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Wastewater Treatment Plants & Bacteria Strategies for Compliance Best Practices for Effluent Sampling TANNY BUSBY & LAURA BONJONIA ENVIRODYNE LABORATORIES, INC. April 26, 2011 Where Does the Analytical Testing Begin? Container Preparation Sample Collection Chain of Custody Preservatives Field QC 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 The sample reflect characteristics of the Effluent Identification of Sampling Points Effluents Influents Process Control Collection Systems DEFENSIBLE DATA Sample Collection Grab e-Coli Fecal Coliform Oil & Grease *DO *pH Cyanide VOC *Chlorine Residual Composite Metals BNA Pesticides TPH Minerals Nutrients (Nitrogen) BOD TSS (Solids) DEFENSIBLE DATA QUALITY CONTROL Calibrate Field Equipment - (Document in field log) ~ pH, DO & Cl2 Meters Field Blanks Trip Blanks Rinseates Field and Lab Duplicates Field and Lab Matrix Spikes (MS) Laboratory control samples (LCS) Method Numbers WHAT ARE COLIFORM Gram stain • Gram negative (turns red) Do not produce spores Bacilli (rod shaped) Aerobic (require oxygen) or Facultative Anaerobic WHERE ARE COLIFORM(s) FOUND? Surface Water Decaying Plants Soil Intestinal Tract of Warm-blooded Animals TYPES OF COLIFORM BACTERIA Total Coliform E. coli e.g. Salmonella (Typhoid Fever) Shigella Pseudomonas Fecal Coliform Enterobacter aerogenes Free in the environment e.g. soil & plants Fecal streptococcus Clostridium ~ Microbes ~ Commonly Regulated in Wastewater Effluents Fecal E. Coliform 200 CFU/100ml ~ Monthly Geo-Mean (Avg.) 400 CFU/100ml ~ 7 Day Geo-Mean (Avg.) 800 CFU/100ml ~ Single Grab Max. coli ~ (Escherichia coli) 126 CFU/100ml ~ Monthly Geo-Mean (Avg.) 394 CFU/100ml ~ Single Grab Max. Enterococci 36 CFU/100ml ~ Monthly Geo-Mean (Avg.) 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 E. coli Membrane filtration m-ColiBlue 24 • Total coliform and E. coli simultaneously • E. coli – Blue colonies • Total coliform – Red or blue colonies Other methods are approved ENTEROCOCCI PLATE Membrane filtration SM 9230 C CFUs/100 ml Blue colonies Other methods Enterolert • MPN (Most Probable Number) COLILERT- QUANTITRAY Incubate 18-24 hours- Most Probable Number TOTAL COLIFORMS E.Coli (UV Light) 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 Micro Samples How to Collect Effluent Directly into Sterile Testing Container How to Collect and Split Samples 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)