1. Freshwater Mussel Collaborative Study for Wastewater Treatment Plants May 23, 2016 May 23, 2016 Environmental DNA Pilot Study Sacramento-San Joaquin Delta, CA

2. Sacramento-San Joaquin Delta Largest watersheds in the Central valley/CA Largest watersheds in the Central valley/CA Drains 40% of CA’s land mass Drains 40% of CA’s land mass Fresh to brackish water habitat Fresh to brackish water habitat Drinking water for 22 million people Drinking water for 22 million people Water for millions of acres of agriculture and industry Water for millions of acres of agriculture and industry Up to a billion gallons of wastewater discharged daily Up to a billion gallons of wastewater discharged daily Municipal sources Municipal sources Commercial operations Commercial operations

3. Project Background Waste water dischargers are required to comply with number of regulations Waste water dischargers are required to comply with number of regulations 2013 EPA released stringent ammonia criteria 2013 EPA released stringent ammonia criteria Based on the most sensitive endpoints-freshwater mussels Based on the most sensitive endpoints-freshwater mussels May affect waste water dischargers ammonia limits to the Delta if mussels present May affect waste water dischargers ammonia limits to the Delta if mussels present Component of larger CVCWA pilot study Component of larger CVCWA pilot study Few historic records of mussels in Delta Few historic records of mussels in Delta Unknown if mussels are present-difficult to detect in large water bodies Unknown if mussels are present-difficult to detect in large water bodies If present how widespread? If present how widespread? Ubiquitous or scattered? Ubiquitous or scattered?

4. Aquatic Animals Species Distribution Knowledge of species distribution critical to ecological management/conservation Knowledge of species distribution critical to ecological management/conservation Especially rare or invasive species Especially rare or invasive species Some environments are particularly difficult to monitor Some environments are particularly difficult to monitor Different sampling strategies required Different sampling strategies required http://www.jsonline.com/

5. Environmental DNA (eDNA) The retrieval and analyses of genetic material obtained directly from environmental samples The retrieval and analyses of genetic material obtained directly from environmental samples Allows for aquatic species monitoring within different environments Allows for aquatic species monitoring within different environments Greater sensitivity than traditional survey methods Greater sensitivity than traditional survey methods Useful for detecting rare species and determining distribution Useful for detecting rare species and determining distribution http://fishbio.com/field-notes/conservation/traces-left-behind

6. Delta Pilot Study Goal Characterize relative distribution of mussel eDNA in the Sacramento-San Joaquin Delta Characterize relative distribution of mussel eDNA in the Sacramento-San Joaquin Delta Anodonta (Family Unionidae) Anodonta (Family Unionidae) Gonidea (Family Unionidae) Gonidea (Family Unionidae) Margaritifera (Family Margaritiferidae) Margaritifera (Family Margaritiferidae) Assess efficacy of using eDNA methodology in tidal waters where DNA may be distributed in a non-uniform manner Assess efficacy of using eDNA methodology in tidal waters where DNA may be distributed in a non-uniform manner

7. Delta Sampling Locations 10 Locations One sampling location Anadonta previously documented (2009) by the Nature Conservancy

8. Methods All samples collected in duplicate All samples collected in duplicate 50% samples collected 1 ft below surface 50% samples collected 1 ft below surface 50% samples collected 1 ft and 8 ft below surface 50% samples collected 1 ft and 8 ft below surface Field blanks (distilled water)– 2 locations Field blanks (distilled water)– 2 locations Samples analyzed using qPCR Samples analyzed using qPCR

9. Results Potential for False-Negatives By determining ability of the qPCR to detect mussel eDNA where mussels confirmed visually By determining ability of the qPCR to detect mussel eDNA where mussels confirmed visually Analyses of field blanks yielded no false-negatives Analyses of field blanks yielded no false-negatives eDNA successfully detected mussels at all sites where presence was confirmed visually eDNA successfully detected mussels at all sites where presence was confirmed visually

10. Results Potential for False-Postives Potential for false-positive determinations evaluated by analyzing 7 field blanks Potential for false-positive determinations evaluated by analyzing 7 field blanks 2 Delta field blanks 2 Delta field blanks All field blanks passed QC and were negative for mussel eDNA, thus no cross-contamination or false-positives All field blanks passed QC and were negative for mussel eDNA, thus no cross-contamination or false-positives Strict adherence to clean sampling techniques and QA/QC protocols is imperative to minimize potential for false-positives Strict adherence to clean sampling techniques and QA/QC protocols is imperative to minimize potential for false-positives

11. Results No Gonidea or Margaritifera detected No Gonidea or Margaritifera detected Anodonta detected at four (40%) locations Anodonta detected at four (40%) locations http://www.xerces.org/wp- content/uploads/2010/12/xerces-status-review- anodonta-californiensis-and-nuttalliana.pdf

12. Anodonta Presence Anodonta Presence Anodonta presence visually confirmed at 1 sampling location (Old River/San Joaquin River confluence) Anodonta presence visually confirmed at 1 sampling location (Old River/San Joaquin River confluence) USFWS crew member indicated live mussels and shells frequently collected in trawl USFWS crew member indicated live mussels and shells frequently collected in trawl Spent shell retrieved from nets Spent shell retrieved from nets

13. Results LocationDepth (ft) Anodonta eDNA Load (Log(Sequences/Liter)) Sacramento River @ I Street Bridge 1ND 88.96 Disappointment Slough @ Bishop Cut 18.75 8ND Old River/San Joaquin River Confluence 111.08 * 110.91 * San Joaquin River near Vernalis 19.57 19.84 * Anodonta confirmed present

14. Conclusions Replicate grab samples collected at 1 ft and 8 ft below surface Replicate grab samples collected at 1 ft and 8 ft below surface Yielded conflicting presence/absence determinations at 2 locations Yielded conflicting presence/absence determinations at 2 locations Likely attributable to non-uniform distribution of mussel eDNA in the water column Likely attributable to non-uniform distribution of mussel eDNA in the water column Recommendation: collect samples at multiple locations and depths across a transect at each location in large water bodies of the Delta Recommendation: collect samples at multiple locations and depths across a transect at each location in large water bodies of the Delta

15. Key Findings Mussel eDNA distribution may be non-uniform in large channels due to incomplete mixing Mussel eDNA distribution may be non-uniform in large channels due to incomplete mixing Collection of a small number of samples in large waters may result in: Collection of a small number of samples in large waters may result in: Increased potential for false-negatives Increased potential for false-negatives Underestimation of the actual eDNA load Underestimation of the actual eDNA load An adequate number of samples and sampling locations is necessary to minimize false-negative determinations An adequate number of samples and sampling locations is necessary to minimize false-negative determinations Gonidea and Margaritifera appear to be absent Gonidea and Margaritifera appear to be absent Based on preliminary results eDNA is a cost effective method to determine mussel presence/absence Based on preliminary results eDNA is a cost effective method to determine mussel presence/absence

16. Future Work Determine appropriate number and volume of samples Determine appropriate number and volume of samples Investigate best sampling depths and locations Investigate best sampling depths and locations Determine distance between sampling locations Determine distance between sampling locations eDNA attenuation eDNA attenuation Sampling design for wastewater treatment plants to determine mussel presence/absence Sampling design for wastewater treatment plants to determine mussel presence/absence

17. http://www.hillsboroughgc.org/the-delta-and-water/ Questions?