Wastewater Treatment Wastewater Treatment Plants & Bacteria Strategies Plants & Bacteria Strategies

for Compliancefor Compliance

Best Practices for Effluent Best Practices for Effluent SamplingSampling

TANNY BUSBY&

LAURA BONJONIA

ENVIRODYNE LABORATORIES, INC.April 26, 2011

Where Does the Analytical Testing Where Does the Analytical Testing Begin?Begin?

Container PreparationContainer Preparation Sample CollectionSample Collection Chain of CustodyChain of Custody PreservativesPreservatives Field QCField QC

What is a Representative What is a Representative Sample?Sample?

DefinitionDefinitionEvery Member of the Population has an Every Member of the Population has an

“Equal Chance” of being Selected“Equal Chance” of being Selected

Typically a Population is very large

A Sample is a Subset of the Population

The sample reflect characteristics The sample reflect characteristics of the Effluentof the Effluent

EffluentsEffluents InfluentsInfluents Process ControlProcess Control Collection SystemsCollection Systems

Identification of Sampling PointsIdentification of Sampling Points

DEFENSIBLE DATADEFENSIBLE DATASample CollectionSample Collection

GrabGrab e-Colie-Coli Fecal ColiformFecal Coliform Oil & GreaseOil & Grease *DO*DO *pH*pH CyanideCyanide VOCVOC *Chlorine Residual*Chlorine Residual

CompositeComposite MetalsMetals BNABNA PesticidesPesticides TPHTPH MineralsMinerals Nutrients (Nitrogen)Nutrients (Nitrogen) BODBOD TSS (Solids)TSS (Solids)

DEFENSIBLE DATADEFENSIBLE DATAQUALITY CONTROLQUALITY CONTROL

Calibrate Field Equipment - (Document in Calibrate Field Equipment - (Document in field log) ~ pH, DO & Clfield log) ~ pH, DO & Cl2 Meters Meters

Field Blanks Field Blanks Trip BlanksTrip Blanks RinseatesRinseates Field and Lab DuplicatesField and Lab Duplicates Field and Lab Matrix Spikes (MS)Field and Lab Matrix Spikes (MS) Laboratory control samples (LCS)Laboratory control samples (LCS) Method NumbersMethod Numbers

WHAT ARE COLIFORMWHAT ARE COLIFORM

Gram stain Gram stain • Gram negative Gram negative

(turns red)(turns red)

Do not produce Do not produce sporesspores

Bacilli Bacilli (rod shaped)(rod shaped) Aerobic Aerobic (require (require

oxygen)oxygen) or or Facultative Facultative AnaerobicAnaerobic

Surface WaterSurface Water Decaying PlantsDecaying Plants

SoilSoil Intestinal Tract of Warm-blooded Intestinal Tract of Warm-blooded

AnimalsAnimals

WHERE ARE WHERE ARE COLIFORM(s) FOUND?COLIFORM(s) FOUND?

TYPES OF COLIFORM TYPES OF COLIFORM BACTERIABACTERIA

E . co lie .g . S a lm on e lla (Typ h o id F ever)

S h ig e llaP seu d om on as

F eca l s trep tococcu s C los trid iu m

F eca l C o lifo rm E n terob ac ter aerog en esF ree in th e en viron m en t

e .g . so il & p lan ts

Tota l C o lifo rm

~ Microbes ~ ~ Microbes ~ Commonly Regulated in Commonly Regulated in

Wastewater EffluentsWastewater Effluents Fecal Coliform 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)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.

EnterococciEnterococci 36 CFU/100ml ~ Monthly Geo-Mean (Avg.)36 CFU/100ml ~ Monthly Geo-Mean (Avg.)

COLIFORM LAB TESTING COLIFORM LAB TESTING METHODSMETHODS

- Multiple Tube Fermentation (Quantifies)Multiple Tube Fermentation (Quantifies)- Reported as MPN (most probable number)Reported as MPN (most probable number)

- Membrane Filtration (Quantifies)Membrane Filtration (Quantifies)- Reported as CFU’s/100 mL (colony forming units)Reported as CFU’s/100 mL (colony forming units)

- Quanti-tray (Quantifies)Quanti-tray (Quantifies)- Colilert – 18-24 hoursColilert – 18-24 hours- Reported as MPNReported as MPN- Positive sample turns yellow; E.coli fluorescencePositive sample turns yellow; E.coli fluorescence

- Presence/Absence (Qualifies)Presence/Absence (Qualifies)- Colilert, ColisureColilert, Colisure- Positive sample turns yellow (Colilert); turns magenta Positive sample turns yellow (Colilert); turns magenta

(Colisure)(Colisure)- E.coli fluorescenceE.coli fluorescence

E. coliE. coli

Membrane filtrationMembrane filtration m-ColiBlue 24m-ColiBlue 24

• Total coliform and Total coliform and

E. coli simultaneouslyE. coli simultaneously• E. coli – Blue E. coli – Blue

coloniescolonies• Total coliform – Red Total coliform – Red

or blue coloniesor blue colonies Other methods are Other methods are

approvedapproved

ENTEROCOCCI PLATEENTEROCOCCI PLATE

Membrane filtrationMembrane filtration SM 9230 CSM 9230 C CFUs/100 mlCFUs/100 ml Blue coloniesBlue colonies

Other methodsOther methods Enterolert Enterolert

• MPN (Most Probable MPN (Most Probable Number)Number)

COLILERT- QUANTITRAYCOLILERT- QUANTITRAYIncubate 18-24 hours- Most Probable Number

TOTAL COLIFORMS

E.Coli (UV

Light)

SAMPLING HOLD TIMESSAMPLING HOLD TIMES

Fecal Coliform, E. coli and EnterococciFecal Coliform, E. coli and Enterococci Grab (Not Composite)Grab (Not Composite) Sterile container Sterile container Hold Time ~ Hold Time ~ Not to Exceed a Total of 8HrsNot to Exceed a Total of 8Hrs

6 hours transport + 2 hours set up 6 hours transport + 2 hours set up

Micro Samples Micro Samples How to Collect Effluent How to Collect Effluent

Directly into Sterile Testing Container Directly into Sterile Testing Container

How to Collect and Split SamplesHow to Collect and Split Samples

Total Coliform Qualitative Analysis Total Coliform Qualitative Analysis (PRESENCE~ABSENCE)(PRESENCE~ABSENCE)

COLILERT-18

-Incubate 18-22 hours

COLILERT

-Incubate 24-28 hoursCOLISURE

-Incubate 24-48 hours

E.coli positive

(UV Light)