Laboratory Issues with TKN and TP Digestion and Analyses

1. Laboratory Issues with TKN and TP Digestion and Analyses With some discussion on laboratory ideas for low level phosphorus and LOQ Our SOPs and information are all online at:tiaer.tarleton.edu/laboratory.html

2. The Texas Institute for Applied Environmental Research (TIAER) is a multi-component, environmental science and public policy entity as established by the state legislature. TIAER operates a laboratory on the campus of Tarleton State University for analyses of samples under various state, federal and private industry contracts and projects. TIAER Lab is NELAP/TNI accredited for certain analyses on non-potable water and solid/chemical matrices.

3. TIAER Lab is not large The majority of our samples are streams and reservoirs (grabs or storm composites), but many are WWTP influents and effluents, CAFO runoff and industrial waste. The latter are often highly complex matrices. • 2 full time chemist analysts • 2-3 technicians • Lab QAO who can fill in on some tests • Member of Small Lab Advocacy Group (SLAG) Clients include the EPA, TCEQ, USDA, USGS, TSSWCB, TPWD, several WWTPs, river authorities and private entities.

4. TNI/NELAP Accreditation and methods of interest to this presentation: Total phosphorus by EPA 365.4 for non-potable water and EPA 365.3 for solid/chemical matrices. Total Kjeldahl nitrogen by SM 4500-NH3G (automated phenate) with preparation by SM 4500-Norg B/D Both TP and TKN are run on a LaChat Autoanalyzer (now Hach) (QuikChem 8000 flow injection analyzer) We analyze TP & TKN from the same digestate. Normal Range TKN: 0.2-10.0 mg/L Normal range TP: 0.06 – 10.0 mg/L Low range TP: 0.01-0.5 mg/L

5. Spikes and Standards Desired Analyte Range mL of Spike (spike mg/L) Solution Concentration Low range Total Phosphate 1.0 ( 0.2) 100 Normal range TKN and TP 1.0 (4.0) 100 High range TKN and TP and solid samples 1.0 (40) 1000 Calibration, check standards and spikes are made from ammonium chloride and potassium dihydrogen phosphate. All preserved with the same acid concentration as samples. Laboratory Control Samples are made from separate sources than calibration standards. LCS for TKN is L-lysine; for TP we use AMP (adenosine monophosphate).

6. Federal Register / Vol. 72, No. 47 / Monday, March 12, 2007, 40 CFR Part 122, 136, et al. The final rule approves the replacement of the mercuric sulfate catalyst with copper sulfate in methods approved for determination of total Kjeldahl nitrogen (TKN), but mercuric sulfate is still allowed.SM 4500-Norg D states to use copper sulfate. For TP, Federal Register: September 23, 2010 (Volume 75, Number 184) Proposed rule, Final Action December 2011 says copper can be substituted for mercury. SM 4500-P H is chemically similar and does not use mercury. We’ve done extensive studies on Cu vs Hg. We use copper in place of the mercury catalyst…

7. 25 mL samples/standards in 100 mL tubes. All standards and blanks digested the same as samples.

8. Preparation logbook map of blocks

9. 3- 40 place block digesters with programmable heating modules: Tecator 2040 block digester, BD-40 and Tecator 2000 series control pad, CU-10 (or equivalent as in Fisher)

10. Programming the block: 160°C for 3 hours, 380°C for 3 hours, 98°C until reconstituted in DI water.

11. The next day they are heated for over an hour in about 10 mL DI at 98°C, mixed and rinsed x3, then reconstituted to 25 mL original volume.

12. Hengar ® granules for boiling stones, not Teflon®

13. Snap vials for digested samples save screwing lids on and off and free a hand

14. XYZ autosampler

15. Analyst at LaChat workstation

16. Dual manifolds- Heaters at 37°C for TP, 60°C for TKN

17. Sampler, pump module and dual manifolds

18. Set up of a Tray Table

19. Close up view of calibration standard peaks

20. Close up view of TP calibration standard peaks

22. TP run peaks

26. Special TP/TKN LOD comparison study in 2011 • Does the background noise level increase as values get closer to the lower limit of quantitation? • What is the overall picture of percent recoveries in TP vs TKN LOQ? • Some History Some type of low level standard has been analyzed for TP and TKN for over 20 years at TIAER. Until some time in 2008, this standard was analyzed in the calibration curve only and required a ± 25% detection variance from the true value in order to be acceptable. After that time, the low standard was analyzed both as the lowest point on the calibration curve (all standards requiring ± 25% detection variance) and as a calibration verification check standard.

27. The nomenclature for the low calibration and calibration verification standard used at TIAER has changed through the years. • Formerly the Reporting Limit (RL) standard, and the Ambient Water Reporting Limit (AWRL) standard before that (at TIAER). • MDL/PQL >>> AWRL >>> RL >>> LOQ/LOD • The relationship of the MDL (determination defined by 40 CFR 136) to the LOQ is an approximate ratio of 2:5, MDL: LOQ. SM 1030 C also states “Although the LOQ is useful within a laboratory, the practical quantitation limit (PQL) has been proposed as the lowest level achievable among laboratories within specified limits during routine laboratory operations”. The ratio of the PQL to the MDL is about 5:1. • We confirm calibration at both the LOQ (TCEQ) and at the midpoint of the calibration curve (SM). For compliance with 40 CFR 136 and other project clients, the TIAER lab also determines and reports some non-TCEQ data at the LOD and uses the PQL to screen some levels of quality control (duplicate/field split precision) and data acceptance criteria.

28. For the comparison study, several sites on the Bosque River were run in replicates. The average versus the relative standard deviation (STD/Mean) for the TKN and TP laboratory duplicates and triplicates were computed and graphed.  • Figures 1 & 2 show the relative standard deviations compared to concentration for duplicate and triplicate analyses of actual field samples. One set of outlier triplicates was thrown out for TKN (sample 85218, 15Aug2011) where sample results ranged from 2.07 to 5.12 mg/L.  One set of TP outlier duplicates (sample 84994, 20Jun2011 values 0.119 & 4.04 mg/L) was also not included.  It is possible that this sample was accidently spiked. • Figures 3 & 4 show TP & TKN LOQ recoveries over a time period studied with the 75-125% acceptance level bars. These exclude a gap in 2009 where reruns were not clearly identified in the data storage spreadsheet and LIMS of that time. The policy has since been changed to identify reruns separately and distinctly.  As required by the QAPPs and SOPs, if required quality control parameters do not pass, the batches and/or samples are rerunor the data is flagged as unacceptable. Some project managers may still want failed data for various purposes and often request it. Data from more recent years were evaluated where the low level calibration standard (AWRL, RL or LOQ) or the LOQ check standard was documented electronically and easily retrievable. Older paper logbooks were not mined for this data due to time and financial restraints.

29. Figure 1- Sample duplicate/triplicate precision for TKN

30. Figure 2- Sample duplicate/triplicate precision for TP

31. Figure 3- Accuracy: TP LOQ recoveries over time

32. Figure 4- Accuracy: TKN LOQ recoveries over time

33. Figures 1 & 2 showed marked variability at the lower concentration levels as expected. • Figures 3 & 4 indicate that TKN recoveries are generally tighter than TP. Batches are not generally separated into clean and dirty sample matrices, only into solid or aqueous groups. A clean, ambient water sample or a feedlot runoff sample may be the one to receive duplicate and spike quality control, but they are selected at random by the order received at the analysis station.

34. Some we things learned that you may already know: • 1. We combine the preparation steps of TP & TKN into one digestate and analyze them separately. • 2. No distillation is performed for automated phenate TKN or TP (SM 4500 N-org B, “Selective electrode methods and automated colorimetric methods may be used for measurement of ammonia in digestate without distillation. Follow equipment manufacturer’s instructions.” • 3. Selenized Hengar granules are used rather than Teflon, which tend to float rather than sink. • 4. A primary chemist doing the sample preparation has better results than technicians. • 5. We now use Class A graduated cylinders, routinely replace the filter on the analyzer to achieve a steadier baseline, and digest samples of more difficult matrices at a lower initial temperature to make digestions more complete and more consistent. • For TKN: • The sodium hypochlorite (bleach) must be fresh. A few years ago the stores changed from about 5% to 6% hypochlorite as standard bleach, and the reagent concentration had to be adjusted. • Working buffer solution for TKN may need to be filtered through a 0.45 µm filter. • Reduce exposure to air as this may tend to absorb ammonia from the atmosphere. 4500-NH3 G. Section 3b-f.  We use these solutions as described in the Lachat method.  The solutions are similar, the primary differences are concentrations. 4500-Norg B. Section 2.  We use the apparatus setup as described in EPA 365.4 section 4.0 for Total Phosphorus and in the instrument manual. • Section 3a.  We use the digestion reagent described in EPA 365.4 section 5.2 with the exception of substituting copper for mercury. Section 4a.  We use 25mL for most samples, digestion tubes instead of Kjeldahl flasks Section 4b.  We do not perform the ammonia removal step.

35. Specific for TP: • EPA 365.4 • Sections 5.1-5.2.  We substituted copper for mercury in the digestion solution many years ago. First with a variance letter from Region 6 EPA, then from changes in methods approved in the Federal Register. • Sections 5.6-5.7.  The reagents NaCl and H2SO4 reagents are combined into one in the Lachat instrument method. • Section 7.1.  Our instrument uses peak area rather than peak height. • EPA 365.3 • Section 6.2.  We use hot 1 N HCl rather than hot 1:1 HCl for washing glassware.  Additionally we do not treat glassware with all the reagents to remove traces of phosphorus in order to reduce hazardous waste generation, but we have not seen contamination problems in the blanks identified as caused by this. • Additional for Low Level TP: • Aliquots of about 50 mL are added to each digestion tube and heated at 160°C until reduced in volume. Aliquots are added and reduced for a total volume of 500 mL for each sample, standard and blank. One sample per QC batch is spiked with 1.0 mL of WS for a 0.2 mg/L spike.

36. Some last points: 1. Samples are identified at login as field or lab preserved (due to submission to TCEQ by different parameter codes for each), but are all analyzed together for TP/TKN. 2. Spike is to 4 normally, but 40 or more if matrix is obviously complex as sample (and spike) will need to be diluted significantly in order to read concentration. Low TP spike is 0.2 mg/L. We have had mixed success with higher spiking of difficult matrices for TP & TKN, but very good success on low TP spikes. 2. For LOQ calibration standard failures, we calibrate and reanalyze. For LOQ check standard failures, our policy is to rerun the standard once. Upon passing, the run is accepted. If failed, the data is flagged as failing. We don’t keep rerunning until we get a passing LOQ check standard for a batch.

37. 0.1 mg/L TKN standard LOQ is 0.2 mg/L LOD for this run was 0.045 mg/L

38. Extra LOQ not used

39. LOQ cal std fails 10 & 0.5 stds removed, LOQ cal passes

40. Raw data from Low TP run

41. Thanks! murphy@tarleton.edu tiaer.tarleton.edu