Department of Civil Engineering, University of Minnesota, Minneapolis, Minnesota

Transcription

1 Antibiotic Resistance Gene Concentrations in Duluth-Superior Harbor and in Tertiary-Treated Municipal Wastewater Discharged into Duluth-Superior Harbor Tucker R. Burch, Timothy M. LaPara, Patrick J. McNamara, David T. Tan, Mi Yan & Jessica J. Eichmiller Department of Civil Engineering, University of Minnesota, Minneapolis, Minnesota Department of Bioproducts and Biosystems Engineering, University of Minnesota, St. Paul, Minnesota Department of Soil, Water, and Climate, University of Minnesota, St. Paul, Minnesota Minnesota Water Resources Conference, October 16, 2012, St. Paul, Minnesota

2 Remember two things: 1. Antibiotic resistance genes (ARGs) = an emerging contaminant 2. Treated municipal wastewater is an environmental source of ARGs

3 The problem posed by ARGs: Clatworthy AE, Pierson E, Hung DT Nature Chemical Biology 3(9):

4 ARG Reservoirs:

5 Approach: 1. Collect surface water and sediment samples; extract DNA 2. Quantify ARGs and other gene targets using real-time PCR Gene Target 16S rrna gene Total Bacteroides Human-Specific Bacteroides tet (A) tet (W) tet (X) inti1 Function "biomass" fecal "biomass" human-specific fecal "biomass" efflux pump, tetracycline ribosomal protection protein, tetracycline metabolic degradation, tetracycline integrase, class 1 integron (potential for multiple resistance)

6 Study Area: Western Lake Superior Sanitary District Wastewater Treatment Plant Tertiary Treatment (Mixed Media Filters) LaPara TM, Burch TR, McNamara PJ, Tan DT, Yan M, Eichmiller JJ Environmental Science & Technology 45(22):

7

8 Microbial community analysis: LaPara TM, Burch TR, McNamara PJ, Tan DT, Yan M, Eichmiller JJ Environmental Science & Technology 45(22):

9 16S rrna gene concentrations: gene copies per ml LaPara TM, Burch TR, McNamara PJ, Tan DT, Yan M, Eichmiller JJ Environmental Science & Technology 45(22):

10 gene copies per ml ARG concentrations: gene copies per ml gene copies per ml gene copies per ml LaPara TM, Burch TR, McNamara PJ, Tan DT, Yan M, Eichmiller JJ Environmental Science & Technology 45(22):

11 Fecal indicator concentrations: gene copies per ml gene copies per ml LaPara TM, Burch TR, McNamara PJ, Tan DT, Yan M, Eichmiller JJ Environmental Science & Technology 45(22):

12 Concentrations in sediment: LaPara TM, Burch TR, McNamara PJ, Tan DT, Yan M, Eichmiller JJ Environmental Science & Technology 45(22):

13 Remember two things: 1. Antibiotic resistance genes (ARGs) = an gene copies per ml emerging contaminant 2. Treated municipal wastewater is an gene copies per ml environmental source of ARGs gene copies per ml gene copies per ml Tertiary Treatment

14 Thank you! Questions? Acknowledgments: R/V Blue Heron Bill Arnold and students Dan Engstrom and students NSF Award

15 The Distribution and Persistence of Genetic Markers of Fecal Pollution in Sand and Sediment Jessica Eichmiller 1,2, Michael Sadowsky 1, and Randall Hicks 2 1 University of MN Twin Cities 2 University of MN Duluth

16 Acknowledgments Funding Water Resources Center Biotechnology Institute NIH Biotechnology Training Grant WLSSD Staff Tim Tuominen Joe Mayasich Sample Collection and Processing Qinghong Ran Erik Smith

17 Contamination of recreational beaches is widespread and has negative impacts on human health and local economies.

18 Worst beaches in the Great Lakes 1. Hearding Island, Duluth, MN 2. Winnetka Park Beach, IL 3. St. Clair Shores, MI 4. Edson Creek, OH 5. Lakeview, OH 6. Edgecliff, OH 7. Villa Angela, OH 8. Wisconsin Point, WI 9. Jeorse Park, IN 10.Moss Point, OH This beach is currently under water contact advisory NRDC, 2012

19 Culturable fecal indicator bacteria are used to determine the safety of water contact, but this method is problematic.

20 Quantitative PCR for molecular indicators EPA will publish recommendations for qpcr for Enterococci in 2013 Benefits: Pitfalls: Source-specific; rapid Unknown persistence; few studies consider sand and sediment matrices

21 Beach sand poses health risks

22 WRC Project Goals 1. Characterize the distribution of genetic markers of fecal pollution in water, sand, and sediment on a beach with continuous treated effluent inputs 2. Determine the effect of temperature and moisture on persistence genetic markers in water, sand, and sediment 3. Compare the decay rates of genetic markers and bacterial pathogens in sand

23 Project aims 1. Characterize the distribution of molecular markers. 2. Determine the persistence of molecular markers in the field.

24 Project aims 1. Characterize the distribution of molecular markers. 2. Determine the persistence of molecular markers in the field.

25 Site Lake Superior Duluth-Superior Harbor

26 Quantitative PCR (qpcr) for molecular indicators Entero1: Enterococcus spp.; 23S rrna gene AllBac: Total Bacteroides; 16S rrna gene HF183: Human-specific Bacteroides; 16S rrna gene

27 Effluent controls water column marker concentrations Genetic marker Treated effluent Beach water column Entero1 5.8 ± ± 0.4 AllBac 6.4 ± ± 0.5 HF ± ± 0.8 Mean ± standard deviation log 10 gene copies 100 ml 1

28 Effluent controls water column marker concentrations Beach markers (Log 10 copies/100 ml) Entero1 AllBac HF183 R 2 = 0.77 R 2 = 0.46 R 2 = Effluent markers (Log 10 copies/100 ml)

29 Effluent controls water column marker concentrations Effluent markers (log 10 copies/100 ml) R 2 = 0.80 R 2 = 0.22 R 2 = Entero1 Entero1 3 AllBac AllBac HF183 HF Turbidity (NTU) R 2 = 0.40 R 2 = 0.21 R 2 = Beach markers (Log 10 copies/100 ml)

30 Distribution of culturable enterococci in sand and sediment Sand Sediment

31 The distribution of markers in sand Entero1 AllBac HF183 Depth (cm) 0 2 A B Copies/g dw C Depth (cm)

32 The distribution of markers in sediment Entero1 AllBac HF D E F 0 2 Depth (cm) Depth (cm) Entero1 (markers/g dw) AllBac (markers/g dw) Copies/g dw HF183 (markers/g dw)

33 Concentration of markers in sand and sediment could not be explained solely by water column loading Entero1 negatively correlated to sand markers at 3, 5, 7, and 9 cm negatively correlated to sediment at 1 cm depth AllBac not correlated to sand or sediment markers HF183 positively correlated to 1 and 7 cm depths in sediment

34 Culture and qpcr methods for enterococci are not equivalent Enterococci (CFU or CE per 100 ml or g) 10 6 CE CFU Water Sand Sediment

35 Aim 1 summary Effluent controls fecal marker concentration in the water column. The distribution of genetic markers is similar to that of culturable fecal indicator bacteria. Water column loading does not explain marker distribution in sand and sediment. qpcr and culture assays of enterococci are not equivalent.

36 Project aims 1. Characterize the distribution of molecular markers. 2. Determine the persistence of molecular markers in the field.

37 Field Incubations

38 Water column Entero1 AllBac HF183 Culturable enterococci No change No change Decreased to below detection Decreased to below detection

39 Culturable enterococci in sand and sediment Overall, the increase in percentage of samples below the detection limit was 26% in sand 57% in sediment.

40 Sand and Sediment Percent increase or decrease in copy number Sand Sediment Increase Decrease Increase Decrease Entero AllBac HF Sand: Deceases occurred in August Sediment: Increases occurred in June

41 Aim 2 summary Entero1 and AllBac persist for extended periods of time in water, sand and sediment. HF183 is a good candidate for tracing recent contamination events.

42 Conclusion Sand and sediment can act as a reservoir of molecular markers of fecal pollution on freshwater beaches, given sufficient water column loading. Resuspension of sand and sediment Water quality exceedances Issues for the shift from culturable to molecular indicators

43 Questions

44 Endocrine Disruption in Minnesota Lakes: Past Results and Future Directions Sarah Elliott and Richard Kiesling U.S. Geological Survey Minnesota Water Science Center Minnesota Water Resources Conference October 16, 2012 U.S. Department of the Interior U.S. Geological Survey

45 What is an Endocrine Active Compound? A Compound that Interacts with the Endocrine System Hypothalamus Spleen Ovaries/Testes Pituitary Thyroid Pancreas The Endocrine system is a Chemical Based Communication System

46 Examples of EACs Hormones Alkylphenols Bisphenol A PCBs Pesticides Synthetic Musks Phytoestrogens Pesticides Numerous Potential Sources

47 National stream and ground water reconnaissance studies U.S. Streams U.S. Ground Water Kolpin et al. (2002) Barnes et al. (2008) What about lakes?

48 On-site septic systems a potential source of OWCs to lakes: Approximately 80% of compounds analyzed detected in at least one wastewater sample from residential septic tanks (Conn et al., 2006). Lopid Indocin Aleve Carrara, et al. (2008)

49 Effects on Wildlife Reproductive effects at exposure to low levels (1-10 parts per trillion) of 17α-ethinylestradiol (Kidd, et al. 2007) Prevelance of intersex occurrence in male bass (Hinck, et al. 2009) Kidd, et al. (2007)

50 Objective Identify the occurrence of OWCs (and EACs) in Minnesota lakes and determine to what extent these compounds have an effect on wild fish populations

51 MN Statewide Survey OWCs & EACs present? YES Evidence of endocrine disruption? YES Lake Septic Survey Mesocosm Exposure What are sources of EACs in lakes? Do endocrine disruption and septic influences correlate? MAYBE Population effects in natural settings?

52 2008 Statewide Survey Writer, et al. (2010) Oligotrophic Mesotrophic Eutrophic Water, sediment, passive samplers analyzed for nutrients and OWCs Resident and caged fish processed for signs of endocrine disruption

53 Compounds Analyzed: Hormones (water) Alkylphenols Bisphenol A PCBs Pesticides Synthetic Musks PAHs Pesticides Pharmaceuticals (sediment)

54 Compound Detections in Water Number of Compounds Detected OWC Adapted from Ferrey, et al. (2010) Urban Septic Reference Hormones Commonly Detected Compounds: DEET (100%) Cholesterol (73%) Caffeine (64%) Androstenedione (64%) Estrone (55%) 17β-estradiol (55%) Bisphenol-A (45%)

55 Compound Detections in Sediment Number of Compounds Detected OWC Pharms Adapted from Ferrey, et al. (2010) Urban Septic Reference Commonly Detected Compounds: Cholesterol (100%) Coprostanol (100%) Estrone (91%) 17β-estradiol (91%) Bisphenol-A (82%) Acetaminophen (50%) 4-(tert)octylphenol (45%) 4-octylphenol diethoxylate (36%) Carbamazepine (36%)

56 Biological Results 120 Mean Male Vitellogenin/Mean Female Vitellogenin (%) Sunfish Fathead ND = No Data ND ND ND ND A lot of variation!! No correlation between endocrine disruption and land use or trophic status

57 What Do These Results Mean for Resident Fish Populations? Lepomis macrochirus Pimephales promelas

58 Fish Biomarker Response Fecundity - daily Body Condition Factor (BCF) Gonadosomatic Index (GSI) Hepatosomatic Index (HSI) Miles-Richardson and others 1999 Secondary Sexual Characteristics Plasma Vitellogenin Liver and Gonad Histopathology Wolf and others 2004

59 Evidence of Reproductive Effects F0 FHM Daily Average Cumulative Total Eggs Aug-10 Control 10-Aug-10 E2 17-Aug Aug Aug-10 7-Sep-10

60 Fathead Minnow Results 2.1 F0 Fathead Minnows 2010 Difference driven by dorsal pad thickness and color Body Condition Factor Control E2 * Control E2 * * = significant difference from control (p<0.01) Secondary Sexual Characteristics Index F0 Fathead Minnows Control E2 *

61 Bluegill Results Male Bluegills Gonadosomatic Index Control E2 * *= significant difference from controls (p<0.025)

62 Mesocosm Take Home Only saw what appear to be reproductive effects and body condition differences in parental fatheads; gonad development in bluegills Exposures to a naturally occurring hormone produced only subtle effects What about wild fish exposed to other compounds?

63 Septic Influenced Groundwater in Lakes Present Hormones Steroids Detergents Plastics PAHs Fire Retardants Disinfectants Fragrances Insecticides

64 Study Methods: groundwater and biology HYDRO - GEOLOGY Temperature differential TEMPERATURE SURVEYS Strong Conductivity Gradient Mini-piezometer Isotope Survey Proximity to active fish population ACTIVE FISH

65 Temperature Survey Perry Jones MN WSC USGS

66 Water & Sediment Sampling In-lake SC: ~300 us/cm 566

67 Biological Sampling Analyzed for multiple biomarkers indicative of endocrine disruption Native bluegills collected from nesting area during spawn

68 Preliminary Results from 16 Samples: Frequency of Detection of Waste Indicator Compounds in Water Compound Frequency (%) in MN Lakes Cholesterol 100 DEET 94 beta-sitosterol 88 beta-stigmastanol 63 Atrazine 50 Isophorone 50 Camphor Dimethylnaphthalene 38 2-Methylnaphthalene 31 Bisphenol A 31 Metolachlor 25 1-Methylnaphthalene 25 4-Nonylphenol diethoxylate 19 Benzophenone 19 Pyrene 19 Triphenyl phosphate 19 No significant differences in detection frequency between sites within lakes Higher frequency of NP and BPA at septic sites

69 Compound Frequency (%) Septic Lakes Cholesterol 100 DEET 94 beta-sitosterol 88 beta-stigmastanol 63 Atrazine 50 Isophorone 50 Camphor 38 2,6-Dimehtylnaphthalene 38 2-Methylnaphthalene 31 Bisphenol A 31 Metolachlor 25 1-Methylnaphthalene 25 Compound Frequency (%) MN Lake Water DEET 100 Cholesterol 73 Caffeine 64 Androstenedione 64 Estrone 55 17β-Estradiol 55 Bisphenol A 45 Compound Comparisons Among Frequency (%) Nat'l Stream Survey Coprostanol 86 Cholesterol 84 DEET 74 Caffeine 71 Tri (2-chloroethyl)phosphate 58 Triclosan 58 4-nonylphenol 51 4-NPEO butoxy-phosphate ethanol 46 4-OPEO1 44 Studies Compound Frequency (%) Lake Sediment Cholesterol 100 Coprostanol 100 Estrone 91 17β-estradiol 91 Bisphenol-A 82 Acetaminophen 50 4-(tert)octylphenol 45 4-octylphenol diethoxlyate 36 Carbamazepine 36

70 Summary: OWCs and EACs are being detected in MN lakes Endocrine disruption evidence highly variable, still trying to understand Compounds appear to be accumulating in lake sediments with similar signatures seen in wastewater influenced surface waters

71 Thank You! Sarah Elliott USGS MN Water Science Center

72 Questions? - More Information USGS MN: Follow prompts to /projects/ced/index.html USGS Publications: USGS Information: kiesling@usgs.gov

73 Examples of GW Methods: Perry Jones, Don Rosenberry and others - White Bear Lake GW Inflow Study GW, piezometer, and lake surveys Thermal surveys sediment and water Jones, P Ground-water/surface-water interaction in nearshore areas of Three Lakes on the Grand Portage Reservation, northeastern Minnesota, USGS SIR: Rosenberry, D. And J. LaBaugh (ed) Field Techniques for Estimating Water Fluxes Between Surface Water and Ground Water. USGS Techniques and Methods: 4-D2

74 F0 FHM 2010 F1 Fathead Minnows 2011 Plasma Vitellogenin, parts per billion a Control a E2 b Control b E2 Plasma Vitellogenin Concentration, parts per trillion /0 30/0 30/30 0/30 F0 Bluegills 2011 Some differences, but a lot of variability! Vitellogenin, parts per billion Females Males Control E2 Control E2