1. Luminescent Dissolved Oxygen (LDO) Measurements for Wastewater Applications
Edward C. Craig, Ph.D.*
Cary B. Jackson, Ph.D.
Christopher P. Fair
Hach Company
Loveland, CO
Ed Craig – R & D Chemist
Cary Jackson – Regulatory Chemist
Chris Fair – Applications Chemist Specializing in Wastewater Applications

2. Purpose of this Talk…
Discuss our effort to add Luminescence (LDO) to the List of EPA Approved Methods for Dissolved Oxygen Measurements
Proposed EPA Method 360.3

3. Specifically…
Discuss our In-House Preparation for a External Nationwide Validation Study run as Part of a Tier 3 EPA Approval Process for the Luminescence Method
Also share some of the results from the External Validation Study

4. Current EPA Approved Methods for Measuring Dissolved Oxygen Conc.
Winkler Titration (1888)
EPA Method 360.2
Membrane Electrodes (1959)
EPA Method 360.1

5. Winkler Titration
L.W. Winkler, “The Determination of Dissolved Oxygen in Water”, Ber. Deut. Chem. Ges., 21, 2843 (1888)
EPA “Reference Method” for Dissolved Oxygen Measurement
This is a note

6. Winkler Titration – Reactions
Based on the quantitative oxidation of Mn(II) to Mn(III) in alkaline solution and the subsequent oxidation of I- by Mn(III) in acid solution. The iodine that is liberated is then titrated with Sodium thiosulfate.
Depends on careful control of pH and [I-]
Precisions better than +/- 0.1 mg/L of dissolved oxygen can be realized

7. Winkler Method – Floc Formation

8. Winkler Method - Titration

9. Winkler Titration Pros Cons Direct DO Measurement Easy Clean-up
Samples must be Analyzed in a Laboratory
Subject to Several Chemical Interferences
Very Technique Sensitive
Direct DO Measurement  No Salinity Correction Needed
Easy Clean-up  Pour the BOD bottles out and they’re ready for use next time
Analyzed in Lab  No Field Measurements / Requires Sample Collection and Transport
Chemical Interferences  Any oxidizing or reducing agents in the sample will interfer with the REDOX titration
Notable Interferents: Chlorine, Iron (?)
Technique Sensitive  Trained Laboratory Technicians Required for Analysis

10. Membrane Electrode L.C. Clark “Clark Cell” Patent Nov. 19, 1959
Sometimes called “MPODs” for Membrane covered Polarographic Oxygen Detectors

11. O2 + 2 H2O + 4 e-  4 OH- Clark Cell – Edit3
Cathode Highlighted Yellow/Gold (site of reduction)
O2 + 2 H2O + 4 e-  4 OH-

12. Clark Cell – Edit 4
Anode Highlighted (Red)

13. Clark Cell – Edit 5
Electrolyte Solution Highlighted (Blue)

14. Clark Cell – Edit 6
Membrane Highlighted (Green)

15. Membrane Electrode Pros Cons In-situ Measurements Possible
Easy to Make Measurements
Cons
Frequent Maintenance Required
Must be Polarized Before Use
Must be Calibrated Before Use
Consumes Oxygen
Very Sensitive to Sample Flow
In-situ Measurements  No Sample Collection Necessary
Frequent Mainenance Required
Membranes tear very easily and require replacement Membranes must be kept hydrated at all times (must be hydrated before initial use)
Electrolyte needs periodic replacement – it may become poisoned by metal ions etc.
Electrodes need to be cleaned/polished periodically The quality of the probe response varies with the condition of the electrode surfaces.
Polarization – Depending on the particular brand of instrument it can take anywhere from 10 min. to half an hour for full polarization
Note – the more electrode surface you have the longer it takes to polarize the electrode
Side Note – Larger electrodes generally mean more stable responses and longer periods between electrode cleaning/polishing but they also require longer polarization times.
Calibration – The calibration is actually a simple process. Typically the probe is inserted into a Water Saturated Air environment and the response vs. Dissolved Oxygen is set. Two things cause the calibration to be difficult. First is the polarization, if the electrode isn’t fully polarized when the instrument is calibrated then it will appear to drift over time. Second is the electrode surface, as the electrode operates oxides tend to build-up on the surface of the electrode. This changes the characteristics of the electrode surface and (more importantly) the current flow through the electrode. The result is the response tends to drift over time. The instrument eventually needs to be recalibrated to compensate for gradual changes in the electrode surface.
Consumes Oxygen – the Clark Cell actually consumes oxygen. It is the consumption oxygen that generates the electrical current that is measured. The difficulty is the longer the probe sits in the sample the more oxygen it consumes. If you have a large sample this generally isn’t a problem. If you have a small sample (e.g., BOD bottle with very low DO Conc.) then it may be a very significant problem.
Sensitive to Sample Flow – Because the Clark Cell is “Consumptive” it must replinish the oxygen consumed at the same rate as it is consumed otherwise the DO response drifts downward over time. To offset this effect the sample must be stirred very rapidly. This is even more significant at higher DO concentrations because it becomes progressively more difficult to replinish the oxygen that is consumed. (Mass Transport Limited Process)

16. Luminescent Dissolve Oxygen (LDO)
Completely New Technology
Measures the Quenching of a Luminescent Reaction caused by Oxygen

17. Luminescent Dissolved Oxygen (LDO) - Probe

18. Luminescent Dissolved Oxygen (LDO) Probe Components

19. Luminescent Dissolved Oxygen (LDO)
Probe
LED
Photo Diode
LED

20. Luminescent Dissolved Oxygen (LDO)
Probe
Sensor
LED
Photo Diode
LED

21. Luminescent Dissolved Oxygen (LDO)
LED
Photo Diode
Probe
Sensor
Oxygen
Luminescent
Indicator
Molecules
Gas Permeable
Polymer Matrix
Clear, Gas
Impermeable
Substrate

22. Luminescent Dissolved Oxygen (LDO)
Pros
No Maintenance
Field Measurements Possible
No Polarization
Insensitive to Sample Flow
Rugged
“Essentially” No Maintenance – beyond changing out sensor caps and wiping them occasionally with a rag to clean them there’s no maintenance (no membranes / no electrolyte / no electrode surfaces)

23. Luminescent Dissolved Oxygen (LDO)
Pros (cont.)
Accurate
Precise
Very Stable Calibrations
No Chemical Interferrents
Lower Day-to-Day Cost than Winkler Titration

24. Luminescent Dissolved Oxygen (LDO)
Cons
Higher Initial Cost than Winkler Titration
Not Currently EPA Approved
Higher Initial Cost than Winkler Titration – You have to buy a meter and a probe but once you have the probe and meter your day-to-day cost is significantly less than that of running Winkler Titrations
Initial Cost are comparable to that for the Membrane electrodes
It isn’t EPA Approved yet but we expect to have full EPA approval within 1 to 1 ½ years and we expect that we’ll be granted provisional approval within a few months (verify this with Cary)

25. Process LDO… Cost Savings John Anderson
Electricity, Labor, and Chemicals
John Anderson
City of Longmont, WWTP Superintendent
Process LDO has been out for a little more than a year longer than Lab unit.
One person to talk to about the Process LDO is John Anderson (WWTP Superintendent, City of Longmont) whose been using them for over a year now.
John has documented savings in Labor and Chemicals. The Electricity savings are estimated from reduced blower usage times.

26. Process LDO – Longmont WWTP
Savings Over 3 Month Period
Electricity ($ 28,000)
Labor ($ 15,000)
Chemicals ($ 36,000)
Total Savings ($ 70,000)
Displayed values were given to me by Devin Standard at Hach company.
John Anderson says the Electricity savings are estimated from blower usage times. Labor and Chemical savings are directly documented.

27. External Validation Study…
12 Wastewater Laboratories Nationwide
Accuracy & Precision (IPR)
Method Detection Limit (MDL) Determn.
5 Day BOD
Side-by-side Comparisons with Other DO Methods
A total of 12 wasterwater (including us as one) laboratories from all over the nation were involved.
Accuracy & Precision (at 2 ppm and ASW) --- “IPR’s” Initial Recovery & Precision Samples
[we sent them the 2 ppm std.s and the protocol to prepare ASW]
MDL – the data from each lab was used to determn. The MDL for that Lab and the resulting MDL’s were then pooled to obtain a “nationwide” MDL
BOD5 – Each lab did a full 5-day BOD determination with 2 Effluent and 2 Influent samples plus controls (GGA, blanks, & multiple dilutions)
Side-by-side Method Comparisons – Each lab was tasked to make comparison measurements using Winkler Titrations and Membrane Measurements.

28. In-House Preparation for External Validation Study…
Preparation of Replicate DO Standards
Shelf-Life Experiment
Shipping Experiment
Ruggedness Testing
Use Model Comparisons
Accuracy & Precision Determinations
MDL Determination
BOD5 Demonstration
Side-by-side Comparison with Other Methods
Preparation of Replicate DO Standards – We had to actually develop all new technology to allow us to prepare and ship replicate DO standards
Shelf-Life Experiment – We ran experiments to verify that the prepared DO samples would be stable long enough to ship them to the external validation laboratories and long enough for those laboratories to make the required DO measurements.
Shipping Experiment – verified the prepared samples would survive shippment to the external validation laboratories
Ruggedness Testing – demonstrated that the LDO instrument will produce reliable DO measurements on a day-to-day basis over a period of several days.
Use Model Comparisons – we compared different Use Models to determine which one was most appropriate for BOD applications in particular and Wastewater App.s in general
Accuracy & Precision Determinations – We looked at two DO conc.s in particular (nominal 2 ppm and ASW) as part of the In-House preparation but I will share data from across the whole range of DO Conc.s
In-house MDL Determination – Complete Dress Rehearsal of the External MDL (Method Detection Limit) Determination. To get a “best case” determination of the MDL.
BOD5 Demonstration – A complete In-house BOD5 determination with Influent and Effluent samples
Side-by-side Comparisons w/ Other EPA Approved Methods for DO Measurements
Clearly I don’t have time to tell you about everything we did so I decided to focus on a few of the more significant experiments – Namely the ones I’ve highlighted in Yellow.

29. Accuracy & Precision Determinations

30. 5 LDO Probes Replicate Measurements w/ dry sensor caps (data collected on probe test stand with NIST traceble gas mixtures)
Key Points –
Linear Response across the entire concentration range.
Calibration Curve passes through zero.
LDO Response is exactly correlated with the Theoretical DO Conc. in ASW
Additional Point
Precision actually improves at lower end of the concentration range (this is because the S/N is higher at the lower concentrations because the signal is actually quenched “less”)

31. Same LDO data as in previous slide.
I’ve superimposed side-by-side data collected with an electrochemical probe and by Winkler Titration
Winkler – is linear and for the most part scattered around the calibration curve.
Response from Membrane Electrode matches the Theoretical values prettly well up to Air-saturated Water ( ~ 7 ppm) and then it starts to drop off and get more erratic at higher concentrations. This happens because the membrane electrode becomes more sensitive to the sample flow rate. It essentially can’t replenish the O2 from the bulk solution at a faster rate than it consumes it.

32. Percent Recovery at 1.69 mg/L
8 replicates in BOD bottles for each of the three methods LDO, Clark Cell (YSI), and Winkler Titration
The error bars correspond to 1 standard deviation about the mean.

33. Percent Recovery in Air-saturated Water
8 replicates in BOD bottles for each of the three methods LDO, Clark Cell, and Winkler Titration
The error bars correspond to 1 standard deviation about the mean.

34. LDO – Histogram Air-saturated Water
Replicate DO Samples in BOD Bottles (a total of 48 replicates, 4 each measured with 12 different LDO meter/probe/sensor combinations calibrated vs. WSA)

35. MDL Determination

36. LDO – In-House Method Detection Limit
49 Replicated DO Samples (Theoretical DO Conc mg/L)
7 LDO Instruments (calibrated vs. WSA and “preconditioned” prior to measurements)
7 Replicates with each instrument (actually there were 8 replicates for LDO #4)
2 operators
Avg. = (+/ ) mg/L
MDL ~ 3 x Std. Dev. = 3 x = mg/L
Source: LDO EPA Approval / DataSummary.xls / Expt 4 - Analysis -- In-House MDL Determination

37. Pooled MDL and ML Results (mg/L)
Data Source
Pooled MDL ML
In-house Study
0.02
0.07
Inter-laboratory Study
0.06
0.18
Pooled
0.05
0.16
Method Detection Limit (MDL is essentially 3 x the Std. Dev. at zero conc. --- The Std. Dev. is actually measured at a concentration that is 3 to 5 times that of the estimated MDL
The Method Limit (ML) is more of a practical limit representing 3 x the MDL (or ~ 9 X Std. Dev. at zero conc.)

38. BOD5 Demonstration

39. BOD5 Comparison
This slide summarizes the BOD data obtained for replicate samples. A total of 3 dilutions was used for each of the G&GA’s (Glucose and Glutamic Acid), Effluent and Influent Wastewater samples.
After correcting for dilution, the G&GA BOD’s should be 198 mg/L

41. Method Comparison – DO Measurements Influent and Effluent Samples from 12 Different Wastewater Facilities
This data is small part of the data that was collected from the External EPA Approval.
What is shown is the combined influent and effluent DO measurements made at 12 different wastewater facilities nationwide.
Each facility measured parallel influent and effluent samples using each of the different methods for measuring DO (Winkler Titrations, Membrane (Clark Cell) Electrodes, and LDO).
What is plotted is the measured DO Conc. from either the Winkler Titration or Membrane Electrode measurement vs. the Measured DO concentration obtained for a parallel sample with the LDO instrument.
Note: the correlation between the LDO, and the other methods is good throughout the concentration range (from ~ 0 to 9.5 mg/L)
The other thing that came out of this study was that the LDO was completely insensitive to the type of sample matrix. That ultimately means you can use the same quality control criteria for LDO for both influent and effluent samples.

42. What’s Next?
Report for External Validation Study has been submitted to EPA
Next the results are opened to the public for Review and Comments (~ 3 mo.)
Expect Full EPA Approval in 12 to 18 months
Currently Partitioning EPA for Provisional Approval (6 to 12 mo.)
Soliciting/Helping WW Labs seeking Tier 1 Approvals

43. Contacts… Edward C. Craig, Ph.D. Cary B. Jackson, Ph.D.
Research & Development Chemist
Cary B. Jackson, Ph.D.
Regulatory Chemist
Christopher Fair
Wastewater Applications Chemist