Grantee Research Project Results
2005 Progress Report: Fate and Effects of Fluoroquinolone Antibacterial Agents in Aquatic Ecosystems
EPA Grant Number: R829008Title: Fate and Effects of Fluoroquinolone Antibacterial Agents in Aquatic Ecosystems
Investigators: Graham, David W. , deNoyelles, Frank J. , Lydy, Michael J. , Larive, Cynthia K. , Smith, Marilyn S.
Current Investigators: Graham, David W. , deNoyelles, Frank J. , Lydy, Michael J. , Larive, Cynthia K.
Institution: University of Kansas
EPA Project Officer: Page, Angela
Project Period: August 20, 2001 through August 19, 2004 (Extended to August 19, 2006)
Project Period Covered by this Report: August 20, 2004 through August 19, 2005
Project Amount: $520,976
RFA: Drinking Water (2000) RFA Text | Recipients Lists
Research Category: Drinking Water , Water Quality , Water
Objective:
The objective of this research project is to assess the fate, attenuation, and ecotoxicity of selected fluoroquinolone (FQ) antibiotics on surface water quality. These compounds were selected for specific investigation because: (1) they are potent antibacterial agents and possible genotoxins; (2) they are used in agriculture and medicine; and (3) little is known about their environmental fate or impact. The goal of the research project is to assess FQs under both laboratory- and field-scale conditions to permit the extension of fundamental results to a practical scale. Emphasis is being placed on developing new methods for detecting and quantifying FQs and their degradation products at low concentrations and developing new molecular techniques for monitoring antibacterial resistance to FQs in exposed microorganisms.
Progress Summary:
Early work on the project focused on the development of methods for quantifying ciprofloxacin (cipro), enrofloxacin (enro), and breakdown products at concentrations typical of environmental exposures. This task was achieved in the early years of the project, primarily using refined extraction and concentration protocols and liquid chromatography-nuclear magnetic resonance and liquid chromatography-tandem mass spectroscopy analytical methods.
Work in Year 4 of the project focused on assessing factors at both the laboratory- and field-scale that impacted the fate and persistence of resistant bacteria and resistance genes in aquatic systems (after release from a resistance-generating source). Rather than focusing on FQs, these studies focused on the fate and persistence of different tetracyclines in aquatic systems because our previous results indicated that FQs were exceedingly photosensitive and adsorptive in aquatic systems. Tetracyclines were used because they are considered a better model antibiotic for persistence studies, especially related to resistance migration in receiving waters.
To pursue the tetracycline resistance in the environment, a novel method was developed using real-time PCR for quantifying tetracycline resistance genes in the environment (Smith, et al., 2004). Technical work on tetracycline fate and persistence is focusing on three basic experiments; the first two assessing the affect of tetracycline loading rate on the in situ development of tetracycline resistance, changes in community structure, and macrophyte growth in exposed surface waters, and the third assessing resistance persistence in flowing waters in a natural watershed. The first experiment, which was performed using 15 field-scale mesocosms, indicated that tetracycline loading rate had minimal impact on in situ resistance development in exposed aquatic bacteria, suggesting that resistant bacteria observed in the environment more likely are enriched at the point of use rather than after release into the environment (Knapp, et al., submitted). Figure 1 shows that only at oxytetracycline (oxytet) levels greater than 50 μg/L was there a proportional increase in resistance genes in the mesocosms after 42 days exposure, and this increase largely was related to lower total levels of bacteria in the systems, presumably as a result of the antibacterial action of oxytet in the waters. This is a practically significant result because most surface waters typically have less than 10 μg/L tetracycline levels, even in highly exposed sites such as feedlot wastewater treatment lagoons. A similar result was observed in complementary analysis, using whole-organism techniques, where only approximately 9 percent more resistance organisms were found in systems exposed to greater than 50 μg/L oxytet over 6 weeks, and this result was not statistically significant.
Figure 1. Resistance Gene Retention Over Time in Mesocosm-Scale Systems as a Function of Ambient Oxytetracycline Level in the Water Column and Total Bacterial Community 16S rDNA Level
Given these observations, the watershed study is ongoing, focusing on monitoring of resistance genes and tetracycline levels in the Sumas River watershed that straddles Washington State and British Columbia (Canada). This watershed was selected for study because it has well-defined and variable land use, ranging from pristine mountain streams to areas of high agriculture, and because it has a 20-year historic water quality record that precisely defines zones of high and low known agricultural impact. This historic record is available from Dr. Ken Hall at the University of British Columbia and provides a perfect guide for field monitoring because it identifies locations of high and low agricultural influence a priori,allowing the focused sampling of areas that provide clear contrasts in agricultural exposure. Early data indicate that the type of land use has a massive effect on the number of resistance genes in the river, although gene numbers do not correlate with measured tetracycline levels, implying that resistance genes and tetracycline (itself) have different mass transport traits in the environment. Further, observed resistance gene numbers have been highly seasonal, ranging from below detection in the summer to levels as high as feedlot lagoons during the autumn when manure is released onto the field surfaces. This sampling program will continue through the spring of 2006.
Future Activities:
Three main tasks still are ongoing. First, new laboratory experiments are underway to assess the impact of longer-term exposure to a suite of antibiotics and FQs in bench-scale laboratory bioreactors simulating biological wastewater treatment units. These experiments are being undertaken because early results indicated that many antibiotics can be quite short-lived in light-exposed surface waters; however, such conditions are not always present in wastewater facilities. As such, these systems may be breeding grounds for antibacterial resistance development. This experiment, therefore, will monitor antibacterial resistance using molecular and traditional culturing methods under five different antibiotic loading conditions over more than 3 months (tentative) to assess chronic exposure effects.
The second ongoing task is to assess other factors that might cause increased resistance persistence of antibacterial impacts in surface waters. Real-time PCR methods are being expanded (new probe-primer sets) and will be used to continue studying mechanisms of resistant development during and after use and gene transport through the environment. Specifically, a new mesoscale experiment is underway that assesses the influence of native environmental microorganisms as vectors for resistance migration from fecal organisms released at a point source (e.g., a feedlot) through gene transfer (e.g., integrons) in natural systems. This experiment is being coupled with ongoing monitoring of the Sumas River and other local watersheds, using similar and complementary tracking methods for assessing in situ gene migration in the environment.
Journal Articles on this Report : 8 Displayed | Download in RIS Format
| Other project views: | All 43 publications | 19 publications in selected types | All 18 journal articles |
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Cardoza LA, Almeida VK, Carr A, Larive CK, Graham DW. Separations coupled with NMR detection. Trends in Analytical Chemistry 2003;22(10):766-775. |
R829008 (2002) R829008 (2003) R829008 (2004) R829008 (2005) R829008 (Final) |
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Cardoza LA, Korir AK, Otto WH, Wurrey CJ, Larive CK. Applications of NMR spectroscopy in environmental science. Progress in Nuclear Magnetic Resonance Spectroscopy 2004;45(3-4):209-238. |
R829008 (2003) R829008 (2004) R829008 (2005) R829008 (Final) |
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Cardoza LA, Knapp CW, Larive CK, Belden JB, Lydy M, Graham DW. Factors affecting the fate of ciprofloxacin in aquatic field systems. Water, Air and Soil Pollution 2005;161(1-4):383-398. |
R829008 (2003) R829008 (2004) R829008 (2005) R829008 (Final) |
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Hanson ML, Knapp CW, Graham DW. Field assessment of oxytetracycline exposure to the freshwater macrophytes Egeria densa Planch and Ceratophyllum demersum L. Environmental Pollution 2006;141(3):434-442. |
R829008 (2005) R829008 (Final) |
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Knapp CW, Cardoza LA, Hawes J, Wellington EMH, Larive CK, Graham DW. Fate and effects of enrofloxacin in aquatic systems under different light conditions. Environmental Science & Technology 2005;39(23):9140-9146. |
R829008 (2004) R829008 (2005) R829008 (Final) |
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Knapp CW, Caquet T, Hanson ML, Lagadic L, Graham DW. Response of water column microbial communities to sudden exposure to deltamethrin in aquatic mesocosms. FEMS Microbiology Ecology 2005;54(1):157-165. |
R829008 (2005) R829008 (Final) |
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Robinson AA, Belden JB, Lydy MJ. Toxicity of fluoroquinolone antibiotics to aquatic organisms. Environmental Toxicology and Chemistry 2005;24(2):423-430. |
R829008 (2003) R829008 (2004) R829008 (2005) R829008 (Final) |
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Smith MS, Yang RK, Knapp CW, Niu Y, Peak N, Hanfelt MM, Galland JC, Graham DW. Quantification of tetracycline resistance genes in feedlot lagoons using real-time PCR. Applied and Environmental Microbiology 2004;70(12):7372-7377. |
R829008 (2005) R829008 (Final) |
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Supplemental Keywords:
antibacterial agents, antibiotics, antiseptics, aquatic ecosystems, chemical contaminants, microbial contamination, microbial effects, toxicokinetics,, RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Water, Waste, Ecological Risk Assessment, Health Risk Assessment, Fate & Transport, Environmental Chemistry, Ecosystem/Assessment/Indicators, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Drinking Water, Ecology and Ecosystems, anticeptics, toxicokinetics, ecological effects, microbial contamination, antibiotics, water quality, stressors, microbial risk management, monitoring, chemical contaminants, fate, microbial effects, drinking water contaminants, fate and transport, exposure, ecological exposure, antibacterial agents, pharmaceuticals, fluoroquinolone, human health effects, aquatic ecosystems, exposure and effectsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.