Eagle Creek Reservoir 2009-2015 Nicolas Clercin, CEES-IUPUI December, 9th 2015 nclercin@iupui.edu

outline Eagle Creek Watershed Characteristics Reservoir Hydrology Seasonal Algae dynamics Taste-and-odor compounds Risk assessment Freshwater bacteria Fate of t&O compounds

Eagle Creek Watershed CHaracteristics

Eagle Creek Watershed * Tedesco, L.P., Pascual, D.L., Shrake, L.K., Hall, R.E., Casey, L.R., Vidon, P.G.F., Hernly, F.V., Salazar, K.A., Barr, R.C., 2005. Eagle Creek Watershed Management Plan: An Integrated Approach to Improved Water Quality. Eagle Creek Watershed Alliance, CEES Publication 2005-07, IUPUI, Indianapolis, 182p.

Reservoir Characteristics Eagle Creek Units Surface Area 5 km2 Reservoir Volume 21 million m3 Mean Depth 4.2 m Watershed Area 420 Watershed : Reservoir Area Ratio 84 % Agriculture/ % Urban 49% / 18%   Trophic Status Eutrophic Samples collected biweekly: From April to October, each year Composite (photic zone) + bottom samples Physico-chemical parameters

Eagle Creek Reservoir Trophic Status Index (TSI) TSI (Carlson 1977) TROPHIC STATUS INDEX & WATER QUALITY (for additional information, go to: MPCAs Citizens Monitoring Handbook) < 30 Oligotrophic; clear water; high DO throughout the year in the entire hypolimnion 30-40 Oligotrophic; clear water; possible periods of limited hypolimnetic anoxia (DO =0) 40-50 Moderately clear water; increasing chance of hypolimnetic anoxia in summer; fully supportive of all swimmable/aesthetic uses 50-60 Mildly eutrophic; decreased transparency; anoxic hypolimnion; macrophyte problems; warm-water fisheries only; supportive of all swimmable/aesthetic uses but "threatened" 60-70 Blue-green algae dominance; scums possible; extensive macrophyte problems 70-80 Heavy algal blooms possible throughout summer; dense macrophyte beds; hypereutrophic > 80 Algal scums; summer fish kills; few macrophytes due to algal shading; rough fish dominance   TSI - P = 14.42 * Ln [TP] + 4.15 (in ug/L) TSI - C = 30.6 + 9.81 Ln [Chlor-a] (in ug/L) TSI - S = 60 - 14.41 * Ln [Secchi] (in meters) Average TSI = [TSI-P + TSI-C + TSI-S]/3 no data

Hydrology Eagle Creek Reservoir 2008-2015

2008-2015 Precipitations 14.3 15.4 16.0 0.91 0.92 Jan Feb Mar Apr May   Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Monthly Avg (cm) 0.35 0.45 0.61 0.88 0.76 0.91 0.92 0.80 0.66 0.60 0.53 0.44 # Rainy days 17.9 15.3 12.9 14.3 15.4 16.0 12.1 10.0 11.9 13.0 12.5 17.4

2008-2015 Inflow Discharge 53.38 58.13 69.13 49.25 75.40 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Mean (m3/s) 37.46 53.38 58.13 69.13 49.25 75.40 21.90 3.15 3.17 4.64 15.01 38.21 N 248 226 240 239 217 95th Pct 137.73 181.11 182.75 246.99 217.75 331.63 78.28 7.93 8.79 14.73 58.40 141.66

2008-2015 Water Elevation Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Mean 240.48 240.31 240.33 240.95 241.05 241.12 241.02 240.82 240.50 240.26 240.29 240.37 N 248 226 240 239 217 95th Pct 240.76 240.69 240.91 241.23 241.22 241.30 241.31 241.26 241.09 240.59 240.64 240.73 Max. Reservoir Water Elevation = 790 ft = 240.79 meters

2008-2015 Retention Time 3-6 months 1-6+ years 118.06 142.53 176.09 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Mean 220.01 202.79 118.06 142.53 176.09 393.68 1079.90 1923.37 2258.08 1539.64 1138.10 460.29 N 248 226 240 239 217 5th Pct 19.78 17.69 18.70 10.11 15.70 10.31 43.70 421.35 377.14 159.54 53.43 20.82 95th Pct 928.34 1,005.70 323.17 389.95 524.71 2,514.25 3,893.04 5,485.65 4,161.53 4,022.81 1,885.69 3-6 months 1-6+ years

Hydrology - Summary Precipitations: High monthly discharge: Highest rainfall in June/july (>0.90 cm) rainy days between april and june High monthly discharge: >50 m3/s: February/june Water elevation Above 790ft (max Elev.): Full capacity between April and August Retention time: 3-6 months (Jan/May): Riverine system 1-6+ years (June/Dec): Lacustrine system

Seasonal Algal Dynamics Cyanobacteria Eagle Creek Reservoir

Bacterial Metabolites Central to many source-water issues; Algal Toxins: Water soluble Non-volatile and Non-odorous Virtually undetectable by consumers Potentially harmful WHO standards: <10 ug/L in recreational waters (Indiana: <6 ug/L) <1 ug/l in drinking waters Taste-and-Odor (T&O) Compounds: Volatile and Odorous No known effects on human health No EPA standards

Challenges In aquatic ecosystems, it is difficult to link T&O occurrences with specific taxa; Diverse sources: planktic, epiphytic or benthic assemblages; Co-occurring taxa may produce similar compounds; Cyanobacteria (=aquatic bacteria); Actinobacteria (=‘soil’ bacteria); Species abundance or dominance may show no apparent relationship with T&O levels; Peak production and population density can be asynchronous; Sub-dominant species might be the producers; Biosynthesis of each metabolite can vary significantly within and among taxa.

inter-Annual Variability 2009-2015 Cyanobacteria No data Dotted Lines: Low risk (blue) 20,000 cells/mL Moderate risk (green) 100,000 cells/mL High Risk (red) 500,000 cells/mL Very High Risk 1,000,000 cells/mL Probability of threshold exceedance Risk Threshold ECR dam ECR 56th >20,000 0.547 0.617 >100,000 0.217 0.325 >500,000 0.003 0.029

Monthly Variability 2009-2015 Cyanobacteria Dotted Lines: Low risk (blue) 20,000 cells/mL Moderate risk (green) 100,000 cells/mL High Risk (red) 500,000 cells/mL Very High Risk 1,000,000 cells/mL Risk Threshold May Jun Jul Aug Sep Oct >20,000 cells/mL 0.50 0.57 0.83 1.00 0.96 >100,000 c./mL 0.17 0.18 0.34 0.79 0.90 0.40 >500,000 c./mL 0.00 0.02 0.11 0.03

Monthly variability 2009-2015 microcystins Drinking water LOQ Dotted Lines: LOQ (blue) 0.15 ug/L (ppm) Drinking water (green) 1 ug/L (ppm) Raw/Recreation (red) 6 ug/L (ppm) Method: ELISA test (Abraxis) Risk Threshold April May Jun Sep Nov Detections 0.29 0.35 0.26 0.34 0.38 >1 ug/L 0.00 0.04 >6 ug/L

Seasonal dynamics – summary cyanobacteria and microcystins Higher probability to get a severe bloom near the intake (vs. dam) Upper areas more vulnerable than the dam risk of cyanobacterial blooms: Moderate in Aug/Sept; High in august; Microcystins are detected 21% of the time Highest probability to exceed 1 ug/l in RAW Water is less than 4% (in November)

Taste and Odor Compounds Production and occurrences Eagle Creek reservoir

After Peter and Von Guten. Environ. Sci. Technol. 2007, 41: 626-631

MIB – Inter-annual variability OTC MIB 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015* #samples 65 30 39 49 56 164 210 218 155 180 245 135 98 %MIB>2 46.2 26.7 28.2 34.7 60.7 91.5 71.4 91.3 98.1 65.0 77.6 69.0 49.6 86.7 OTC = 10 ng/L (ppt)

GSM – Inter-annual variability OTC GSM 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015* #samples 65 30 39 49 56 164 210 218 155 180 245 135 98 %GSM>2 1.5 16.7 43.6 53.1 66.1 95.1 90.5 98.6 89.7 88.3 95.9 100.0 75.6 98.0 OTC = 10 ng/L (ppt)

Taste-and-Odor Episodes No data * * Algaecide treatment OTC OTC Long-term patterns show that T&O episodes are highly variable in: Frequency: 0-4 events/year Duration: MIB <3 months; GSM >3 months Intensity: 10 ppt (>OTC) – several hundreds ppt.

Monthly variability mib+gsm 2009-2015 eagle creek reservoir OTC Spring Fall OTC 3,297* OTC = 10 ng/L (ppt)

T&O compounds - summary T&o episodes are highly variable every year MIB and GSM do not necessarily co-occur However, both compounds tend to occur during the spring and/or fall Risk of bloom increases (>60%) between june and November Meanwhile, Toxin production also increases but barely exceeds 1 ug/L in raw water! Gsm has a high rate of detections (>80%) all year round Major Risk of outbreak (>50%) in may and Dec/Jan Most mib detections occur between may and October Major risk of outbreak (>50%) in may and September

Cyanobacteria and metabolites Risk assessment Cyanobacteria and metabolites Eagle Creek Reservoir 2009-2015

Cyanobacteria Risk assessment Microcystins

MIB Geosmin

Probability of Metabolite co-occurrences 2009-2015 MIB and GSM tend to co-occur >70% MIB and GSM have same chance to co-occur with Microcystin MC/MIB and MC/GSM are similar ~20% All metabolites together co- occur ~20% of the times % Co-Occurrences Reservoir Dam 56th %MC/MIB 20.5 19.7 21.4 %MC/GSM 21.6 20.8 22.4 %MIB/GSM 70.4 73.1 67.2 %MC/MIB/GSM 19.8 19.4 20.1 # samples 668 360 308

Freshwater Bacteria Eagle Creek Reservoir 2013 Dam

2013 Bacterial Populations Metagenomic Analysis (MiSeq, Illumina Inc.) 16S rRNA ~ 1,500 base pairs long Primer pair sequence for V3 and V4 regions Amplicon ~ 460 bp

2013 Bacteria in the reservoir Samples collected between May and October 2013 Near the dam (seasonal stratification); Discrete depths: 0, 3, 6 and 10 meters; Spatial and temporal analysis: Bacteria in the water column; MIB and geosmin.

T&O Producers in the reservoir Algaecide treatment

T&O producers (Genus-level) MIB Georgenia (p<0.01) Cryobacterium (p<0.01) Sanguibacter (p<0.01) Streptomyces (p<0.01) [Jüttner and Watson, 2007] Saccharomonospora (p<0.01) Negative correlation with all cyanobacterial taxa Geosmin Calothrix (p<0.05) [Höckelmann et al., 2009] Cyanobacterium (p<0.05) Microcoleus (p<0.01) [Jüttner and Watson, 2007] Nostoc (p<0.01) [Giglio et al., 2008] Phormidium (p<0.01) [Jüttner and Watson, 2007] Planktothrix (p<0.001) [Jüttner and Watson, 2007] No correlation with Actinomycetales ! Producers MIB GSM Cyanobacteria Aphanizomenon -0.60** -0.16 Calothrix -0.31 0.32 Cyanobacterium -0.27 0.37 Microcoleus -0.17 0.45* Microcystis -0.58** -0.18 Nostoc -0.22 0.44* Phormidium -0.15 0.42* Planktothrix 0.53** Pseudanabaena -0.59** 0.04 Actinomycetales Georgenia 0.40* Cryobacterium -0.05 Sanguibacter 0.41* -0.06 Streptomyces -0.13 Saccharomonospora -0.11 Significant correlation in yellow p<0.05, * p<0.01 and ** p<0.001

T&o degraders in the reservoir Algaecide treatment

T&O Degraders (Genus-level) MIB Novosphingobium (p<0.05) [Ho et al., 2007] Sphingobium (p<0.001) [Ho et al., 2007] Sphingomonas (p<0.001) [Ho et al., 2007] Comamonas (p<0.05) [Guttman and van Rijn, 2012] Variovorax (p<0.05) [Guttman and van Rijn, 2012] Pseudomonas (p<0.001) [Ho et al., 2007] Flavobacterium (p<0.001) [Ho et al., 2007] Geosmin Potential degraders Sphingomonas (p<0.01) Leptothrix (p<0.01) Chryseobacterium (p<0.01) [Zhou et al., 2011] Known degraders [Hoefel et al., 2006]: Novosphingobium Sphingopyxis Pseudomonas No correlation observed here Degraders MIB GSM Sphingomonadales Novosphingobium 0.31 -0.02 Sphingobium 0.57** 0.06 Sphingomonas 0.56** 0.37* Sphingopyxis 0.21 -0.30 Burkholderiales Acidovorax 0.38 -0.13 Comamonas 0.34 Curvibacter 0.33 -0.10 Leptothrix 0.05 0.43* Rhodoferax 0.42* -0.12 Variovorax 0.32 -0.25 Janthinobacterium 0.22 Pseudomonadales Pseudomonas 0.51** 0.02 Flavobacteriales Chryseobacterium Flavobacterium 0.48** 0.08 Myroides 0.46* 0.01 Psychroflexus -0.14 Tenacibaculum 0.35 Significant correlation in yellow p<0.05, * p<0.01 and ** p<0.001

Temporal Distribution of Samples Legend: Zmix= Mixing Depth kT = light extinction coefficient RTRM= Relative Thermal Resistance to Mixing Summer Stratification Bottom Anoxia T&O Event Reservoir Turnovers

Temporal Distribution of Bacteria Legend: Zmix= Mixing Depth kT = light extinction coefficient RTRM= Relative Thermal Resistance to Mixing Reservoir Turnovers Summer Stratification Bottom Anoxia T&O Event

Freshwater bacteria - summary Taste-and-Odor producers: Highly influenced by hydrology: High discharge periods; Nutrient inputs from watershed; Water column: Fully/partially mixed; Organisms involved: Cyanobacteria; Actinomycetes. TASTE-AND-ODOR DEGRADERS: CO-OCCUR ALONG WITH PRODUCERS; HYPOLIMNION: SEDIMENT; WATER/SEDIMENT INTERFACE; ORGANISMS INVOLVED: SPHINGOMONADALES; BURKHOLDERIALES; PSEUDOMONADALES; FLAVOBACTERIALES. Sphingobium sp. Streptomyces sp.

Eagle Creek reservoir sediments Fate of T&O Compounds Sorption Experiments Eagle Creek reservoir sediments

Fate of T&O compounds There is currently no knowledge about the fate of MIB and GSM once released into the water: Naturally after cellular death; Chemically after algaecide treatment. Investigations: Do metabolites sorb onto bottom sediments? Can sediments desorb these compounds?

Eagle Creek Sediments Grain Size ANalysis % Clays % Silts % Sands Total Dam 46.0 52.6 1.4 100 56th 31.5 49.3 19.2 Upper 25.6 70.9 3.5 Sediment cores were collected in May 2015 Sorption experiments conducted on Upper Basin sediments Silty – 71%

Desorption of MIB and GSM Sediment in DI water Glass jars Teflon caps Spme-GC/mS analysis No significant increase of both Mib and GSM after 1 day Possible bacterial degradation in sediment

Sorption of mib and gsm Sediment in DI water, spiked with mib/gsm solution at 100 ng/L (ppt) Preliminary results show: Both compounds can sorb; Kinetics of the reactions is slower than expected: >1 day

Percent Removal from Sediments day 5 day 3 day 1 Removal Rates after 5 days: >80% for GSM; 16.6 ng/L.day-1 >30% for MIB; 7.9 ng/L.day-1 % Removal MIB GSM 1 hr 0.07 18.39 2 hrs 2.20 16.34 3 hrs 1.59 17.19 6 hrs 4.60 15.92 1 day 14.49 46.60 3 days 30.63 82.67 5 days 37.46 88.67

Future steps? Sorption experiments Keep investigating sorption capacities of MIB+GSM onto natural sediments Way to mitigate T&O in the water? Remediation? Mining of the metagenomics dataset Looking for gene sequences coding for: Genes responsible for MIB/GSM production Identification of producers Adapted treatment? Gram+ vs gram- Genes involved in the degradation Identifications of degraders Bioremediation?

Recommendations Treatments can be planned accordingly with: Weather predictions: blooms breakup with heavy rains (ex. June) Blooms flushed during short retention times (<15 days) Reservoir hydrology: Riverine system: production of MIB + Gsm Lacustrine system: sporadic production due to punctual blooms Manipulating mixing of the water column may enhance t&O production. Low production (<OTC) during reservoir stratification Watershed inputs are critical for the nutrient supply to the reservoir and t&o production: NH3 and TP correlated with T&O events Treatment optimization based on specific microbial taxa: Cyanobacteria = algaecide effective, with potential release of toxins but low toxicity risk Actinobacteria = algaecide less effective, high G+C content (~70%), nitrogen addition with copper- amine treatment = problem! How do we best optimize treatment when actinobacteria are dominant?

Questions? REFERENCES Cane, D.E., He, X., Kobayashi, S., Omura, S. and H. Ikeda. 2006. Geosmin Biosynthesis in Streptomyces avermitilis. Molecular Cloning, Expression, and Mechanistic Study of the Germacradienol/Geosmin Synthase. The Journal of Antibiotics, 59(8): 471–479. Giglio, S., Jiang, J., Saint, C., Cane D.E. And P.T. Monis. 2008. Isolation and Characterization of the Gene Associated with Geosmin Production in Cyanobacteria. Environmental Science &Technology, 42(21): 8027–8032. Guttman, L. and J. Van Rijn. 2012. Isolation of Bacteria Capable of Growth with 2-Methylisoborneol and Geosmin as the Sole Carbon and Energy Sources. Applied and Environmental Microbiology, 78(2): 363–370. Höckelmann, C., Becher, P.G., von Reuß, S.H. and F. Jüttner. 2009. Sesquiterpenes of the Geosmin-Producing Cyanobacterium Calothrix PCC 7507 and their Toxicity to Invertebrates. Zeitschrift für Naturforschung, 64(1-2): 49-55. Hoefel, D., Ho, L., Aunkofer, W., Monis, P.T. , Keegan, A., Newcombe, G. and C.P. Saint. 2006. Cooperative biodegradation of geosmin by a consortium comprising three gram-negative bacteria isolated from the biofilm of a sand filter column. Letters in Applied Microbiology, 43: 417–423. Jüttner, F. and S.B. Watson. 2007. Biochemical and Ecological Control of Geosmin and 2-Methylisoborneol in Source Waters. Applied and Environmental Microbiology, 73(14): 4395–4406. Ho, L., Hoefel, D., Bock, F. Saint, C.P. and G. Newcombe. 2007. Biodegradation rates of 2-methylisoborneol (MIB) and geosmin through sand filters and in bioreactors. Chemosphere, 66(11): 2210-2218. Peter, A. and U. Von Gunten. 2007. Oxidation Kinetics of Selected Taste and Odor Compounds During Ozonation of Drinking Water. Environmental Science & Technology, 42(2): 626-631. Wang, C-.M. and D.E. Cane. 2008. Biochemistry and Molecular Genetics of the Biosynthesis of the Earthy Odorant Methylisoborneol in Streptomyces coelicolor. Journal of American Chemical Society, 130: 8908–8909. Zhou, B., Yuan, R., Shi, C., Yu, L., Gu, J., and C. Zhang. 2011. Biodegradation of geosmin in drinking water by novel bacteria isolated from biologically active carbon. Journal of Environmental Sciences, 23(5): 816-823.