Final Report: Fate and Effects of Fluoroquinolone Antibacterial Agents in Aquatic Ecosystems

EPA Grant Number: R829008
Title: Fate and Effects of Fluoroquinolone Antibacterial Agents in Aquatic Ecosystems
Investigators: Graham, David W. , Larive, Cynthia K. , Lydy, Michael J. , deNoyelles, Frank J.
Institution: University of Kansas
EPA Project Officer: Page, Angela
Project Period: August 20, 2001 through August 19, 2004 (Extended to August 19, 2006)
Project Amount: $520,976
RFA: Drinking Water (2000) RFA Text |  Recipients Lists
Research Category: Drinking Water , Water Quality , Water

Objective:

The purpose of this study was to assess the fate, attenuation, and ecotoxicity of fluoroquinolone (FQ) antibiotics on surface water quality.  These compounds were chosen for 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. A further goal of this study was to test FQ fate and impacts at both the laboratory- and field-scale to extend the results to practical applications. The work initially focused on the development of new methods for detecting and quantifying FQs and their degradation products at very low concentrations, and also the development of molecular techniques for quantifying antibiotic resistance to FQs and other antibiotics in exposed organisms. To fulfill this latter aim, the fate and effects of tetracyclines and resistance genes in the aquatic systems was also performed because FQs were found to be unstable in most receiving waters and the tetracycline class of antibiotics was better suited to studying the migration of antibiotic traits in nature.

Approach:

Four tasks will be performed: (1) develop analytical methods for detecting FQs and possible breakdown products in environmental samples; (2) determine attenuation rates and mechanisms of FQs in aquatic systems; (3) assess toxicological impacts of FQs on selected invertebrate species; and (4) determine the impact of FQs on microbial community conditions, co-contaminant fate, and antibiotic resistance development in exposed organisms. Tasks will be performed using both laboratory- and field-scale systems. In the first two years of the study, laboratory experiments will be used to develop analytical techniques for FQs and potential breakdown products, and establish general relationships between FQ fate and water chemistry conditions. Molecular biological and other monitoring methods also will be developed and tested for assessing FQ exposure impacts, including antibiotic resistance development in exposed organisms. In the final year, mesocosm-scale experiments will be performed to field-validate laboratory results. In particular, experiments will be used to verify in situ FQ transformation rates and mechanisms, and relationships between FQ exposure level, water chemistry, and microbial community conditions.

Summary/Accomplishments (Outputs/Outcomes):

Analytical Method Development: Early work on the project focused on developing new methods for analyzing ciprofloxacin (cipro), enrofloxacin (enro), and their breakdown products at environmentally relevant levels (i.e., very low). This task was largely achieved by the end of the first year, developing improved extraction and concentration protocols that were optimized for LC-NMR and LC-MS/MS detection methods (Cardoza et al., 2003b; Cardoza et al. 2004). A particular focus was placed upon the removal of background organic matter from environmental samples during pre-extraction, which permits higher sensitivity in detection of FQs due to the removal of masking compounds, such as humic and other complex organic acids. Five previously unidentified cipro and enro breakdown products were found, structures were determined, and new methods for their potential quantification developed (Cardoza et al., 2003a).
 
Trial Experiments on FQ Detection Methods: Analytical methods were trialed on samples collected from preliminary laboratory and field studies on the fate of cipro and enro in aquatic receiving waters. The new methods worked well, and initial results on FQ fate indicated that organic particulate matter (POC) and light supply were most important to FQ disappearance rate in aquatic systems (Cardoza et al., 2005). Photodegradation reactions completely destroyed both cipro and enro at half-lives (t1/2) as low as 1.2 hr under simulated ambient sunlight and quasi-natural aerobic water conditions. However, FQs were also found to readily adsorb onto POC at high rates (t1/2 < 15 min). Neither dissolved organic carbon (DOC) level nor the presence or absence of microorganisms had impacts on cipro and enro disappearance rates, although acid pH conditions did enhance the extent of adsorption onto POC. Overall, the half-lives of cipro and enro were very short in aquatic systems with light exposure or even moderate POC levels, suggesting the FQs tend not to prevail at any significant level or any significant time in most aquatic environments. One exception to this generalization was identified, which was when POC levels were low, light level was low, and water conditions are anaerobic, such as in a sediment zone. Despite some effort, FQ quantification in sediment samples was largely unsuccessful so we cannot verify this observation absolutely. In general, FQs adsorbed readily and strongly onto POC, which has implications to whether FQs actually remain active as antibiotics once they are adsorbed onto such surfaces or in sediment.
 
These laboratory observations were verified at the field-scale by trial experiments with cipro using 11.3 m3 aquatic mesocosms (Cardoza et al., 2005). Cipro was provided to open and tanks with black lids (in triplicate; see Figure 1), and similar results to lab studies were observed in field as  the lab; i.e., cipro fate was associated light exposure, although mesocosm waters in this experiment were low in POC, therefore it was not possible to field verify POC observations in this field experiment.
 
 
 


Figure 1:  Mesocosm Array for the FQ
 
Development and Testing of FQ Antibiotic Resistance Testing Methods: Concurrent to work assessing FQ detection methods and the overall fate of FQs in aquatic systems, additional method development was performed on molecular biological techniques for monitoring and quantifying FQ resistance in environmental organisms. This work was performed in collaboration with Dr. Elizabeth Wellington at the University of Warwick in the United Kingdom (U.K.). New non-quantitative methods were successfully developed using density gradient gel electrophoresis (DGGE) as a method for tracking mutations in the gyrA gene sequence (the QRDR region) that confers resistance in selected bacteria (e.g., Pseudomonas aeruginosa). This method was trialed in a subsequent mesocosm experiment assessing the fate and effects of enro in aquatic systems, and it was found that enro exposures at ~ 100 times levels usually observed in the environment had no detectable affect on either community biodiversity or the appearance of resistance traits (Knapp et al., 2005a). However, the developed methods were not considered ideal because they were specific to relatively narrow classes of bacteria and they were not quantitative. Further, it was concluded that developing a set of quantitative measures for tracking FQ resistance was going to be prohibitively expensive and it was decided that another class of antibiotics should be considered for subsequent studies assessing resistance-related effects of antibiotics in the environment.
 
As an ancillary component to assessing molecular biological methods for tracking antibiotic resistance in environmental samples, considerable effort was placed into creating broader general tools for characterizing aquatic microbial ecosystems for comparison with resistance traits. Specifically, quantitative rDNA gene hybridization techniques were developed to segregate bacterial and plastid signals in aquatic samples (Knapp and Graham, 2004), which were tested and validated in various parallel laboratory and field studies on other trace contaminants in the environment (Knapp et al., 2003; Knapp et al., 2005b; Knapp et al., in press). These new molecular tools allowed the discrimination between bacterial and non-bacterial signals, and also photosynthetic and non-photosynthetic signals in mixed microbial aquatic communities. This was a significant discovery because plastid DNA in photosynthetic eukaryotes closely resembles bacterial chromosomal DNA, which made it previously impossible to segregate these two different signals in natural samples.  
 
Laboratory Testing of FQ Toxicity to Selected Aquatic Organisms: Classical toxicity tests were separately performed on seven FQ antibiotics, cipro, lomefloxacin, ofloxacin, levofloxacin, clinafloxacin, enro, and flumequine, on five aquatic organisms (Robinson et al, 2005). Overall toxicity values ranged from 1,100-23,000 µg/L, which are well above typical levels of environmental concern. Microcystis aeruginosa, the cyanobacteria, was the most sensitive organism tested to FQ exposure.  Based on 5-day growth and reproduction studies, effective concentrations (EC50) ranged from 7.9 to 1,960 µg/L with a median level of 49 µg/L. Duckweed (Lemna minor) and the green algae, Pseudokirchneriella subcapitata, were less sensitive than M. aeruginosa.  Results from toxicological testing on the crustacean Daphnia magna (48-h survival) and fathead minnows (Pimephales promelas, 7-d early life stage survival and growth) showed limited toxicity with “no observed effect concentrations” at or near 10 mg/L.  However, fish dry weights observed under high ciprofloxacin, levofloxacin, and ofloxacin conditions (10 mg/L) had significantly higher masses compared with control fish.
 
Antibiotic Resistance Migration in the Environment: FQ studies indicated that FQs degraded rapidly in the environment and had minimal acute toxic effects; therefore, latter work on the project shifted to study factors that more generally affected the persistence of antibiotic resistant bacteria and genes in aquatic systems. A previous study had shown that the fate and persistence of tetracycline resistance genes differed than FQs in aquatic systems, and molecular biological methods had also been partially developed on this class of antibiotics. Specifically, tetracyclines differed in that resistance could be conferred by the explicit acquisition of individual resistance genes, and that those genes could be conditionally promiscuous, often associated with mobile gene cassettes called transposons and integrons. This different resistance mechanism provides a more explicit target for tracking resistance in original hosts (say humans or other animals that have been fed antibiotics) and genes released to nature, especially as it relates to practical quantitative studies.  
 
As such, a novel method for tracking tetracycline resistance genes using real-time PCR (qPCR) was developed for quantifying resistance gene abundances in environmental samples (Smith et al., 2004). This tool (and expanded versions of this tool) were subsequently used in many experiments within this project, including studies on the relationship between antibiotic use-practices at cattle feedlots (e.g., organic vs. large commercial-scale operations) and resulting resistance gene abundances in waste lagoons (Peak et al., 2007); resistance gene disappearance rates and their effects after release into different types of receiving waters (Engemann et al., 2006; Engemann et al., submitted; Hanson et al., 2006), and; the relationship between transposon abundances, antibiotic levels, and the selected retention of specific resistance genes, such as tet(W) and tet(M), after release to aquatic systems (Knapp et al., submitted).
 
Specifically, Peak et al (2007) showed that significantly higher resistance gene abundances (3 to 5 orders of magnitude) were observed in commercial vs. organic cattle feedlot lagoons. Second, Engemann et al. (2006, submitted) showed that light supply strongly regulated the retention time of resistance genes in a receiving waters with gene retention being prolonged in dark compartments, including peripheral biofilms (Figure 2). Finally, Figure 3 from Knapp et al. (submitted) shows that prolonged exposures of antibiotics in the environment, even at low tetracycline levels (< 50 days at 5 to 20 ug/L oxytetracycline), can result in insignificantly increased levels of resistance in exposed organisms.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Figure 2:  Distribution of resistance genes over time in the water column and peripheral biofilms. 
                  Percentages refer to the proportion of tetR genes in the biofilm relative to the water column on  
                  each   day.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Figure 3:  First-order rates of increase of: A) resistance genes and B) Tn916/Tn1545 genes both 
                   normalized to 16S-rRNA genes over oxytetracycline concentrations.
 
These latter observations are of great practical importance because they show that not all resistance genes are transported equally in the environment; that some genes selectively migrate on transposons into biofilms and sediments, and; that others are enriched in situ at very low antibiotic exposure levels. As such, this work is being highlighted in a feature article in the March 2008 issue of Discover magazine (the popular science magazine) as benchmark work in establishing relationships between antibiotic use in agriculture and the loss of efficacy of critical drugs in medicine. Finally, new manuscripts on this broader topic are still in preparation, especially new work that links increases in resistance genes across nature to both local- (land use at the watershed level) and national-scale events (annual antibiotic production rates to increases in resistance genes at un-exposed sites) using contemporary and historic samples collected over the past 60 years.

Expected Results:

Through this study, we will develop clear, field-tested data on the fate and impact of FQ antibiotics in aquatic systems.


Journal Articles on this Report : 17 Displayed | Download in RIS Format

Other project views: All 43 publications 19 publications in selected types All 18 journal articles
Type Citation Project Document Sources
Journal Article 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)
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  • Journal Article 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)
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    Journal Article 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)
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  • Journal Article Engemann CA, Adams L, Knapp CW, Graham DW. Disappearance of oxytetracycline resistance genes in aquatic systems. FEMS Microbiology Letters 2006;263(2):176-182. R829008 (Final)
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  • Journal Article Engemann CA, Keen PL, Knapp CW, Hall KJ, Graham DW. Fate of tetracycline resistance genes in aquatic systems: migration from the water column to peripheral biofilms. Environmental Science & Technology 2008;42(14):5131-5136. R829008 (Final)
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  • Journal Article 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)
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  • Journal Article Knapp CW, Graham DW, Berardesco G, deNoyelles Jr. F, Cutak BJ, Larive CK. Nutrient level, microbial activity, and alachlor transformation in aerobic aquatic systems. Water Research 2003;37(19):4761-4769. R829008 (Final)
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  • Journal Article Knapp CW, Graham DW. Development of alternate ssu-rRNA probing strategies for characterizing aquatic microbial communities. Journal of Microbiological Methods 2004;56(3):323-330. R829008 (Final)
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  • Journal Article 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)
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  • Journal Article 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)
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  • Journal Article Knapp CW, Engemann CA, Hanson ML, Keen PL, Hall KJ, Graham DW. Indirect evidence of transposon-mediated selection of antibiotic resistance genes in aquatic systems at low-level oxytetracycline exposures. Environmental Science & Technology 2008;42(14):5348-5353. R829008 (Final)
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  • Journal Article Knapp CW, Findlay DL, Kidd KA, Graham DW. A comparative assessment of molecular biological and direct microscopic techniques for assessing aquatic systems. Environmental Monitoring and Assessment 2008;145(1-3):465-473. R829008 (Final)
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  • Journal Article Lorphensri O, Intravijit J, Sabatini DA, Kibbey TCG, Osathaphan K, Saiwan C. Sorption of acetaminophen, 17 α-ethynyl estradiol, nalidixic acid, and norfloxacin to silica, alumina, and a hydrophobic medium. Water Research 2006;40(7):1481-1491. R829008 (Final)
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  • Journal Article Lorphensri O, Sabatini DA, Kibbey TCG, Osathaphan K, Saiwan C. Sorption and transport of acetaminophen, 17α-ethynyl estradiol, nalidixic acid with low organic content aquifer sand. Water Research 2007;41(10):2180-2188. R829008 (Final)
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  • Journal Article Peak N, Knapp CW, Yang RK, Hanfelt MM, Smith MS, Aga DS, Graham DW. Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies. Environmental Microbiology 2007;9(1):143-151. R829008 (Final)
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  • Journal Article Robinson AA, Belden JB, Lydy MJ. Toxicity of fluoroquinolone antibiotics to aquatic organisms. Environmental Toxicology and Chemistry 2005;24(2):423-430. R829008 (2003)
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  • Journal Article 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)
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  • Supplemental Keywords:

    fluoroquinolones, tetracyclines, aquatic systems, photodegradation, adsorption, DGGE, ecotoxicology, antibiotic resistance, quantitative real-time PCR, QRDR regions, horizontal gene transfer, transposons , RFA, Scientific Discipline, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Ecosystem/Assessment/Indicators, Health Risk Assessment, Fate & Transport, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Ecological Risk Assessment, Ecology and Ecosystems, Drinking Water, ecological effects, fate, fate and transport, microbial contamination, monitoring, ecological exposure, human health effects, pharmaceuticals, stressors, antibacterial agents, antibiotics, exposure and effects, exposure, chemical contaminants, fluoroquinolone, microbial effects, microbial risk management, aquatic ecosystems, water quality, drinking water contaminants, other - risk management, toxicokinetics

    Progress and Final Reports:

    Original Abstract
  • 2002 Progress Report
  • 2003 Progress Report
  • 2004 Progress Report
  • 2005 Progress Report