1. Eagle Creek Reservoir 2009-2015
Nicolas Clercin, CEES-IUPUI
December, 9th 2015

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

3. Eagle Creek Watershed
CHaracteristics

4. 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., Eagle Creek Watershed Management Plan: An Integrated Approach to Improved Water Quality. Eagle Creek Watershed Alliance, CEES Publication , IUPUI, Indianapolis, 182p.

5. 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

6. 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 = * Ln [TP] (in ug/L) TSI - C = Ln [Chlor-a] (in ug/L) TSI - S = * Ln [Secchi] (in meters) Average TSI = [TSI-P + TSI-C + TSI-S]/3
no data

7. Hydrology
Eagle Creek Reservoir

8. 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

9. 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

10. 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 = meters

11. 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
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

12. 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

13. Seasonal Algal Dynamics
Cyanobacteria
Eagle Creek Reservoir

14. 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

15. 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.

16. 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

17. 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

18. 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

19. 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)

20. Taste and Odor Compounds
Production and occurrences
Eagle Creek reservoir

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

22. 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)

23. 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)

24. 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.

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

26. 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

27. Cyanobacteria and metabolites
Risk assessment
Cyanobacteria and metabolites
Eagle Creek Reservoir

28. Cyanobacteria
Risk assessment
Microcystins

29. MIB
Geosmin

30. 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

31. Freshwater Bacteria
Eagle Creek Reservoir
2013 Dam

32. 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

33. 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.

34. T&O Producers in the reservoir
Algaecide treatment

35. 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

36. T&o degraders in the reservoir
Algaecide treatment

37. 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

38. 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

39. 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

40. 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.

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

42. 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?

43. 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%

44. 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

45. 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

46. 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

47. 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?

48. 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?

49. Questions?
REFERENCES
Cane, D.E., He, X., Kobayashi, S., Omura, S. and H. Ikeda 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 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 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 Sesquiterpenes of the Geosmin-Producing Cyanobacterium Calothrix PCC 7507 and their Toxicity to Invertebrates. Zeitschrift für Naturforschung, 64(1-2):
Hoefel, D., Ho, L., Aunkofer, W., Monis, P.T. , Keegan, A., Newcombe, G. and C.P. Saint 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 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 Biodegradation rates of 2-methylisoborneol (MIB) and geosmin through sand filters and in bioreactors. Chemosphere, 66(11):
Peter, A. and U. Von Gunten Oxidation Kinetics of Selected Taste and Odor Compounds During Ozonation of Drinking Water. Environmental Science & Technology, 42(2):
Wang, C-.M. and D.E. Cane 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 Biodegradation of geosmin in drinking water by novel bacteria isolated from biologically active carbon. Journal of Environmental Sciences, 23(5):