1. Wastewater Treatment Plants & Bacteria Strategies for Compliance Best Practices for Effluent Sampling TANNY BUSBY & LAURA BONJONIA ENVIRODYNE LABORATORIES, INC. April 26, 2011

2. Where Does the Analytical Testing Begin?  Container Preparation  Sample Collection  Chain of Custody  Preservatives  Field QC

3. What is a Representative Sample? Definition Every Member of the Population has an “Equal Chance” of being Selected Typically a Population is very large A Sample is a Subset of the Population

5. The sample reflect characteristics of the Effluent

6.  Effluents  Influents  Process Control  Collection Systems Identification of Sampling Points

7. DEFENSIBLE DATA Sample Collection  Grab e-Coli e-Coli Fecal Coliform Fecal Coliform Oil & Grease Oil & Grease *DO *DO *pH *pH Cyanide Cyanide VOC VOC *Chlorine Residual *Chlorine Residual  Composite Metals BNA Pesticides TPH Minerals Nutrients (Nitrogen) BOD TSS (Solids)

8. DEFENSIBLE DATA QUALITY CONTROL  Calibrate Field Equipment - (Document in field log) ~ pH, DO & Cl Meters  Calibrate Field Equipment - (Document in field log) ~ pH, DO & Cl 2 Meters  Field Blanks  Trip Blanks  Rinseates  Field and Lab Duplicates  Field and Lab Matrix Spikes (MS)  Laboratory control samples (LCS)  Method Numbers

9. WHAT ARE COLIFORM Gram stain Gram stain Gram negative (turns red)Gram negative (turns red) Do not produce spores Do not produce spores Bacilli (rod shaped) Bacilli (rod shaped) Aerobic (require oxygen) or Facultative Anaerobic Aerobic (require oxygen) or Facultative Anaerobic

10.  Surface Water  Decaying Plants  Soil  Intestinal Tract of Warm-blooded Animals WHERE ARE COLIFORM(s) FOUND?

11. TYPES OF COLIFORM BACTERIA

12. ~ Microbes ~ Commonly Regulated in Wastewater Effluents  Fecal Coliform 200 CFU/100ml ~ Monthly Geo-Mean (Avg.) 200 CFU/100ml ~ Monthly Geo-Mean (Avg.) 400 CFU/100ml ~ 7 Day Geo-Mean (Avg.) 400 CFU/100ml ~ 7 Day Geo-Mean (Avg.) 800 CFU/100ml ~ Single Grab Max. 800 CFU/100ml ~ Single Grab Max.  E. coli ~ (Escherichia coli) 126 CFU/100ml ~ Monthly Geo-Mean (Avg.) 126 CFU/100ml ~ Monthly Geo-Mean (Avg.) 394 CFU/100ml ~ Single Grab Max. 394 CFU/100ml ~ Single Grab Max.  Enterococci 36 CFU/100ml ~ Monthly Geo-Mean (Avg.) 36 CFU/100ml ~ Monthly Geo-Mean (Avg.)

13. COLIFORM LAB TESTING METHODS - Multiple Tube Fermentation (Quantifies) -Reported as MPN (most probable number) - Membrane Filtration (Quantifies) -Reported as CFU’s/100 mL (colony forming units) - Quanti-tray (Quantifies) -Colilert – 18-24 hours -Reported as MPN -Positive sample turns yellow; E.coli fluorescence - Presence/Absence (Qualifies) -Colilert, Colisure -Positive sample turns yellow (Colilert); turns magenta (Colisure) -E.coli fluorescence

14. E. coli  Membrane filtration m-ColiBlue 24 m-ColiBlue 24 Total coliform andTotal coliform and E. coli simultaneously E. coli simultaneously E. coli – Blue coloniesE. coli – Blue colonies Total coliform – Red or blue coloniesTotal coliform – Red or blue colonies Other methods are approved Other methods are approved

15. ENTEROCOCCI PLATE  Membrane filtration SM 9230 C SM 9230 C CFUs/100 ml CFUs/100 ml Blue colonies Blue colonies  Other methods Enterolert Enterolert MPN (Most Probable Number)MPN (Most Probable Number)

16. COLILERT- QUANTITRAY Incubate 18-24 hours- Most Probable Number TOTAL COLIFORMS E.Coli (UV Light)

17. SAMPLING HOLD TIMES  Fecal Coliform, E. coli and Enterococci  Grab (Not Composite)  Sterile container  Hold Time ~ Not to Exceed a Total of 8Hrs 6 hours transport + 2 hours set up 6 hours transport + 2 hours set up

18. Micro Samples How to Collect Effluent  Directly into Sterile Testing Container

19. How to Collect and Split Samples

20. Total Coliform Qualitative Analysis (PRESENCE~ABSENCE) COLILERT-18 -Incubate 18-22 hours COLILERT - Incubate 24-28 hours COLISURE - Incubate 24-48 hours E.coli positive (UV Light)