The Influence of Wastewater Reuse on the Occurrence of Antibiotics, Antibiotic Resistant Bacteria, and Toxicological Impacts in the Environment
Open Access
- Author:
- Franklin, Alison Mary
- Graduate Program:
- Soil Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- April 25, 2019
- Committee Members:
- John Earl Watson, Dissertation Advisor/Co-Advisor
Maryann Victoria Bruns, Committee Chair/Co-Chair
John Patrick Vanden Heuvel, Committee Member
Subhashinie Kariyawasam, Committee Member
Clinton Williams, Outside Member - Keywords:
- Antibiotics
antibiotic resistance
wastewater reuse
water reuse
soil
groundwater - 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 world. 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, specifically for human and animal health, due to selection and development of drug-resistant bacteria that limit the drugs available to fight infections. As cases of resistance increasing, 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 treatment 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 5 cm of effluent per week. Given the continuous application of WWTP effluent, this site offers a unique opportunity to analyze the potential for soil to provide a tertiary treatment of the effluent and remove antibiotics as well as analyze the potential biological impact of low concentrations of antibiotics within the soil and water environment. The objectives of this dissertation were to (i) quantify concentrations of four antibiotics (sulfamethoxazole, trimethoprim, ofloxacin, and lincomycin) in soil and water environments impacted by reuse of WWTP effluent, (ii) analyze for antibiotic resistant bacteria and antibiotic resistance genes in soil irrigated with WWTP effluent, and (iii) provide preliminary analysis of potential toxicological impacts of WWTP effluent. For the first objective, analysis of water, soil, and wheat samples at the Living Filter showed that antibiotic concentrations are present throughout the system from the influent entering the WWTP to the effluent that was spray-irrigated on soil, wheat plants grown on site, and the groundwater at the Astronomy Site. Concentrations of the four antibiotics are highest at the WWTP with concentration ranging from <LOD for lincomycin up to 22 μg L-1 for sulfamethoxazole in the effluent, with overall antibiotics concentration averages of 0.71±1.3 μg L-1 and 1.05±3.8 μg L-1, respectively, for influent and effluent. Groundwater had the lowest overall load of antibiotics with most concentration below 25 ng L-1 and a yearly range of <LOD – 660 ng L-1. Sulfamethoxazole was the antibiotic typically present in influent, effluent and groundwater at the highest concentrations, while trimethoprim, ofloxacin and lincomycin present, but with greater variability in overall abundance. When wheat plants were analyzed, residues of each compound were present on most plant surfaces. Ofloxacin was found throughout the plant with higher concentrations in the straw (10.2 ± 7.05 ng g-1) and lower concentrations in the grain (2.28 ± 0.89 ng g-1). Trimethoprim was found only on grain or straw surfaces, while carbamazepine, an anti-epileptic drug, and sulfamethoxazole were concentrated within the grain (1.88 ± 2.11 ng g-1 and 0.64 ± 0.37 ng g-1, respectively). Within the soil profile, ofloxacin had the highest background concentrations (650±204 ng kg-1) even after 7 months without effluent irrigation. However, after 10 weeks of effluent irrigation, concentrations of sulfamethoxazole (730±360 ng kg-1) were higher than ofloxacin, and trimethoprim was finally quantified (190±71 ng kg-1) in the soil profile. For the second objective, analysis of antibiotic resistant bacteria and antibiotic resistance genes began with a preliminary study showing that Gram-negative bacteria were resistant to trimethoprim above minimum inhibitory concentrations at the cropped location of the Astronomy site but absent at the control site. Additionally, this preliminary work found that copy numbers of SulI, a sulfonamide resistance gene, were significantly higher at the Astronomy site at a depth of 10-15 cm compared to the Control site. Based on this preliminary work demonstrating that spray irrigation of effluent may alter levels of antibiotic resistance in the soil profile, a full-scale assessment of four different antibiotic resistance genes was performed [two sulfonamide resistance genes (SulI and SulII), a macrolide resistance gene (ermB), and an integron associated with antibiotic resistance (Int1L)]. The impacts of spray irrigating with effluent at the Astronomy Site varied by type of antibiotic resistance gene, location (depression versus summit), and soil profile depth. For SulI, 10 weeks of irrigation resulted in significant increases in resistant gene numbers at a depth of 0-5 cm for the depression sites. Regardless of the number of irrigation events (1 week versus 10 weeks versus indirect irrigation), the number of SulI resistance genes at a depth of 35-80 cm at the Astronomy was higher for both depression and summit locations compared to the control site. On the other hand, for SulII, spray irrigation had little impact on resistance gene numbers near the surface regardless of location or number or irrigation events. By depth, differences in sulII resistance gene numbers did occur with higher levels at the Astronomy Site compared to the control site. Similar results occurred with ermB with little differences near the surface, but by depth ermB was present at the Astronomy Site while absent at the control site. Finally, for Int1L, while a few locations at the Astronomy Site had higher numbers of resistance genes near the surface, the most significant trend was that the amount of this resistance gene at the 35-80 cm depth was higher at the Astronomy Site compared to the control site. Overall, antibiotic resistance does appear to be impacted at the Astronomy Site due to spray irrigation of effluent on cropped lands; however, it varies by resistance gene. The most consistent impact was higher numbers of resistance genes by depth at the Astronomy Site compared to the control site. Finally, the initial assessment of potential toxicological impacts of effluent containing complex mixtures of contaminants that is, then, released into the environment did show that biological effects in mammals might be possible. In fact, the highest biological activity found in effluent was endocrine disruption with the total effluent having much higher activity than even the reference compound, 17β-estradiol. At concentrations similar to environmental levels of effluent, the whole effluent had an activity level equivalent to 54.5 μg L-1 of 17β-estradiol, which is higher than most acceptable daily intakes for humans and predicted no effect concentrations for aquatic wildlife. While the contaminant load from the whole effluent would be diluted once it enters the environment, that dilution effect might not be enough to lower the overall biological activity of that contaminant mixture so that it wouldn’t have adverse effects in endocrine systems of exposed organisms. Additional research is necessary to determine the specific receptors within the endocrine system that are being activated by the whole effluent, but this preliminary work demonstrates the possible risk that effluent going out into the environment might pose. In addition, bioassays may be an adequate screening tool for assessing the overall environmental risk of effluent and other complex contaminant mixtures. In conclusion, this research project has shown that antibiotics are present throughout the Living Filter system from the WWTP to the impacted soil and water environments. Antibiotics are found at the highest concentrations in the influent and effluent of the WWTP and those concentrations decrease once in the environment with the lowest concentrations found in the groundwater. The soil does appear to prevent most of the antibiotics from reaching the groundwater system by either retaining those compounds in the soil profile, specifically SMX and OFL, and/or by degradation processes. However, the presence of these antibiotics in the soil profile does lead to update into wheat plants and appears to impact antibiotic resistance with higher numbers of antibiotic resistance genes found to a depth of 80 cm at the Astronomy Site when compared to a control site. Finally, initial toxicity assays demonstrate that effluent containing not only antibiotics but also other contaminants has the potential for negative biological impacts, especially with regard to endocrine disruption. Overall, by spray irrigating effluent on cropped lands the soil does filter the antibiotics to prevent significant groundwater contamination, but the possible biological impacts need to be assessed more carefully.