1. Montecito Sanitary District Ocean Outfall Study Carter Ohlmann 1, Trish Holden 1, Libe Washburn 1, Laurie Van De Werfhorst 1, Bram Sercu 1, Cindy Wu 2, and Gary Andersen 2 1. Institute for Computational Earth System Science, University of California, Santa Barbara 2. Center for Environmental Biology, Lawrence Berkeley National Laboratory, Berkeley, CA Acknowledgements. Funding for this project has come from the California State Water Resources Control Board through the California Clean Beaches Initiative. This work would not have been possible without the support and cooperation of Heal the Ocean and the Montecito Sanitary District.

2. Outline Background and motivation Experimental design, what was measured and why Data from a few specific days to illustrate the design Results: - buoyant plume modeling - surface current trajectories - salinity signal - microbiology of effluent - microbiology following plume motion in the ocean Conclusions

3. Background Historical AB411 beach water quality at Hammonds Beach. Does MSD effluent plume contribute to poor water quality at Hammonds Beach?

4. UCSB proposed a project with two primary goals: Goal #1: Observe pathways of a wastewater plume and plume composition along path. Goal #2: Determine if/how effluent plume contributes to poor water quality at shoreline. Background

5. Experimental design – monitor plume microbiology from source to destination following plume motion 1. Effluent sample and flow rate at source (MSD) November 2007 through November 2008 0 to 4 m 3 sec -1 0 to 18 inches rain Microbiological sampling for: FIB using IDEXX (total coliform, E. coli, enterococci; 50 sampling days) Nutrients (phosphate, nitrate+nitrite, ammonia; 50 sampling days) DNA using TRFLP (10’s of microbes; 26 sampling days) DNA using PhyloChip (1000’s of microbes; 8 sampling days)

6. 2. Mooring at diffuser measures current and temperature profiles to understand plume surfacing. mostly down-coast flow mostly very weak offshore flow near surface Temperature stratification during summer Mostly small short period wind waves

7. 3. Drifting buoys follow surface waters that emanate from a location above the diffuser. Ohlmann et al. 2005, and Ohlmann et al. 2007 Microstar Drifters: tags water between ~0.5 and 1.5 m 10 minute position sampling w/ GPS 1 cm/s slip in 10 m/s wind 6 units deployed at diffuser each week 6 units deployed further offshore each week recovered when reach water depth of ~3 m direct measure of horizontal mixing

8. 4. CTD profiles and water samples following drifter motion to monitor changes in plume salinity and microbiology. Niskin bottle collects depth integrated water sample over top meter of ocean. Conductivity, Temperature, Depth (CTD) profiler to identify fresh plume signal.

9. Observational Plan: coast line boat sampling microbiology and CTD drifter releases mooring measures temp and currents at diffuser + outfall outfall plume shoreline and effluent microbiology Weekly (once per week) sampling for 1 year off Santa Barbara (CA) coast.

10. Observations of total coliform following drifter motion 14.5 in effluent < 10 at all ocean samples following drifter motion Total coliform from water samples collected on June 2, 2008 showing a weak signal from source to shoreline. Diffuser

11. Observations of total coliform following drifter motion. 22.8 in effluent 87 at shoreline < 10 for ocean samples except shoreline following drifter motion Total coliform from water samples collected on April 7, 2008 showing a weak signal and disconnect with shoreline. Diffuser

12. Observations of total coliform following drifter motion. 5.2 in effluent 369 at shoreline < 21 for ocean samples except shoreline following drifter motion Total coliform from water samples collected on February 20, 2008 showing drifters do not move to the shoreline, but large shoreline value. Diffuser

13. Observations of total coliform following drifter motion. 7.5 in effluent 573 at shoreline 354  171 following drifter motion Disconnect between effluent (7) and ocean water sampled above diffuser (354). Coliform source presumably upstream of diffuser as concentration shows steady decrease following drifter motion. Sampling after large rain event. These were max concentrations observed. Jan 28, 2008 - large rain event

14. Buoyant Plume Model to determine distance and time of plume to surface. Inputs: moored stratification and currents, effluent flow rate Distance from diffuser where plume reaches the surface is 59.56 ft, with a std of 13.855 ft (left) Time for plume to surface is 56.2 sec, with a std of 3.3 sec (right). Plume reaches surface in all realizations.

15. Starting location in green. Ending locations in red. ~300 tracks, hours in length. Few move to Hammonds Beach All drifter tracks starting at diffuser location Hammonds Beach ~3 km

16. - after 3 hours drifters reach the “surf zone” within ~1500 m alongshore - after 3 hours there are more “surf zone” encounters upcoast - bathymetry is relatively coarse for this application Distribution of along coast location where drifters reach surf zone within 3 hours of release from diffuser location. -2 km 0 1 km

17. Salinity Signal at Diffuser and Following Drifters Salinity increases toward background value with mixing. 1. S(offshore1000,1 m) = 33.82 ppt 2. S(diffuser,1 m) = 33.63 ppt Presumed plume signal 3. S(Lagrangian1,1 m) = 33.70 ppt Mixing w/ ocean increases S. 4. S(Lagrangian2,1 m) = 33.73 ppt More mixing further increases S. 5. S(Lagrangian3,1 m) = 33.78 ppt More mixing, greater S. Verify plume tracking with salinity from CTD casts CTD casts following plume drifter tracks and locations of salinity data

18. Water sampling for plume microbiology/chemistry Water sampling following plume (w/ CTD casts) Effluent samples are variable in time and distinct from ocean samples. Site 6 = Effluent Multi-dimensional scaling (MDS) plot of all samples grouped by site from the 26 selected sampling events for DNA-based TRFLP analysis.

19. E. coli summary statistics by location Site 6 = Effluent E. Coli summary statistics (MPN/100ml) by sampling location showing values at shoreline are much larger than at other sampling locations.

20. Nutrient summary statistics by location Phosphate (top) and Nitrate+Nitrite (bottom; μM) summary statistics by sampling location showing large effluent values compared with ocean.

21. TRFLP DNA yield summary statistics by location DNA yield (ng/l) summary statistics by sampling location showing values at shoreline are larger than at other sampling locations.

22. PHYLOCHIP OTU DISTRIBUTIONS Table 2. Number of stable OTUs shared between sampling locations. Total number of stable OTUs are indicated between parentheses for each location. Total number of stable OTUs shared between two locations are shown in bold; number of stable OTUs belonging to each family are added between parentheses. Lachnospiraceae is the only OTU family that appears to consistently and uniquely characterize the effluent. PHYLOCHIP OTU DISTRIBUTIONS Table 2. Number of stable OTUs shared between sampling locations. Total number of stable OTUs are indicated between parentheses for each location. Total number of stable OTUs shared between two locations are shown in bold; number of stable OTUs belonging to each family are added between parentheses. Lachnospiraceae is the only OTU family that appears to consistently and uniquely characterize the effluent. PHYLOCHIP OTU DISTRIBUTIONS Table 2. Number of stable OTUs shared between sampling locations. Total number of stable OTUs are indicated between parentheses for each location. Total number of stable OTUs shared between two locations are shown in bold; number of stable OTUs belonging to each family are added between parentheses. Lachnospiraceae is the only OTU family that appears to consistently and uniquely characterize the effluent. PhyloChip Technology - The Berkeley Lab PhyloChip probes samples for the 16S rRNA gene, which is involved in making proteins and is found in all bacteria. The microarray quickly and accurately identifies known and unknown organisms. Unknown organisms are classified based on their similarities to known microbes.

23. Effluent samples are variable in time and distinct from ocean samples. 1 = offshore 3 = diffuser 4 = drifters 5 = shoreline 6 = effluent Multi-dimensional scaling (MDS) plot of PhyloChip results (8 days) some samples are distinct on rain day

24. PhyloChip analysis of water samples collected above the diffuser (8 days) Number of microbial OTUs, families, and classes found with consistency. The nearshore community varied considerably from week to week. no consistent ocean tracer was found using PhyloChip

25. Lachnospiraceae OTUs distributions from PhyloChip analysis offshore effluent diffuser drifter track shoreline drifter track OTUs in the family Lachnospiraceae are consistently found in the effluent and at the shoreline. No Lachnospiraceae OTUs are found at the offshore site.

26. Table shows Lachnospiraceae is most dominant taxa in human waste. “ sewage contained between 2000 and 3000 unique V6 sequences for the taxonomic groups Bacteriodes, Lachnospiraceae, and Feacalibacterium (Table 3); however, fewer than 20 unique V6 sequences accounted for the majority of tags for each of these taxonomic groups.”

27. Conclusions Novel design for observing dilution and transport of a variety of plume tracers. Microbial communities within effluent are highly variable in time. Treated effluent reaches the surf zone along ~3 km span of shoreline directly inshore of the diffuser with a dilution > 250. No apparent relationship exists between bacterial concentrations in the effluent and sampled in ankle-deep water at the shoreline. Effluent fertilizes the near shore environment. The highly diverse set of OTUs distinguishable through PhyloChip analysis provide an improved mechanism for identifying effluent. Lachnospiraceae OTUs are found in effluent, at the diffuser, following drifters, and at the shoreline; but not beyond.