BOD/CBOD: Options, Tips& Tricks and

Upcoming Method Changes

Presented by: Christina Baker, Chesterfield County Wastewater;

Robin Parnell and Kerri Williams, Hampton Roads Sanitation District

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Any reference to product or company names does not constitute endorsement by

Chesterfield County or HRSD.

Tell us about you!

Write on a sticky note: •Municipality/Laboratory affiliation

•Most problematic concern with the analysis

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Workshop Goals

• Present examples of different ways laboratories can meet the requirements of Standard Methods for the Examination of Water and Wastewater 22nd Edition, section 5210B (2011) for biochemical oxygen demand/ carbonaceous biochemical oxygen demand (BOD/CBOD).

•Discuss questions and concerns regarding BOD/CBOD analyses.

•Discuss upcoming changes to SM5210B.

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Purpose of BOD/CBOD Analysis • Determine how organic matter affects the concentration of

dissolved oxygen (DO) in a stream or lake is integral to water-quality management.

• Environmental laboratories conduct BOD/CBOD testing for the purpose of: Process control for treatment of wastewater Reporting data for compliance with permits such as VPDES Monitor/regulate industrial users

• Used as an indication of the pollutant removal from wastewater.

• Represents the amount of oxygen consumed by bacteria and other microorganisms while they decompose organic matter under aerobic conditions at a specified temperature.

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Laboratory Analysis/ Reporting Requirements • Analyze wastewater samples for the purpose of reporting compliance

data-must be VELAP certified or accredited for BOD/CBOD in non-potable water.

• Implement QC requirements stated in Chapter 45/46 or TNI 2009, and the QC requirements stated in SM5210B-2011. This includes blanks, glucose glutamic acid (GGA), and matrix duplicate samples.

• Meet VPDES permit requirements such as the BOD or CBOD “Quantification Level” (usually defined as 2 mg/L or 5 mg/L).

• Report data as specified in the VPDES permit, such as using at least the same number of significant digits as the permit limit.

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Analysis Limitations

• Analysis is not easy: One time shot: 48 hr holding time-5 day analysis Demands consistency Years of experience

• Takes too long: Not a great measure for process control After 5 days hard to troubleshoot problems

• Accuracy is difficult to measure: The GGA standard is at a target concentration of 198 mg/L, while most final effluents

range 2-45 mg/L

• Blank contamination: Difficult to pinpoint; so many moving pieces to the puzzle

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Why Not Replace With COD and TOC ?

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• COD- Chemical oxygen demand is a measure of oxidizable organic and inorganic material

• TOC-Total organic carbon is a measure of organic carbon

• Not an exact replacement for BOD/CBOD

• Can find trends in BOD/CBOD/COD/TOC of a specific sample using multiple data points over a period of time

• No other test provides a better assessment of bioavailability of a waste stream

How To Make BOD/CBOD Work For You

• Equipment

•Reagents

•Dilution water

• Seed

•Analysis technique

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Equipment- Introduction • DO meter

Analog or digital; must be suitable for the selected probe

• DO probe

Polarographic or galvanic membrane

Optical (also referred to as luminescent)

• Air incubator (or water bath), 20 ± 1°C, excluding all light

• BOD bottles, stoppers, caps

• pH meter

• Chlorine meter/ test strips/ titration equipment

• Thermometer(s)

• Carrying racks for BOD bottles

• Micropipettors and/or pipettes

• Analytical Balance

• Robotic instrument 10

DO Probe Types

Images from www.fondriest.com

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DO probe/ Meter Use

• Calibrate right before use (and document) Most labs calibrate in air

May also calibrate in air-saturated water or by using Winkler titration

• May or may not have a built-in sensor to adjust for barometric pressure

• To ensure that the calibration is still good, blot the probe, allow to stabilize, and check to see that calibration value (100% as an example) is within about ± 2% of the original value

• Verify and document the temperature correction for the meter and probe at least annually with NIST reference thermometer

• Document SN of meter and probe 12

DO Probe/Meter Use

• Auto-stabilization may reduce accuracy

• Watch for signs of maintenance needed

• Some meters will alert when maintenance is due

• Short-term probe storage: a clean, moist environment

• Probe zero check

• Bubbles cause interference

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DO Probe/Meter Maintenance Tips

• Samples with excess hydrogen sulfide, a weekly membrane change is recommended (polarographic/galvanic)

• Electrolyte (polarographic/galvanic)

• Polarographic, periodically wet sand the silver anode and gold cathode May soak overnight in household ammonia cleaner or 2-3 minutes in

14% ammonium hydroxide and follow with wet sanding.

• Galvanic, periodically wet sand the cathode and anode

• Optical probe, the sensor is good for one year but may last longer

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Electrochemical Probe Troubleshooting

• If having probe stability issues, check the following: Sufficient stirring

Wrinkles, scratch, or hole in membrane

Bubbles under membrane (may need to refill with fresh solution if bubble present)

Blot/ shake off excess water, allow to re-stablize about 10 minutes, and recalibrate

RE-READ samples if stability issue occurs during an analysis. remember to record original values, never delete data.

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Optical Probe Troubleshooting

• If having probe stability issues, check the following: Scratch on cap

Blot/ shake off excess water, allow to stabilize about 10 minutes, and recalibrate

Change cap and i-button together

RE-READ samples if stability issue occurs during an analysis. remember to record original values, never delete data.

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Air Incubators

• Store samples in the dark at 20 ± 1°C (no windows)

• Document temperature at least daily

• Desired air recirculation to facilitate temperature stability

• Some labs have walk-ins and/or back-up incubators

• May purchase with a hi/lo alarm and/or power failure alarm

• If using a liquid thermometer, place at same level as samples

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Bottles • Method requires 60 mL capacity or greater; 300 mL

preferred

• Glass bottles

- Inspect for internal scratches or cracks

- Inspect for build-up of residue on bottom surface

- Meticulous cleaning

- May use laboratory dishwasher. Occasional acid-rinse of bottles can eliminate residue issues

• Disposable plastic bottles

- Can be recycled

- Can eliminate contamination issues

- Save time and resources on glassware washing

- At set-up, watch for bubbles which sometimes cling to the plastic.

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Pipettes

• Pipettes/ micropipettors - Measuring seed and small sample aliquots - Adding dechlorinating agent to samples

• Glass or disposable plastic acceptable - Disposable pipettes-document an accuracy check for each lot prior to use - Reusable glass pipettes-class A is recommended

• Use a clean pipette or micropipettor tip for each sample

• If re-using pipettes, wash with an automatic pipette washer

• Check glass pipettes for chips or cracks; make sure graduations legible

• For both Chapter 45 and Chapter 46 labs, quarterly verification of micropipettors/ digital pipets is required

Laboratory Balance

• Analytical or top-loading balance: used if your lab prepares chemical solutions in-house (buffer solutions/ dechlorinating agent/ GGA)

• Top-loading balances are not as accurate as analytical balances (use an analytical balance for GGA preparation!)

• Must calibrate or verify calibration at least annually

• Calibration verification “working weights” must bracket the range in which the balance is used, and must be done prior to use on each day of use

• “Working weights” must be verified against NIST “reference weights” on an established schedule

• If you have issues with static electricity, try adding a “ionizer” or anti-static unit to your balance

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pH Equipment/ Residual Chlorine Equipment • pH

pH meter and probe

Hand held portable pH probe that can read 0.1 su

• Residual Chlorine- Class A glass burette glass or digital

Pocket colorimeter or spectrophotometer

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Robotics

• Many different manufacturers

• Performs: Seed addition Dilution water addition CBOD inhibition chemical addition Probe movement to bottle positions Data reduction

• Low maintenance

• Durable

• Helpful addition if your lab has high sample throughput

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Questions on Equipment?

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Reagents- Introduction

May prepare solutions “from scratch” OR purchase commercially prepared

• Dilution water reagents

• GGA

• Supporting reagents-residual chlorine check, pH buffers

• CBOD inhibition reagents

• Winkler reagents

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Dilution Water Reagents • Ensure all reagents are within expiration date and free from growth

before use

• Weigh individual solid chemicals as suggested in the method

Top Loading balance can be used

• Nutrient buffer solution available in pre-measured pillows: 300ml, 3,4,6 and 19L

• Bulk ready-made reagents in 500mL or 1L

Phosphate buffer- pH 7.2

Magnesium sulfate, MgSO4, 2.25%w/v

Calcium chloride, CaCl2, 2.75%w/v

Ferric chloride FeCl3, 0.025%w/v

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GGA Standard Reagent

• Must run 3 bottles per batch

• In-house prepared GGA Dry reagent-grade glutamic acid and glucose at 103°C for one hour

Cool in desiccator

Weigh 0.150g of each on an analytical balance

May store in vials in a desiccator until use

Dilute 0.150g of glutamic acid and 0.150g of glucose to 1L with distilled or DeI water

• Commercially prepared GGA Available in pre-measured vials or bulk bottles

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Supporting Reagents

• pH buffers

• pH adjustment Sulfuric acid/ sodium hydroxide

• Residual chlorine detection/removal Sodium sulfite 1:1 acetic acid 1:50 sulfuric acid Potassium Iodide (KI) Starch indicator

• Electrolyte solution

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CBOD Inhibition Reagent

• 2-chloro-6 (trichloromethyl) pyridine-TCMP Delivered in solid form Does not dissolve readily Preferred nitrification inhibitor

• Allylthiourea-ATU Delivered in liquid form Stable for 2 weeks Used in Polyseed NX

Inhibits bacteria from reactions taking place

• NH3 + 1.5 O2 + Nitrosomonas bacteria → NO2-

• NO2- + 0.5 O2 + Nitrobacter bacteria → NO3

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Winkler Reagents • Manganous sulfate

• Alkali-iodide-azide

• Concentrated Sulfuric acid

• Standardized 0.025M sodium thiosulfate

• 2%w/v Starch solution

• 0.0021M Potassium bi-iodate solution

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Questions on Reagents?

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Dilution Water Source • Method specifies “distilled, tap, or receiving water” and discourages

deionized water. VELAP makes “recommendations” regarding this language.

• May purchase either water type from a water vendor, lab supply vendor, or even grocery store If purchased, track lot #

• Perform required water testing (at least check chlorine and metals); establish frequency and criteria; keep records

• If having issues with DeI, try modifying the system to include U/V sterilization and extra filtration at the tap.

• If distilling in-house, build-up of lime scale in the boiling chamber and on the heating element must be cleaned and disinfected; a water softener may be used to reduce scale build-up.

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Dilution Water Preparation

• Estimate how much water needed for analysis

• Check source water DO prior to use, and make adjustments if needed Too high: boil aliquots, add and mix to drive off

excess DO

Too low: aerate using a filtered air pump and stone or shake excessively

• Day of use, add buffer and minerals and ensure DO is above 7.5 mg/L

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Seed Suspension

• If CBOD, must add seed per the method. If pH adjusted, must add seed. If BOD, may or may not add seed depending on presence of a microbiological population.

• Method states that domestic wastewater (unchlorinated/ undisinfected) or commercial seed sources may be used.

• If using commercial seed such as Polyseed, follow manufacturer’s instructions!

• If using wastewater, it is recommended to use fresh sample to prepare the seed for each analysis.

• Technique for preparing seed AND pipetting technique are very important for obtaining consistent test results.

• Seed correction “generally should be 0.6-1.0 mg/L”

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Questions on Dilution Water or Seed?

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Preparation and Analysis

• Sample Temperature

• Sample pH

• Residual chlorine detection/removal

• Dissolved oxygen

• Automated set-up/ analysis

• QC elements

• Processing data

• Reporting

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Preparation of Sample: Temperature

• Refrigerate samples at ≤ 6°C until ready to begin preparation

• Warm samples in a water bath/ sink to 20 ± 3°C

• Mix when checking the temperature

• Document the serial number of the thermometer used

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Preparation of Sample: pH

• If not within 6.0 to 8.0, adjust to range of 7.0 to 7.2

•Don’t dilute sample by >0.5% when adjusting pH

•Document pH before and after adjustment

•Always seed samples that have been pH adjusted

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Preparation of Sample: Residual Chlorine

• Chlorine residual check: May use test strips, colorimeter, or titration method to check for presence

• Sodium sulfite must be prepared fresh daily

• To determine amount of sodium sulfite needed, titrate using a glass burette, digital titrator, or even consecutive aliquots of sodium sulfite delivered by micropipettor

• Treat a fresh portion of sample with just enough sodium sulfite to remove the chlorine

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Preparation of Sample: Dissolved Oxygen

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• Use SM Table 4500-O:I as guidance to assess sample DO saturation

• If initial DO < 7.5 mg/L, pour sample into another container, cap and shake to aerate

• If initial DO too high, degas samples using a vacuum pump fitted with a hose and rubber stopper. Tap the bottom of the bottle on the counter top to generate air bubbles that were in solution, the vacuum will remove the bubbles without aspirating the sample

• DO can also be reduced by vigorous, extended shaking or by using a laboratory blender (must allow headspace)

Preparation of Sample: Other Notes

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• Dilution selection based on history; at least 3 bottles

• For bottles with more than 67% of the sample after dilution (more than 201 mL in a 300 mL bottle), must add nutrient, mineral, and buffer solutions directly to the diluted sample at a rate 0.3mL/300 mL bottle.

• Be sure inhibitor is added after sample bottles are at least 2/3 full (i.e., after initial dilution but before final filling with dilution water)

• Add seed suspension after diluting samples but before final filling with dilution water

• When reading initial DO’s make sure to rinse the probe thoroughly after each sample set

Automated Analysis

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• Samples are manually entered into software or imported from a LIMS system

• Analyst plants samples and places in racks • Analyst partially fills bottles with dilution water • Probe and meter are calibrated before use • Auto sampler moves probe/seed/dilution water housing to each bottle

Dispenses selected volume of seed Fills bottles using an ionic sensor Reads DO after stabilizes

• Calculates seed correction and BOD of samples meeting depletion requirements

Frequency of QC Elements

For every batch of 20 or fewer samples, the following are required: • Method blank (1 or more bottles)

• GGA (3 bottles)

• Matrix duplicate (3 sample dilutions analyzed twice)

• Seed control (3 bottles)

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Capping and Incubation

• Top off any bottles with additional dilution water if level is too low.

• Add a stopper and cap to each bottle to create a water seal.

• Check each bottle for bubbles after capping.

• Document incubation start time.

• Incubate 5 days ± 6 hours.

• After 5 days, inspect bottles for air bubbles

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Data processing

• Requirements for a bottle to be used in a calculation: Must have depletion of 2.0 mg/L or greater Must have final DO of 1.0 mg/L or greater

• Average all bottles which meet the above rules. • Qualify results if toxicity is evident:

When dilutions show more than 30% difference between high and low values. Example: 5mL bottle produces BOD of 315 mg/L, 25 mL bottle produces BOD of 300 mg/L, and 50 mL bottle produces BOD of 150 mg/L.

• When all dilutions result in final DO <1 mg/L, select the bottle with lowest DO or greatest dilution and calculate using that result. Report as “>”.

• When all dilutions fail to produce a depletion of 2.0 mg/L select the bottle with the greatest volume and calculate assuming a depletion of 2. Report as “<“.

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Data Processing

• Method blanks average must be ≤ 0.20 mg/L.

• GGA average must be 198 ± 30.5 mg/L.

• Matrix duplicate RPD: May use limits provided by VELAP Fixed Limits Table:

o either ≤20% for all duplicates

o or ≤25% for “low level duplicates” and ≤10% for “high level duplicates”

May develop limits using internal data based on SM5020B.f-2010 (but can’t be wider than Fixed Limits Table options above)

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Questions on Analysis and Sample Preparation?

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Troubleshooting Blanks and GGA

• Is there a contamination issue? - Algae or slime growth in carboy - Film in bottles (look closely at the bottom) - Growth in tubing - Dilution water source - Air stones or air lines

• Is there a technique issue? - Pipetting issue - Proper preparation of GGA

• Is there an equipment issue? - Balance verified - Incubator consistently in range - Meter/ probe issue - Robotic volume delivery

• Adjust the seed! - For GGA issues (no suspicion of contamination), adjust the strength or

amount of seed used

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Troubleshooting Matrix Duplicates (precision)

• Ensure that samples are well-mixed prior to withdrawing each aliquot

• Check for meter/probe stability issues

• Ensure that all sample bottles are thoroughly cleaned

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Questions on Troubleshooting?

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“Standard Methods” for the Examination of Water and Wastewater (SM) Introduction • How do methods get updated?

• How do methods get approved for regulatory use?

• Changes in the most recent BOD update – what is coming – (currently not approved.)

• SM Part 5000 23rd Edition Corrections

• SM BOD Common Questions

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How do Standard Methods Sections get Updated?

Standard Methods Structure • Joint Editorial Board (WEF, AWWA, APHA) •Part Coordinators for each Section (1000 – 10000) • Joint Task Group Chair • Joint Task Group Members • Standard Method Committee Members

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How do Standard Methods Sections Get Updated?

1. Joint Task Group Formed

2. Joint Task Group revises method with leadership of Joint Task Group Chair

3. Joint Task Group ballots the revised method within the group

4. Joint Task Group works out any issues with “No” votes within group if they occur

5. Part Coordinator and Joint Editorial Board member review the method

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How do Standard Methods Sections Get Updated?

6. Method is sent out to Standard Methods Committee for ballot

7. Ballots are reviewed by the Part Coordinator and the Joint Task Group Chair

8. “No” votes and comments are addressed. Comments should not be editorial in nature and must be backed up with explanation and evidence.

9. If major changes are made to the method that are not editorial it may have to go back to Joint Task Group for further review and/or the method will be re-balloted by the Standards Methods Committee again.

10. Once any “No” votes are addressed, the method is finalized and approved.

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How does the Updated “Standard Methods” get EPA approval?

•Before a new version of a method can be used for regulatory purposes, EPA must review the updates and changes and include the new version of the method in a Method Update Rule (MUR) which is published in 40 CFR Part 136 for wastewater.

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Upcoming Changes in SM5210B

• 23rd Edition – A new version of SM5210B was approved by Standard Methods Committee in 2016.

• This version is not currently approved for use but should be included in the next MUR (Method Update Rule).

• Current approved version of SM5210B is from 2001 with QC Editorial Revisions in 2011.

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Upcoming Changes in SM5210B (cont.)

• Disposable plastic BOD bottles have been added to the method. (EPA already allows approval for these bottles but it is not in the current method.)

• Oxygen-sensitive optical probe has been added to the method. (EPA already allows approval for these DO probes but they are not in the current method.)

• Distilled water is changed to reagent-grade water in the reagents section. Also removed preferably sterilized from the reagent water requirement.

(Reagent Grade Water Reference SM1080B/SM1080C)

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Upcoming Changes in SM5210B (cont.)

• ATU Solution – storage 2 weeks or less at <6oC without freezing

• GGA – prepare fresh unless solution is maintained in a sterile container – store at <6oC without freezing • Discard solution if there is evidence of contamination (growth or failing test

results)

• Dilution water – addition of reagent grade water as possible source

• Sample Holding Time – Addition of EPA allows for a 48 hour holding time on samples

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Upcoming Changes in SM5210B (cont.)

• pH adjustment of samples– if sample is not between pH of 6.0 – 8.0, adjust pH to 6.5 – 7.5 (currently 7.0 – 7.2).

• Source water criteria for dilution water Copper (<0.05 mg/L)

Chlorine (<0.10 mg/L)

Deionized Water is acceptable as long as dilution water blanks consistently meet QC limits.

• Number of Sample Dilutions – 2 dilutions are allowed if experience with a sample source results in at least 1 bottle meeting depletion requirements.

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Upcoming Changes in SM5210B (cont.)

Clarified Sample Dilution Criteria • Dilutions greater than 1:300 require a primary dilution (previous 1:100)

• If more than 67% sample remains after dilution, add additional nutrient, mineral and buffer solutions directly at a rate of 1 mL/ 1L.

• Can use commercially prepared solutions.

• BOD Bottle – mix sample well immediately before pipetting

• Fill BOD bottle with approximately two-thirds full with dilution water and/or sample without entraining air.

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Upcoming Changes in SM5210B (cont.)

• DO Calibration • Perform daily by following manufacturer calibration procedure

• Make frequent calibration checks daily to ensure accurate DO readings

• GGA • Add nitrification inhibitor to CBOD GGA standards.

• Average BOD/CBOD GGA must fall within 198 ± 30.5 mg/L or control limit range established in SM5210B.8a

• Establish control limits for BOD or CBOD by analyzing 25 GGA standards and calculating limits for the mean ± 3 standard deviations.

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Upcoming Changes in SM5210B (cont.)

• Dilution Water Check (Blank) • 2 or more bottles required (previous 1)

• Average results

• Criteria is <0.2 mg/L (previous <0.20 mg/L)

• Calculation • Added calculation for less than values for diluted samples.

• If all dilutions result in a DO depletion of <2.0 mg/L.

• Use bottle with largest volume of sample.

• Calculate the report as if the dilution had depleted 2.0 mg/L.

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SM Part 5000 Corrections

• BOD – SM5210B-2016 One error has been found in regards to a reference.

Section 5210B.6.c states the following: “Determine initial and final DO for each bottle (5210B.5 e and i), and average result.” The correct reference is the following: “Determine initial and final DO for each bottle (5210B.5 g and i), and average result.”

• COD – SM5220B and C – 23rd Edition publication The formula for calculation is listed as B - A instead of A - B.

Errata has been released on SM website for this error already.

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Common Questions

1. BOD Holding Time

2. CBOD GGA Acceptance Criteria

3. Do you have to standardize sodium sulfite solution?

4. Blank Issues

5. GGA Issues

6. Seed Issues

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Questions?

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Christina Baker Wastewater Laboratory Manager Chesterfield County (office) 804-768-7744 bakerch@chesterfield.gov

Kerri Williams HRSD Laboratory Manager Hampton Roads Sanitation District (office) 757-460-4209 kwilliams@hrsd.com

Robin Parnell HRSD Laboratory Manager Hampton Roads Sanitation District (office) 757-460-4203 rparnell@hrsd.com

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Fictitious batch set up on 7/19/18 by Chesterfield County WWTP Laboratory

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EXAMPLE ANALYSIS ISSUES: 1. Times do not include AM/PM or military time format.

2. The serial number or unique identifier of the D.O. meter is not documented.

3. Sample pH for PC EFF was not adjusted to within the required range of 7.0 to 7.2 (Standard

Methods 21st and 22nd Edition requirement.)

4. Chlorine check and pH not documented for some samples. (If only used for internal process control purposes, the laboratory may choose to omit.)

5. One obliteration in seed uptake column (must use one line through a correction and initial/date the correction).

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EXAMPLE ANALYSIS ISSUES: 6. For sample fINF-071818, bottle 10 should not have been included in the average because it did not meet

the minimum depletion of 2.0 mg/L.

7. Bottle #4 should have been omitted from the average of the seed control bottles because it did not meet the minimum depletion of 2.0 mg/L.

8. The analyst did not consistently round data in the same manner. For GGA bottle #7, the result of 166.5 was rounded to 167, while for GGA bottle #8, the result of 172.5 was rounded to 172. The lab must establish a rounding convention to be used consistently by all analysts. Note also that your VPDES permit may have language requiring that the permittee uses the same rounding convention used by its subcontracted laboratories.

9. Bottle #27 for fEFF-071818 had an initial D.O. of 9.86 mg/L. The sample should have been degassed to reduce the D.O. to below saturation prior to reading the initial D.O. According to Table SM4500-O:I, if the sample was at 20.0°C, the D.O. would need to be less than 9.09 mg/L. (If 23.0°C, saturation is 8.58 mg/L. If 17.0°C, saturation is 9.66 mg/L