2007 Eastern South Dakota Water Conference 52 nd Annual Midwest Groundwater Conference Sioux Falls, SD October 31, 2007

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1 Impact of antimicrobial compounds Tylosin and Chlorotetracycline during swine manure treatment James Stone Assistant Professor South Dakota School of Mines and Technology 2007 Eastern South Dakota Water Conference 52 nd Annual Midwest Groundwater Conference Sioux Falls, SD October 31, 2007 Special thanks to: National Science Foundation CBET (grants and ) National Science Foundation RET program National Science Foundation MRI program (DBI grant ) South Dakota Governor s 2010 Individual Seed Grant

2 Environmental Impacts from the Custer National Forest Abandoned Uranium Mines Harding County, South Dakota Tuombe See Emmanuel Tuombe POSTER Lake Isabel Lake Hurley Corson Harding Perkins Dewey Butte Ziebach Meade Lawrence Stanley Haakon Campbell Walworth Potter Sully Hughes W HW81 Twin Lakes McPherson Marshall Brown Roberts Edmunds Day Grant Faulk Spink Codington Clark Deuel Hamlin Hyde Hand Beadle Kingsbury Brookings Buffalo Jerauld Pennington Sanborn Miner Lake Moody Jones Lyman Bitter Lake Development of Mercury TMDL for South Dakota lakes with current fish consumption advisories Custer Fall River Shannon Jackson Bennett Mellette Todd Tripp Brule Gregory Roosevelt Lake Aurora Hanson Minnehaha Davison McCook Douglas Hutchinson Turner Lincoln Charles Mix Bon HommeYankton Clay Union North Island

3 Sources of EDCs into eastern South Dakota waterways Big Sioux River Basin National Atlas.gov 125 organic wastewater compounds monitored Human Rx Human and vet antimicrobials Ag herbicides PAHs Sterol compounds Common household organics 45 compounds had conc > analytical reporting limits Sando,, S.K., Furlong, E.T., Gray, J.L., Meyer, M.T.,., and Bartholomay,, R.C., 2005, Occurrence of organic wastewater compounds in wastewater effluent t and the Big Sioux River in the Upper Big Sioux River Basin, South Dakota,, : 2004: USGS Scientific Investigations Report

4 Sources of EDCs into eastern South Dakota waterways Big Sioux River Basin Brookings, SD findings: National Atlas.gov WWTF discharge did not substantially contribute to total organic conc. Upstream non-point ag runoff and EDC detected Human and vet antimicrobial commonly found upgradient,, at conc significantly greater than US average Sando,, S.K., Furlong, E.T., Gray, J.L., Meyer, M.T.,., and Bartholomay,, R.C., 2005, Occurrence of organic wastewater compounds in wastewater effluent t and the Big Sioux River in the Upper Big Sioux River Basin, South Dakota,, : 2004: USGS Scientific Investigations Report

5 Swine production Cattle/dairy production National Atlas.gov With increased confined feedlot operations, manure handling has dramatically increased. In US 62 million swine currently in production, and increasing 1 million swine/yr (faster than ethanol plants?) In addition, the population base of eastern South Dakota has increased, straining existing water and wastewater treatment facilities.

6 Antibiotic/Antimicrobial Use therapeutically to treat existing disease conditions subtherapeutical doses in presence of pathogens or high stress environments subtherapeutically to enhance growth predominant use of tetracyclines and bacitracin in the swine industry poorly absorbed in the gut and consequently, substantial amounts of these compounds and their breakdown products are excreted

7 Antibiotic/Antimicrobial Use Chlorotetracycline (CTC) Tylosin Feed additive for growth promotion, > 68% swine CAFO usage Affect wide range of gram-positive and gram- negative bacteria Interrupt aminoacyl trna interaction resulting inhibition of the protein synthesis Inhibit acetoclastic methanogens,, a primary intermediate for anaerobic methane production Macrolide class, most commonly administered (>48% swine CAFOs) Inhibits protein synthesis by binding to the 23S rrna Tylosin D (relomycin( relomycin), and Tylosin A (tylosin)) most commonly excreted

8 92% swine CAFOs reported antibiotic use (USDA 2002) Significant discrepancies in the amount antibiotic or antimicrobial ial usage: Union of Concerned Scientists (2001) 24.6 million pounds of antibiotic were used for non-therapeutic purposes, and 50 million pounds total Animal Health Instituite (2001) 20.5 million pounds of antibiotics sold for all animal use Lack of publicly available data, however in United States, it is clear that antibiotics are in heavy use in animal agriculture

9 Environmental Pathways Antimicrobials only partially metabolized up to 75% excreted The land application of manure large areal scale of antimicrobials Excretion of waste products by grazing animals Atmospheric dispersal of feed and manure dust containing antimicrobials Incidental release of products from spills or discharge Transported either in a dissolved phase or (ad)sorbed to colloids or soil particles in surface- and ground-water

10 Environmental Mobility Log K ow (measure of hydrophobicity) typically < 5 relatively non-hydrophobic Water solubility generally exceed 1 g/l relatively hydrophilic K d values are wide ranging (0.2-6,000 L/kg) Cationic exchange, surface complexation, and hydrogen bonding are the likely mechanisms for sorption to soils. Tetracyclines can adsorb strongly to clays, soils, sediments immobile Tylosin have a weaker tendency to sorb to soil materials relatively immobile Sulfonamides exhibit weak sorption to soil very mobile Because of the strong sorption tendency of tetracycline and macrolide antibiotics, their mobility may be facilitated by transport with manure and soil colloidal materials

11 Environmental Persistence Half-lives lives typically less than manure storage period significant degradation of the parent compounds prior to land application Tetracyclines most persistent (> 100 days) Tylosin: Tylosin B, and D lost within 30 to 130 hours in anaerobic manure (Kolz etal 2005), likely due to sorption Residual Tylosin and breakdown product (dihydrodesmycosin( dihydrodesmycosin) found in slurries after eight months (Kolz etal 2005) Application of manure to agricultural fields introduces breakdown products and parent compounds into the environment Persistence data for degradation products is unknown

12 Gestation, farrowing,, nursery, finishing facilities Manure waste storage: a a slatted floor over a deep concrete pit a a slatted floor over a shallow pit slatted floor over a shallow pit with outdoor anaerobic lagoon Individual swine typically produce 1.4 tons each of fresh manure in the 5-6 months to reach market weight 185 million head of swine were sold in the U.S. in 2002 producing 1.3x10 8 tons of fresh manure 53.9% of CAFOs were within one mile of another site (USDA 2001). In some regions, CAFO manure likely exceeds regional lands assimilative capacity CAFO Operations / pubs/ip/ip71/ip71.htm

13 Degradation of antimicrobial agents Tylosin and Chlortetracycline during swine manure treatment Study the occurrence and degradation pathways of these emerging environmental contaminants within the growing swine and cattle confined feedlot industry Overarching objectives to determine: changes in treatment efficiency contaminant removal pathways impacts to microbial consortia Dr. J. Stone SDSM&T due to tylosin and CTC usage within swine manure handling systems in cold weather conditions common to upper Midwest US & Canada Dr. S. Clay SDSU

14 Tylosin A Swine metabolism crates containing 9-20kg 9 male pigs 3 treatments: control, + tylosin, + CTC Corn-soybean meal diets identical except antimicrobials 100 g/kg feed and 22 mg/kg body weight = ~ 20 mg/l tylosin

15 50 g anaerobic batch reactors (started( April 07): 33 treatments +/- CTC; +/- Tylosin; +/- Control; o C 26 sacrificed sampling points (~ biweekly monitoring) for ~ 180 days scod,, ph, alkalinity, conductivity, ORP, TSS, VSS, VFA, TOC, nitrate, ammonia (SDSM&T( SDSM&T) Continuous monitoring of CH 4, CO 2, H 2 S (SDSM&T BCF) Residual CTC, Tylosin concentration (SDSU( SDSU) Microbial community dynamics as f(ctc, Tylosin) using T-RFLP for microbial phylogenetic profiling (BHSU)

16 No antimicrobial control, CTC, Tylosin HIGHER MOLECULAR COMPOUNDS FROM SWINE MANURE INSOLUBLE ORGANICS PROTEINS CARBOHYDRATES LIPIDS HYDROLYSIS SOLUBLE ORGANICS AMINO ACIDS SUGAR FATTY ACIDS ALCOHOL FERMENTATION ORGANIC ACIDS Anaerobic lag with CTC ACETATE, HYRDOGEN SULFUR INTERMEDIATE PRODUCTS e.g. PROPIOINC ACID, ISOBUTYRIC ACID, BUTYRIC ACID... HYDROGEN, CARBON DIOXIDE METHANOGENESIS ACETOCLASTIC METHANOGENESIS METHANE, CARBON DIOXIDE HYDROGENTROPHIC METHANOGENESIS FERMENTATIVE BACTERIA-CHEMOHETEROTROPH NONMETHANOGENS ACID FORMAING BACTERIA HYDROGEN-PRODUCING ACETOGENIC BACTERIA Adapted from Masse et al, 2000 ACETOCLASTIC METHANOGENS CARBON DIOXIDE-REDUCING METHANOGENS

17 Control vs Tylosin ph & Alkalinity - Control & Tylosin ph scod [mg/l] Time [days] Control ph Control Alkalinity Tylosin ph Tylosin Alkalinity COD & VSS - Control & Tylosin Alkalinity [mg/l] VSS [mg/l] Tylosin Greater yet consistent alkalinity Tylosin Increasing scod (hydrolysis), similar to control Results similar to literature: no change in scod reduction with Tylosin (Masse 2000) no Time [days] 5000 Control COD Control VSS Tylosin COD Tylosin VSS

18 Control vs Tylosin CO2 (ml/day/g-vss) CH4 (ml/day/g-vss) Control Tylosin Time (days) Control Tylosin Time (days) Tylosin similar CH 4 and C0 2 generation rates as Control Acetoclastic methanogens (Acetate to CH 4 ) and Hydrogentrophic methanogens (H & CO 2 CH 4 ) activity appear unaffected by Tylosin In Literature: (Masse 2000) no change in gas generation due to Tylosin

19 Control vs CTC ph and Alkalinity - Control & CTC ph Time [days] Alkalinity [mg/l] CTC greater acidification (acetate production) and alkalinity Control ph Control Alkalinity CTC ph CTC Alkalinity scod [mg/l] COD & VSS - Control & CTC Time [days] VSS [mg/l] CTC Greater extent of VSS destruction, greater rate of hydrolysis (increased scod) Control COD Control VSS CTC COD CTC VSS

20 CH4 (ml/day/g-vss) CH4 (ml/day/g-vss) CO2 (ml/day/g-vss) CO2 (ml/day/g-vss) Control vs CTC Control Control CTC CTC Time (days) Time (days) Control Control CTC CTC Time (days) Time (days) CTC consistently lower CH 4 and CO 2 generation rates as f(time) Acetoclastic methanogens (Acetate to CH 4 acid conditions??) and Hydrogentrophic methanogens (H & CO 2 CH 4 ) activity appear affected by CTC Similar to (Masse 2000) gas generation reduced 30-40%

21 Volatile Fatty Acids Increased acetate accumulation for CTC Hydrolysis of solid material is occurring Utilization of acetate by methane formers (acetoclastic( methanogens) appears rate limiting Explains drop in ph and reduced CH 4 production Increased proprionate,, butyrate for CTC Fermentation inhibition to acetate and CO 2 Inhibition of hydrogentrophic methanogens (H, CO 2 CH 4 ; Fukazaki 1990) Total VFA elevated for CTC Inhibition of methane formers?

22 Characterization of Methanogenic Population Dynamics rdp8.cme.msu.edu/html/t- rflp_jul02.html T-RFLP: : Profiling microbial diversity, structure, and dynamics Polymerase chain reaction (PCR) amplification targeting 16S rrna Dr. Garth Spellman 3 terminal restriction fragments (T-RFs RFs) ) selected allowing coverage of Archaea individual methanogen groups (Padmasiri 2007) T-RF peaks > 50 florescence units considered operational taxonomic units (OTUs( OTUs), with relative abundance = ratio peak area / sum peak areas

23 Characterization of Methanogenic Population Dynamics Relative Abundance 70% 60% 50% 40% 30% 20% 10% 0% Control Mse103 CTC Mse103 Tylosin Mse Time (days) Methanosactaceae - Msel 103 Acetate substrate Acetoclastic methanogens Control populations highest, confirm acetate utilization and CH 4 production rates CTC and Tylosin populations inhibited 70% Relative Abundance 60% 50% 40% 30% 20% 10% 0% Control Alul341 CTC Alul341 Tylosin Alul Time (days) Methanobacteriales - Alul 341 Hydrogen substrate Hydrogentrophic methanogens Again, Control greatest CH 4 and CO 2 generation What about similarities between Tylosin and Control for scod, VSS, CH 4??

24 Characterization of Methanogenic Population Dynamics Relative Abundance Relative Abundance 70% 70% 60% 60% 50% 50% 40% 40% 30% 30% 20% 20% 10% 10% Control Mse474 CTC Mse474 Tylosin Mse474 Control Mse474 CTC Mse474 Tylosin Mse474 0% 0% Time (days) Time (days) Methanosarcinaceae Alul 474 Acetate, C-1 C substrates Acetoclastic methanogens Tylosin populations highest, but decreasing CTC populations inhibited Similarities in CH 4 generation, and VSS, scod utilization between Control and Tylosin,, however different Acetoclastic methanogens (Alul 474 vs Msel 103) appear responsible

25 Experiments ongoing: 55 sampling events remaining Tylosin and CTC residual concentrations (SDSU) Total organic, inorganic carbon NH 4, TKN C C mass balance

26 Key Findings CTC feed: Inhibition of CH 4, CO 2 production Increased hydrolysis through greater scod production Greater VFA accumulation Decreased Acetoclastic and Hydrogentrophic methanogen populations Tylosin feed: Similar to Control for acetate utilization Decreased Msel 103 Acetoclastic and Alul 341 Hydrogentrophic methanogens,, but increased Alul 474 Acetoclastic methanogen populations No effect on CH 4, CO 2 production or scod consumption

27 The Big Picture Results show antimicrobial usage elicits fundamental changes in methanogen populations Increased land application of antimicrobial-containing containing manure may have profound implications, especially wrt gene transfer with native soil bacteria emergence of new antimicrobial resistant strains? 60 Atrazine Mineralization Study Soil - May % of 14C mineralized Manure Biosolid Soil Bio + Tet Dr. S. Clay SDSU 10 Manure + CTC Sampling day

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