried before extraction, the recoveries of TMP and SMX increased significantly, OFL recoveries only increased marginally, and the recoveries of LIN decreased when compared to ground soils not freeze-dried. The extractability of these compounds was primarily influenced by the pH of the soil or water system and the pKa of the compound. The sorption and desorption behavior of the antibiotics determined in Chapter 3 demonstrated that the type of soil will influence the behavior of the antibiotics. Once ofloxacin entered the soil profile, it was essentially unrecoverable with >99.9% of the original compound adsorbing to the soil profile, as a result adsorption coefficients could not be calculated; however, literature Kd values range from 40,000 – 71,000. For TMP, SMX and LIN, the average Log Kf values determined were 2.8±0.4, 0.92±0.6, and 0.89±0.3 and Kfoc values were 4.8±0.76, 2.96±0.13, and 2.93±0.87, respectively. Trimethoprim exhibited stronger interactions with the soil profile, while SMX and LIN had weaker interactions, as seen by the adsorption coefficients obtained. For TMP the major factor that correlated with its sorption was cation-exchange capacity. Even though when comparing adsorption coefficients the behavior of SMX and LIN appear similar, the key factors that were associated with their sorption were quite different with organic carbon content being correlated with SMX and pH being correlated with LIN. The driving force behind these sorption interactions appeared to be linked to the ionic state of the compound, which would be determined by the pH of the system and pKa of the compound. Based on the sorption analysis of these four compounds, OFL and TMP are expected to be tightly bound to the soil profile and less mobile, whereas SMX and LIN are predicted to have fewer interactions with the soil profile and have higher mobility. Sulfamethoxazole and LIN are the two antibiotic compounds that may have the potential for ecological impacts due to their increased mobility. In Chapter 4, analysis of the concentrations of SMX, TMP, OFL and LIN in the WWTP influent and effluent as well as groundwater at Penn State’s Living Filter during Summer 2013, Spring 2014 and Fall 2014 showed that these four antibiotics are found in the water throughout the year. The highest overall concentrations of these compounds occurred during the spring when the local population and prescription rates of antibiotics would be highest. Concentrations of antibiotics were significantly higher in influent and effluent than in the groundwater. Although, in general, the concentrations in the influent for each antibiotic were higher than the effluent with average annual concentrations of 1.4±2.1 μg L-1 and 2.4±6.2 μg L-1, respectively, in certain instances the effluent concentration was higher than the influent. Yearly average of the antibiotics in groundwater were 37±60 ng L-1. It was notable that the standard deviations were larger than the annual means indicating great variability in concentrations throughout the year. Sulfamethoxazole was the antibiotic found in all samples at the highest concentrations throughout the year with yearly averages of 1.6±2.1 μg L-1, 8.0±12.1 μg L-1, and 72±80 ng L-1 in the influent, effluent, and groundwater, respectively. The other antibiotics while present consistently in the influent and effluent were found variably in the groundwater. Given the low concentrations of antibiotics found in the groundwater, percolation of the effluent through the soil profile appears to remove the compounds and possibly limit the potential ecological impacts. Analysis of the concentrations of these antibiotics in the soil profile would create a more comprehensive picture of the exact fate and transport of these compounds in the Living Filter system as well as provide insight with regard to the potential ecological impacts. For the microbial analyses performed in Chapter 5, optimal culture methods for soil bacteria were determined and preliminary analysis of antibiotic resistance in systems impacted by the application of WWTP effluent with low concentrations of antibiotic compounds was assessed. VL-55 medium with gellan and 0.05% xylan provided adequate total heterotrophic plate counts of bacteria cultured from cropped and forested soils at Penn State’s Living Filter. Additionally, MacConkey and CNA with 5% sheep blood agar for selective culturing of Gram-negative and Gram-positive bacteria provided appreciable plate counts. The preliminary antibiotic resistance analysis showed trends where exposure to low concentrations of antibiotics appeared to alter the incidence of antibiotic resistance compared to a control soil that had not received effluent application or manure application. For the analysis of bacterial isolates, ampicillin resistance was present in all samples and served as a positive control of natural resistance expected in the environment. Ciprofloxacin resistance was not present in any samples and could serve as an indicator of impacts in the future. Resistance to SMX and TMP was present in the soil bacteria previously exposed to low levels of antibiotics, but not present in the bacteria from the control site for Gram-negative bacteria. However, for Gram-positive bacteria, resistance to SMX and TMP was present predominately in control samples. When two SMX resistance genes, SulI and SulII, were quantified, the general trend was for the soil samples that were exposed to WWTP effluent to have higher numbers of the genes compared to the control site. Although this preliminary work demonstrates some trends concerning impacts of exposure to antibiotics, additional work is necessary to fully explain the effects. This thesis work provides adequate methods for the analysis of four antibiotics (SMX, TMP, LIN, and OFL) in soil and water environments. In addition, the behaviors of these antibiotics were analyzed in four different types of soil to determine adsorption coefficients so that sorption and desorption behavior can be estimated for soil environments based on soil characteristics. Understanding sorption and desorption behavior of these compounds will allow estimation of the fate and transport of these antibiotics once they enter a specific soil environment. Quantification of these antibiotics in influent, effluent and groundwater at the Astronomy Site of the Living Filter provided a greater understanding of what is entering the soil system and what compounds reach the groundwater system. Preliminary microbial analysis began to assess the possible ecological impacts of low-level concentrations of antibiotic mixtures on exposed microbial communities and demonstrates that additional work is necessary to fully elucidate the potential effects.