1. What Does the EPA Method 625.1 Round Robin Data Really Tell Us?
National Environmental Monitoring Conference 2018, New Orleans, LA August 9, 2018 Polly S. Newbold, ddms, inc.
Intent is to explore some of the data that was generated during the two Round Robin events. Are there trends based on specific compounds or classes of compounds?
Protective of human health and the environment.

2. OUTLINE General Overview Some data Concluding Remarks What’s Next Q&A
Since the 625 limits were used to determine acceptable data for the 2 studies, we need to look at what’s behind 625 acceptance limits.

3. When Did It Start? First reference in literature to the use of SPE
So the people grumbled at Moses, saying, "What shall we drink?“ Then he cried out to the LORD, and the LORD showed him a tree; and he threw it into the waters, and the waters became sweet. Exodus 15: 24 and 25
Dr. Wells and Bill (Horizon) dates
First reference in literature to the use of SPE
So the people grumbled at Moses, saying, "What shall we drink?“ Then he cried out to the LORD, and the LORD showed him a tree; and he threw it into the waters, and the waters became sweet. Exodus 15: 24 and 25

4. Method 625.1 Acceptance Limits
Table 6
Acceptance criteria are based upon method performance data in Table 7 and from EPA Method Where necessary, limits for recovery have been broadened to assure applicability to concentrations below those used to develop Table 7.

5. Method 625.1 Acceptance Limits (con’t.)
Table 7
Precision and recovery as a function of concentration w/ footnote that says “Regressions based on data from Reference 2”
“EPA Method Study 30, Method 625, Base/Neutrals, Acids, and Pesticides,” EPA 600/ , National Technical Information Service, PB , Springfield, Virginia 22161, June 1984.

6. Method 625.1 Acceptance Limits (con’t.)
Two different columns
1 for acids/ 1 for base neutrals
Two separate calibrations
Packed columns
Separatory funnel extraction

7. Method 625.1 Acceptance Criteria based on 625
Only 6 compounds with lower limit > 50%
Best lower acceptance limit 60%
2-chloronaphthalene
Only 6 compounds with lower limit > 50%
Best lower acceptance limit 60% 2-chloronaphthalene
Approximately 60 compounds
50% isn’t very good.

8. Labs from Method 625 Acurex Corporation
California Analytical Laboratories, Inc.
Envirodyne
Environmental Research Group, Inc.
Environmental Science and Engineering, Inc.
Foremost-McKesson
GCA Corporation
Mead CompuChem
15 labs participated
Trip down memory lane

9. Labs from Method 625 Pedco Rockwell International
Rocky Mountain Analytical
Spectrix
Stewart Labs
The University of Utah Research Institute
West Coast Technical Service, Inc.

10. The Studies 6 or 7 Suppliers Acceptance criteria
Method 625
TNI
Each SPE product was tested in at least three laboratories
ENTER
data compared to that obtained using the specified in EPA Method 625
All know limits in 625 and are huge.
Where 625 didn’t have limits, TNI acceptable analyte recovery ranges were used. The TNI range was defined as being “From the acceptance range generated when the test sample was analyzed by a large number of laboratories using EPA Method 625 during a laboratory proficiency study. “
While one of the laboratories was permitted to be the vendor's in-house laboratory, two of the laboratories had to be independent of the vendor. The laboratories were a combination of commercial, government, and academic laboratories.
While the laboratories followed the particular vendor's directions for using their particular SPE product being evaluated, all product-specific use directions followed the draft SPE protocol and the instrumental analysis described in the draft Method 625A when analyzing the SPE extracts.

11. The Studies Phase 1 Spiked DI water (LCS) ASTM Synthetic Wastewater
Flat light beer
Flour
Sea salts
Kaolin
Triton X-100
DI water
Each lab was to analyze the spike matrices 3X per SPE product.
Each Study was to include 2 LCS
The data were generated from 2 studies, identified as Phase 1 and Phase 2.
ENTER
Phase 1 - For each SPE product, participating laboratories received a container of liquid, a container of solid material, and two or more ampoules of spiking liquid. The liquid was sterilized to ensure adequate shelf life. When the laboratory was ready to perform the experiments, they mixed the solid material into the liquid material, added the spiking solutions, and diluted the mixture with laboratory reagent water to 2 liters.

12. The Studies Phase 2 LCS TCLP acetate buffer solution Limited ASTM WW
Glacial acetic acid
1N NaOH
DI water
Limited ASTM WW
Refer to slide first
The TCLP acetate buffer solution was to be made for the addition of a matrix modifier supplied by Phenova to keep the matrix consistent between laboratories.

13. The Studies - Data Phase 1
Data from 17 labs, but no correlation between supplier and lab was provided
The data I was working with were in spreadsheets compiling all lab recoveries. They did not include any additional studies any of the suppliers may have done outside of Phase 1 and Phase 2. The correlation between suppliers and labs is not known for Phase 1. That is to say, with the Phase 2 data, the data are grouped together by supplier, but the suppliers and the labs are not identified with the data.

14. The Studies Phase 2 24 labs Supplier 1 Labs 1-3 Supplier 2 Labs 4-6
Three different types of media.
Supplier 4 – disks
One supplier – spin bars
All others – cartridges
Note the Supplier 4 used 6 labs identified as Labs 10-15
Watch labs Phase 2 labs 14 and 18

15. The Studies - Assumptions
The samples prior to extraction were all prepared the same from lab to lab.
Each supplier provided the same SPE product(s) to each of their laboratories.
Each laboratory for a supplier implemented that specific-SPE system in the same manner.

16. Data - Phase 1 – LCS (%R)
This is a comparison of the Phase 1 SPE data to liquid-liquid prep by compound class for the LCS data.
Delta overall 4% (acids) % (HCA)
PAHs 9.5%

17. Data - Phase 1 – ASTM (%R) Same comparison but on the ASTM WW data.
Delta overall 1% (Phthalates) -46.7% (Pesticides)
PAHs 16.1%
Acids 9.4%

18. Data - Phase 2 LCS Acids Opted to look at acids and PAHs.
A review of the data indicated that the labeled surrogates and the unlabeled partners did not exhibit consistent recoveries. As the labeled goes, so goes the unlabeled. Only Phase 2 data was provided with surrogate recoveries. The compounds displayed here are unlabeled with labeled partners for acids. Overall average for these 7 compounds is -55% to 1.5%.
2,4-Dichlorophenol -5.02%
2-Chlorophenol -13.3%
2-Methyl-4,6-Dinitrophenol %
2-Nitrophenol %
4-Methylphenol %
4-Nitrophenol 1.53%
Phenol %

19. Data - Phase 2 LCS Acids
Removed 2-Methyl-4,6-Dinitrophenol outlier in order to get a better sense of the variability.
108 %

20. Data - Phase 2 TCLP Acids
TCLP Overall average for these 7 acid compounds is -23% to 1.0%.
2,4-Dichlorophenol -2.77%
2-Chlorophenol -9.49%
2-Methyl-4,6-Dinitrophenol %
2-Nitrophenol %
4-Methylphenol 1.00%
4-Nitrophenol %
Phenol %

21. Data - Phase 2 TCLP Acids
Deleted huge outlier peak.
125%

22. The Data – Phase 2 LCS PAHs
75 %
Looking at PAHs. Overall avg delta for analyte for all lab data
Acenaphthylene %
Anthracene %
Benzo(a)pyrene 8.08%
Fluorene %
Pyrene %
As you can see, there is significant variability when looking at the full data set for these PAHs.
113 %

23. Data – Phase 2 TCLP PAHs
Overall avg delta for analyte for all lab data
Acenaphthylene %
Anthracene %
Benzo(a)pyrene 3.42%
Fluorene %
Pyrene %
As you can see, there is significant variability when looking at the full data set for these PAHs.

24. Acids – Phase 2 MAY TAKE THIS SLIDE OUT
Compound
LCS Average
LCS SD
LCS Min
LCS Max
TCLP Average
TCLP SD
TCLP Min
TCLP Max
ASTM WW Average
ASTM WW SD
ASTM WW Min
ASTM WW Max
2,4,5-Trichlorophenol
83.37%
37.87%
16.63%
162.77%
89.75%
51.25%
17.06%
249.36%
61.52%
28.20%
16.93%
97.44%
2,4,6-Trichlorophenol
90.04%
45.59%
15.75%
206.64%
101.75%
62.94%
26.73%
279.65%
58.88%
24.61%
23.73%
89.68%
2,4-Dichlorophenol
78.19%
45.64%
14.68%
200.63%
78.81%
40.97%
19.92%
212.11%
47.53%
18.30%
19.69%
67.08%
2,6-Dichlorophenol
77.35%
50.52%
0.00%
152.61%
77.09%
49.47%
163.82%
44.16%
34.73%
89.87%
4-Chloro-3-methylphenol
86.04%
46.23%
13.90%
194.38%
85.41%
41.20%
20.90%
208.76%
63.02%
26.64%
18.91%
92.96%
2-Chlorophenol
75.70%
46.21%
14.65%
191.35%
73.51%
37.83%
21.56%
177.07%
44.70%
17.73%
20.66%
64.10%
2,4-Dimethylphenol
86.09%
46.13%
20.97%
196.44%
82.44%
36.55%
20.55%
186.67%
51.81%
20.38%
23.63%
71.01%
2-Nitrophenol
82.82%
51.78%
20.43%
207.99%
89.04%
65.14%
23.23%
347.21%
41.66%
15.13%
21.70%
57.99%
2-Methyl-4,6-Dinitrophenol
151.62%
252.20%
10.75% %
96.68%
109.58%
544.56%
46.57%
16.78%
13.64%
62.37%
Pentachlorophenol
127.72%
187.44%
12.60%
836.86%
93.66%
81.80%
362.53%
46.43%
25.43%
12.58%
89.77%
2-Methylphenol
65.37%
27.98%
22.10%
138.40%
63.98%
28.87%
117.49%
49.48%
21.03%
19.31%
78.35%
4-Methylphenol (and/or 3-Methylphenol)
67.73%
28.26%
18.06%
134.60%
60.56%
27.85%
115.58%
46.82%
17.20%
24.10%
64.54%
2,4-Dinitrophenol
65.98%
62.41%
239.71%
66.12%
86.24%
430.33%
29.07%
17.36%
50.32%
4-Nitrophenol
67.42%
28.79%
16.66%
118.42%
64.50%
33.87%
132.73%
46.51%
19.73%
17.49%
72.89%
Phenol
54.34%
38.92%
6.28%
158.33%
No data
36.80%
15.65%
14.67%
53.66%
Average
83.99%
57.02%
16.62%
272.87%
80.23%
48.66%
19.33%
240.79%
47.66%
17.00%
18.71%
61.37%
MAY TAKE THIS SLIDE OUT
Bottom row “average” is the average recovery of all of the acids.
SD of averages of each compound.
Min = min of avgs
Max = max of all avgs.

25. Acids – Phase 2 LCS Compound LCS Average LCS SD LCS Min LCS Max
LCS Delta Min/Max
2-Methyl-4,6-Dinitrophenol
151.62%
252.20%
10.75% % %
4-Nitrophenol
67.42%
28.79%
16.66%
118.42%
101.76%
This slide and the next few show analytes with the smallest and largest absolute deltas between the max and min values. Also included is the avg over all the labs and the SD in each case.
What kind of acceptance limits would there be with 3 times the SD?

26. Acids – Phase 2 TCLP Compound TCLP Average TCLP SD TCLP Min TCLP Max
TCLP Delta Min/Max
2-Methyl-4,6-Dinitrophenol
96.68%
109.58%
0.00%
544.56%
4-Methylphenol
60.56%
27.85%
115.58%

27. Acids – Phase 2 ASTM WW Compound ASTM WW Average ASTM WW SD
ASTM WW Min
ASTM WW Max
ASTM WW Delta Min/Max
2,6-Dichloro-phenol
44.16%
34.73%
0.00%
89.87%
2-Nitrophenol
41.66%
15.13%
21.70%
57.99%
36.29%

28. PAHs – Phase 2 LCS Compound LCS Average LCS SD LCS Min LCS Max
LCS Delta Min/Max
Phenanthrene
76.12%
48.81%
12.63%
242.68%
230.05%
Fluorene
65.95%
24.39%
12.54%
94.17%
81.62%

29. PAHs – Phase 2 TCLP Compound TCLP Average TCLP SD TCLP Min TCLP Max
TCLP Delta Min/Max
Indeno(1,2,3-cd)pyrene
71.72%
41.04%
5.88%
222.05%
216.17%
Acenaphthene
71.60%
24.62%
17.33%
103.02%
85.69%

30. PAHs – Phase 2 ASTM WW Compound ASTM WW Average ASTM WW SD ASTM WW Min
ASTM WW Max
ASTM WW Delta Min/Max
Pyrene
61.04%
38.11%
11.98%
117.00%
105.02%
Dibenzo(a,h)anthracene
36.72%
14.67%
10.89%
52.37%
41.48%
Again, to be fair this is a limited data set.

31. Acids - Potential Environmental Impact
Phenol
2-Nitrophenol (2-NP)
2,4-Dichlorophenol (2,4-DCP)
2,4,6-Trichlorophenol (2,4,6-TCP)
Pentachlorophenol (PCP)
Phenolics have drawn attention due to their potential toxicity. These are common phenolic compounds which can be introduced into the environment by industrial sewage draining, agricultural irrigation, application of pesticides, and pharmaceutical drugs. Looked at these.

32. Acids – Phase 1 LCS X axis = lab Y axis = %R
Broken lines indicate data gaps. Not sure the cause of the data gaps. Is it labs that didn’t report? Indication that all of the data went through a statistical evaluation. Are the data gaps a result of the evaluation?
Plot of 5 acid compounds from Phase 1. What is striking is that for several labs, the bias seems to be lab-specific not unlike the variability in the labeled vs. unlabeled. Also notice the variability in recoveries.

33. Acids – Phase 2 LCS
Similar here.

34. Acids – Phase 2 LCS Removed pentachlorophenol outlier – lab 14.
Notice how results are still biased high.

35. Acids – Phase 1 ASTM WW
Two suppliers did a Mulligan for the ASTM WW due to poor recoveries. Don’t know if their data from Phase 1 is included or not. Assume no since the data represented 17 labs vs. 24 for Phase 2.

36. Acids – Phase 2 ASTM WW
Supplier 5 Labs 16-18
Supplier 7 Labs 22-24

37. Acids – Phase 2 TCLP

38. PAHs with TEF Benzo(a)pyrene [BaP] Benzo(a)anthracene [BaA]
Benzo(b)fluoranthene [BbF]
Benzo(k)fluoranthene [BkF]
Chrysene [Chry]
Dibenzo(a,h)anthracene [DahA]
Indeno(1,2,3cd)pyrene [I(123cd)P]
Literature seems to agree that these 7 compounds of the 16 carcinogenic have TEFs, with BaP being the most toxic. Only 5 were reported in the 625 study. Looked at these. Chrysene was included in the Phase 1 data.

39. PAHs – Phase 1 LCS

40. PAHs – Phase 2 LCS

41. PAHs – Phase 2 TCLP

42. PAHs – Phase 1 ASTM WW
Chrysene not included in data table.

43. PAHs – Phase 2 ASTM WW
Two suppliers
5 – labs 16-18
7 labs 22-24

44. Finally Averaging Data variability
Large SDs
Orders of magnitude – Min vs. Max
Method or TNI acceptance criteria
Waste water
I started with the question of “What Does the Round Robin Data Really Tell Us?”
Averaging smooths out the data. Can’t ignore the large standard deviations or the orders of magnitude differences in the data. Saw large variations in reported results from lab to lab. What kind of error is introduced?
Method or TNI acceptance criteria – Determined in 1984 with now outdated procedures. Someone said to me that the labs will generate their own limits…not for the 600 series methods, they don’t. how are we protective of “human health and the environment” if we rely on those limits to evaluate acceptable data.
Waste water – synthetic waste water is not real wastewater, and all wastewater is not created equal from day to day within the same system, state to state, or across the country. Method has a provision for the supplier to generate acceptance limits. Seems like a little inappropriate for a supplier to generate acceptance limits for their own product. In addition, the wastewater that the supplier would use could not be representative of all wastewaters. Limits determined on a single waste stream should not be considered valid for all wastewaters.

45. What’s Next? Inconsistent compounds reported from Phase 1 to Phase 2.
Verify SPE products for both phases
Spin bars
Disks
Cartridges
Pair up labs from Phase 1 to Phase 2
Data gaps
Need to get a better understanding of the data. Compounds in the spreadsheets were inconsistent. Ex. BghiP reported Phase 1 data, but not Phase 2 data. Chry in Phase 2, but not Phase 1.
Verify SPE products for both phases – were the same SPE products used for both studies and pair up the data from Phase 1 to Phase 2, including the lab data.
Data gaps – causes

46. What’s Next? Issues with matrices Extractions
Conventional liquid – liquid extraction
Other lab QC
Lab specific excursions
Labeled vs. unlabeled
%Rs
Did the labs have any issues with the matrices – read one place that with a lot wastewaters, with the addition of base, the sample looks like a “snow globe”.
Did any of the labs prefilter the sample? Issues with clogging?
How was the sample spiked before the extraction? I know how it was supposed to have been prepared, but was it.
Conventional liquid – liquid extraction – was the sep funnel or liquid liquid extraction. Data behind that part of the study…how many labs, how many times did they run the sample by this method…
Went into this thinking the issues with recoveries were related to the compound chemistry or the SPE media. Not sure that is true now. Still haven’t resolved why there is a disparity between labeled surrogates and unlabeled paired compounds. Overall, the differences in recoveries to be lab dependent, but why.

47. What’s Next? Extract clean ups
Apply tighter limits to the existing data
Compare recoveries of classes of compounds
Method 625 allows for clean ups. Some labs routinely do clean ups. Were clean ups done on any of the extracts?
Apply limits of 50%-150%...still pretty wide, or 70%-130%.
Compare recoveries of classes of compounds between Phase 1 and Phase 2 and lab to lab…supplier to supplier.

48. Facts do not cease to exist because they are ignored.
Aldous Huxley- author of Brave New World
SPE has advantages and it’s time and place. Agree, but is it appropriate in all places? The data user needs to be aware of the existing bias and the great deal of potential bias in the numbers. We are to be protective of human health and the environment. Are we being protective if we use acceptance limits that are huge? What do you think?

49. We are scientists! We can all figure this out, right?

50. References
“EPA Method Study 30, Method 625, Base/Neutrals, Acids, and Pesticides,” EPA 600/ , National Technical Information Service, PB , Springfield, Virginia , June 1984.
Phase 1 Data
Phase 2 Data
QA/QC Solutions, LLC

51. Acknowledgements NEMC and TNI
You, the audience for your patience and attention
Colleagues, past and present
QA/QC Solutions, LLC
QA/QC Solutions, LLC

52. DISCLAIMER
The views and opinions expressed in this presentation are those solely of the presenters and do not reflect the opinions, official policy, or position of any client or regulatory agency.
QA/QC Solutions, LLC

53. The Studies – SPE Products and Vendors
Agilent Technologies, Inc.
2850 Centerville Road
Wilmington, DE 19808
Bond Elut C18, 6ml, 1 gm, P/N and Bond Elut ENV, 6ml, 500 mg, P/N
Fluid Management Systems
580 Pleasant Street
Watertown, MA 02472
Mixed mode cartridge with a coconut charcoal 2nd cartridge, automated system
Gerstel Corp.
701 Digital Drive; Suite J
Lithicum, MD 21090
GERSTEL Twister, PDMS Phase, 10mm x 0.5mm, P/N
Horizon Technology Inc.
16 Northwestern Drive
Salem, NH 03079
SPE-DEX® 4790 Automated Extractor with One-Pass kit and Envision Controller
47-mm disk holder (Part number ) for 100-mL volume samples and EZ-flow holder for 1-L samples (Part number )
Atlantic® One-pass disk (Part number )
Max Detect Carbon cartridge (Part number )
DryDisk® (Part number HT) used with either the manual drying glassware (Part number SDS /22) or the DryVap in-line drying and evaporation system

54. The Studies – SPE Products and Vendors
Phenomenex Corp.                                        
411 Madrid Avenue
Torrence, CA 90501
Strata™-XL-C 2g/20mL Giga tube (P/N 8B-S044-KEG), Polymer-Based Mixed Mode Strong Cation-Exchange & Reverse Phase,100µm particle size, 300Å pore size
UCT, Inc.
2731 Bartram Road
Bristol, PA
UCT Enviro-Clean 8270 (p/n: EC8270-KIT or EC8270-KIT1L)

55. Thank you! Any Questions?
Contact Information
Polly S. Newbold, Sr. Environmental Chemist
ddms, inc.
Tel: (908)