1

ASTM D 19.06 Sub

- committee

, D

- 19 Water

Inter

- Laboratory Study to Establish Precision Statements for ASTM WK 23240

– Standard Test

Method for Solvent

- Free Membrane Recoverable Oil and Grease by Infrared Determination

Report Considered Preliminary until Results Approved by the D19 Results Coordinator

STUDY REPORT

Technical Contact: Tyler Martin Orono Spectral Solutions

983 Stillwater Ave.
Old Town, ME 04468 Tyler.martin@ossmaine.com

866

- 269

- 8007

1. Introduction

An Interlaboratory Study (ILS) was co nducted to establish a precision statement for WK 23240

-

Standard Test Method for Solvent

- Free Membrane Recoverable Oil and Grease by Infrared

Determination. Accompanying this report, find the list of participating laboratories, description

of samples, s tudy instructions, description of equipment, the data, the data summary, and the

complete analytical method. For completeness, results and information regarding the single

laboratory validation study are included here as well.

2. Test Method

The Test Method used for this ILS is WK23240, 2009. To obtain a copy of WK23240, go to

ASTM website, www.astm.org , or contact ASTM Customer Service at 610

- 832

- 9585 or by

email at service@astm.org

. A copy of the method is also in Appendix E.2

3. Participating Laboratories

Fifteen laboratories were invited to participate in the study. T welve laboratories agreed to

participate

. They are listed below:

1. Massachusetts Water Resources Authority Charlestown Navy Yard, 100 First Avenue
Boston, MA 02129 (617) 242

- 6000

2. Portland Water District 225 Douglass Street PO BOX 3553
Portland, ME 04104

- 3553 (207) 774

- 5961

3. Bangor Wastewater Treatment

760 Main St.
Bangor, ME 04401 (207) 992

- 4470

4. Wright

- Pierce

99 Main Street Topsham, ME 04086 (207) 725

- 8721

5. Katahdin Analytical Services 600 Technology Way Scarborough, ME 04074 (207) 874

- 2400

6. Eastern Analytical, Inc.

25 Chenell Drive
Concord, NH 03301 (603) 228

- 0525

7. A & L Laboratory Inc.

3100 Hote l Road

P.O. Box 1507
Auburn, ME 04211 (207) 784

- 5354

8. Clearwater Labs

153 Main Street Newport, Maine 04953 (207) 368

- 57003

9. Northeast Laboratory Services

227 China Road
Winslow, ME 04901

800

- 244

- 8378

10. Maine Stream Analytical

88 Webster Ave.
Bangor, ME 04401

207

- 577

- 4060

11. Cote Lab Chemistry Dept. Bates College

5 Andrews Road
Lewiston, ME

207

- 786

- 6297

12. Food Chemical Safety and Food Analysis Laboratory University of Maine

5735 Hitchner Hall
Orono, ME 04469

207

- 581

- 1369

4. Description of Samples

Samples for the single laboratory study were used as

- received with no dilution or addition of oil

or grease. Samples were split, preserved and provided to the laboratory for testing.

A Youden Pair study design was followed for the interlaboratory study per ASTM D2777 and

approved by D19.06 subcomittee for the interlaboratory study

. The nature of oil and grease

sample necessitates the use of real

- world samples, not laboratory

- created spike samples,

potentially making it difficult to find samples of the appropriate concentrations for a Youden pair

study. In this case, prior testing was performed to attempt to find three matrices at the

appropriate concentrations. In addition, it was desirable to have a range of types of analyte, i.e.

both a petroleum hy drocarbon sample and a grease containing sample. Also, it was desirable to

test examples of both saltwater and freshwater matrices. With these requirements in mind, t hree

real

- world wastewater samples were chosen that previous testing on trial samples ha d indicated

had the desirable characteristics and concentrations :4

Publicly Owned Treatment Works, Primary Inlet:

~ 1 0 mg/L Dairy:

~ 5 0 mg/L

Ocean

- Going Ship Bilge Water:

~ 100 mg/L

W hen new large volumes of the samples were received for the ILS, at some expense of time and

money, it was found that the Bilge Water sample was lower than the results of testing the trial

sample had led us to expect , and the other two samples were at concentration s higher than

expect ed :

Publicly Owned Tre atment Works, Pr imary Inlet: 20 mg/L Dairy: 65 mg/L

Ocean

- Going Ship Bilge Water: 11 mg/L

As significant resources had already been committed , including twelve laboratories that had been

scheduled, and delaying the study to obtain new samples ( which may or may not have been at or

near the desired concentrations ) would have delayed the study by at least a month, the decision

was made to adjust the concentrations to be closer to the desired levels of oil and/or grease by

diluting with an appropriate diluents or adding an appropriate type of oil and/or grease to the

sample prior to splitting the large volume into smaller bottles for shipping to the laboratories.

Thus, the POTW and Dairy samples were di luted with tapwater to roughly achieve the desired

concentrations. A petroleum oil (lubricating oil) chosen to be similar to the main hydrocarbon

component of the sample was added to the Bilge sample by spiking it from a 10 mg/mL solution

in acetone. Thu s the sample levels were adjusted to the final concentrations:

Publicly Owned Tre atment Works, Primary Inlet: 10.8 mg/L Dairy: 58.8 mg/L

Ocean

- Going Ship Bilge Water: 91.8 mg/L

At this point a number of sample bottles were split to be the higher of the required Youden pair

for each matrix. The lower sample of each Youden pair was created through dilution with the

appropriate diluents for each matrix. The POTW and Dairy samples were further diluted with

tapwater and the Bilge sample was dil uted with art ificial seawater. The lower concentrations of

each Youden pair were thus:5

Publicly Owned Tre atment Works, Primary Inlet: 9.3 mg/L Dairy: 51.6 mg/L

Ocean

- Going Ship Bilge Water: 86.6 mg/L

Additionally, spike samples (for method detection limit (MDL) , in itial precision and recovery (IPR)

, daily laboratory control samples (LCS) , and matrix spikes (MS) to check for interferences) were done with a 1:1 solution of hexadecane:stearic acid at 4 mg/mL in acetone. The MDL, IPR and LCS samples were spiked into la b reagent water.

5. Interlaboratory Study Instructions

Laboratory participants were emailed the test program instructions , provided in Appendix A.

6. Description of Equipment/Apparatus

For information on the equipment/apparatus used by each laboratory, please see Appendix B.

7. Data Report Forms

Each Laboratory was provided with a set of report forms as part of the lab instructions in

Appendix A. Please note that the laboratories were r andomly coded and cannot be identified

herein. A copy of the data for both the single laboratory validation and the interlaboratory

validation is provided in Appendix C

.

8. Statistical Data Summary

A summary of the statistics calculated from the data retu rned by the participating laboratories is

provided below for both the initial single laboratory validation and the subsequent

interlaboratory validation

.

Single L aboratory V

alidation

– Single laboratory validation was carried out according to ASTM

D2777 and E691 as directed by the D19.06 subcommittee of the D19 committee of ASTM. The

results of the single laboratory validation were approved by the Technical Operations Committee

of the D19 Committee of ASTM at the June 2009 meeting, Las Vegas.6

This test m ethod was tested by an independent laboratory for a single laboratory determination of

precision and bias across a range of real

- world matrices, and compared to the result found by

testing the same sample with EPA 1664. Data are shown in Tables 1

- 3. All data were taken

using an MB3000 FTIR manufactured by ABB. Care should be taken if a different instrument is

used, even a different FTIR, as both the method detection limit and upp er limit of detection may

be affected. Results of ASTM WK23240 are compared to average results from testing by EPA

1664. The results of the two methods were compared using the root mean square deviation

(RMSD) , mean square error (MSE) and F

- test critical value on the natural

- log

- transformed

results to test the null hypothesis th at there is no difference between the methods. Grubbs

outliers were removed according to ASTM E178 . The RMSD was found to be 0.091 and the

maximum RMSD for the 95% significance level was found to be 0.100, indicating there is no

significant difference be tween the two methods at this significance level. Details of the

calculation are provided in Appendix D. Laboratory Control Sample ( LCS ) and Matrix Spike

( MS ) recoveries are compared to the known amount of the hexadecane/stearic acid mixture

spiked into the sample. Complete data is provided in Appendix C.7 Matrix Publicly Owned Treatment

Works Gunsmith

Average Recovery by

WK 23240 (mg/L) 29.6 79.7

Average Recovery by

EPA 1664 (mg/L) 32.2 78.9

Average Recovery as % of

EPA 1664 92% 101%

Standard Deviation of

WK 23240 (mg/L) 3.0 7.4

Relative Standard Deviation

as % 10% 9.3%

Matrix Spike Recovery % 102% 69%

Table 1. Single L aboratory Study

- R ecovery and precision data for two real worl d matrices repeated seven times (see also Table C

- 1).

Matrix Auto

Garage Dairy Machine Shop Auto Salvage Yard Fish Processor

Average Recovery by
WK 23240 (mg/L) 19.8 94.9 80.8 5.8 51.8

Average Recovery by EPA 1664 (mg/L) 21.1 108.8 89.4 6.6 54

Average Recovery as %

of EPA 1664 94% 87% 90% 88 % 96%

Standard Deviation of

WK 23240 (mg/L) 1.3 7.8 15.4 0.6 2.0

Relative Standard

Deviation as % 6.7% 8.2% 19% 10% 3.8%

Matrix Spike Recovery

% 101% ** 96% 94% 94%

Table 2. Single L aboratory Study

- R ecovery and precision data for five more real world matrices repeated three times.

** MS not performed on this sample.8

Replicate # Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

Average Recovery by WK 23240 (mg/L) 21.9 28.0 19.3 23.0 21.7 25.7 1.1**

Average Recovery by EPA 1664 (mg/L) 17.0 28.9 20.9 22.9 30.0 29.8 1.2**

Average Recovery as %

of EPA 1664 129% 97% 92% 101% 72% 86% N/A***

Standard Deviation of

WK 23240 (mg/L) 2.2 2.2 3.8 2.1 3.7 3.1 N/A***

Relative Standard
Deviation, % 10% 8% 20% 9% 17% 12% N/A***

Matrix Spike Recovery

% 96% 96% 88%   ** **   110%

Table 3. Single Laboratory Study

- Seven collections from the same POTW spread over a period of time

**  Below MDL *** These recoveries and precision are not calculated because of data below the recommended reporting

limit.

  Matrix spikes not performed on these samples.

Interlaboratory Validation

– The test design of the study meets the requirements of Practice D

2777 for the analytes listed in this test method with one exception. Due to the cost of performing

the analysis, each matrix to be tested will contain only one set of Youden pair concent rations. In

accordance with Section 1.5 of D 2777, an exemption from the requirement for using three

Youden pairs within each matrix was granted by the Technical Operations Committee of D

- 19

on the recommendation of the Results Advisor in order to enable evaluation of the method based

on more than one matrix. The exemption specified that a single Youden pair be used for each

matrix and that the range of concentrations represented by all three Youden pairs thus formed

cover the range of double the expected lower reporting limit to one half the upper limit of

detection. This test method was tested by twelve independent laboratories using the samples

provided to them as described above. Outlier results were removed according to Grubbs or

Tietjen and Moore t esting per ASTM E178. One value below the reporting limit for the

laboratory was not included in summary statistics per ASTM D2777 . One laboratory‘s matrix

spike results were removed for performing the procedure incorrectly, and the technician left the

l aboratory and could not be reached for an explanation or repeat of the test.9

The overall average relative standard deviation (RSD) across all labs/ samples was found to be

13.1%, and the average single

- operator RSD was 9.7%. These show the method to be pr ecise.

Additionally, in the interest of completing the study in a timely manner, several labs were

utilized that do not perform EPA 1664 testing and do not normally handle oil and grease

samples. If the results are examined solely from labs experienced i n handling oil and grease

sample testing (5 out of the 12), we find the overall RSD of 9.2% and single operator RSD of

7.5%, better than the group as a whole. However, as anonymity is required, the  ̳experienced‘

laboratories are not identified below. Sin gle operator standard deviation is calculated according

to Youden statistics for the pair of concentrations of the same matrix. Summarized f igures of

merit data (Method Detection Limit, Initial Precision and Recovery, and Laboratory Control

Sample) data i s shown in Tables 4

- 6 . Summary sample test data is shown in Table 7

.

Parameter MDL

Amount Spiked (mg/L) 4

# Results 12

Average MDL (mg/L) 1.26

Standard Deviation of MDL (mg/L) 0.26

Table 4

. Interlaboratory Study

- Method Detection Limit summary data

Parameter IPR

Mean
Recovery Precision

Amount Spiked (mg/L) 40

# Results 12

Average Recovery (%) 95.5

Standard Deviation of Recovery (%) 5 .1

Average RSD (%) 7.5

Standard Deviation of RSD (%) 2.2

Table 5

. Interlaboratory Study

- Initial Precision and Recovery summary data10

Parameter LCS

Amount Spiked (mg/L) 40

# Results 12

Average % 100%

Standard Deviation of LCS (mg/L) 8.0%

Table 6

. Interlaboratory Study

- Laboratory Control Sample summary data

Matrix POTW 1 POTW 2 Dairy 1 Dairy 2 Bilge 1 Bilge 2

True Value (mg/L) 10.8 9. 3 58. 8 51.6 91.8 86.6

# Sample Results 11 11 12 11 12 10

Average Recovery (mg/L) 11.0 9.1 60.0 53.1 94.8 84.3

Minimum Recovery (mg/L) 8.3 6.6 47.9 43.1 76.5 67.3

Maximum Recovery (mg/L) 13.6 12.4 68.7 62.8 107.1 95.3

Relative Overall Standard
Deviation,% 16.8% 19.6% 11.6% 10.5% 10.2% 10.2%

Relative Single Operator
Standard Deviation, % 16.5% 7.3% 5.2%

# Matrix Spike Results 10 10 9 10 11 9

Average Matrix Spike
Recovery % 109% 117% 91% 108% 87% 87%

Table 7 . Interlaboratory Study

- R esults summary

Method Range and QC Acceptance Limits

Proposed MDL and reporting range are shown in Table 8 and q uality control acceptance limits

were determined from the ILS and are shown in Table 9

. Details of the calculations of these

criteria are shown in Appendix D.11 Analyte MDL (mg/L) Reporting Range (mg/L)

Oil and Grease 1. 0 5

- 200

Table 8. Interlaboratory Study

- Method Detection Limit and Reporting Range

Analyte Initial Precision and Recovery Lab Control Sample Matrix Spike

Test

Concentration (mg/L) Recovery (%) Precision Recovery (%) Recovery (%)

Lower

limit Upper

limit Maximum RSD (%) Lower

limit Upper

limit Lower

limit Upper

limit

Oil and

Grease 40 8 8 10 5 10.5 79 11 3 70 12 6

Table 9. Interlaboratory Study

- QC Acceptance C riteria.

9. Precision and Bias Statement

The determination of precision and bias was conducted according to ASTM D2777

. O il and

grease method validation generally is not performed on laboratory spike samples because they

are not sufficiently representative of real

- world oil and grease samples. Therefore, the

determination of bias is done by comparing results of two methods across a range of real

- world

sample matrices, as was done in the single laboratory validation r esults described above. As

described above, a calculation (based on an F

- test and utilizing the deviation between the results

of the two methods along with the precision of the two methods) indicated that there is no

significant difference between WK 2324 0 and EPA 1664 at the 95% level.

Interlaboratory precision was then determined for WK 23240 by an ILS as described above. The

overall average relative standard deviation (RSD) across all samples was found to be 13.1%, and

the average single

- operator RSD was 9.7%. These show the method to be precise. Additionally,12

in the interest of completing the study in a timely manner, several labs were utilized that do not

perform EPA 1664 testing and do not normally handle oil and grease samples. If the results are

examined solely from labs experie nced in handling oil and grease sample testing (5 out of the

12), we find an overall RSD of 9.2% and single operator RSD of 7.5%, better than the group as a

whole.13 APPENDIX A

– Laboratory Instructions14



### Multi

- Laboratory Validation Study Plan for Determination of Oil and Grease by

ASTM WK23240

– Standard Test Method for Solvent

- Free Membrane

Recoverable Oil and Grease by Infrared Determination

ASTM Interlaboratory Collaborative Study Workplan

Please Read All Instructions Before Proceedi ng with Collaborative Lab Work

Laboratory Name:____ _______________________________________

Operator Name:______ _______________________________________________

Operator Telephone:___ ____________________________________________

Your Lab Code for Identific ation is:____ ____ Date Sent:______ __________

** * Use this code number on all submitted data report forms and questionnaire. ***

Immediately inspect the contents of the collaborative kit for missing items, leaking bottles, etc. If

any discrepancy is note d, call for immediate replacement.

Method Author and Task Group / Collaborative Chairperson

Tyler Martin, Ph.D.

Orono Spectral Solutions

Office: 866

- 269

- 8007

FAX: 866

- 660

- 4759

E

- Mail: tyler.martin@ossmaine.com15 PARTICIPANT‘S INSTRUCTIONS

1.0 Introduction

Orono Spectral Solutions (OSS) is undertaking an ASTM inter

- laboratory collaborative study to

determine inter

- laboratory precision and bias.

The Method is currently under review by ASTM Subcommittee D19.06 on Methods for Analy sis

for Organic Substances in Water . The next action is to have several users evaluate the method for

precision and bias from three sample matrices.

You are asked to be a participant in the collaborative study because of your experience in this

field. Your results and comments will be compiled with several other laboratories performing the

same method, and will be submitted to ASTM for approval.

All submitted results will be considered the property of Orono Spectral Solutions with all rights

to their use; results will not be identified by laboratory in any published reports.

If you have any questions concerning the test method, its performance, calibration, standard or

sample preparation, dilutions, or method interpretation, please contact:

Tyler Martin, Ph.D.

Orono Spectral Solutions

Office: 866

- 269

- 8007

FAX: 866

- 660

- 4759

E

- Mail: tyler.martin@ossmaine.com16

The start of the collaborative study will be determined at a later date and the completed data

package will be due one month later a nd will include:

1) The data report forms.

2) Print out of all standard and sample data, and results.

3) The laboratory notebook documenting your work, calculations, and comments.

4) The completed questionnaire.

5) A back

- up copy of the project data on the enclosed memory stick.

Return to task group and collaborative chairperson:

Tyler Martin, Ph.D.

Orono Spectral Solutions

Office: 866

- 269

- 8007

FAX: 866

- 660

- 4759

E

- Mail: tyler.martin@ossmaine.com

The true values for the spiking solutions, your processed results, and the overall test

- method

performance will be sent to each participant after all participants return their data. Any

corrections to the reported data will not be accepted after the true values have been distributed.

2.0 Packing L ist

2.1 Materials and Reagents to be Supplied by Study Coordinator

- Collaborative Instruction Manual, and the Proposed Test Method

- FTIR spectrometer and data processing system

- syringe pump

- drying system

- OSS Oil in Water Extractors

- infrared

- amenab le solid

- phase extractors

- OSS Calibration Standard Devices

- sample homogenization system

- syringes

- several standard 1 + liter bottles for testing

- One blank memory stick to backup the acquired data for archival purposes.

- Laboratory Notebook

- volumetric pipettes

- spiking solution

– stearic acid, hexadecane, acetone

- sulfuric acid

- pH test strips

- volumetric flasks

- pipette / tips

- graduated cylinder17

2.2 Materials to be Supplied by the Participant (note: if participant has a problem supp lying any

of these items, please contact the study coordinator)

- cleaning materials

- Lab water

3.0 Preliminary Testing Prior to Initiation of the Collaborative

Before initiating the collaborative it is important to ensure that the instrumentation is per forming

according to manufacturers‘ specifications.

3.1 System Preparation

- OSS personnel will install and prepare instrumentation

3.2 Evaluation of Test Standards

- All labs will perform an MDL (Table 1) and an IPR (Table 2) study and several test runs fo r familiarization prior to beginning ILS testing.

4.0 Collaborative Study Determinations

Prior to the analysis of collaborative

- study samples, the system must first be calibrated in order

to verify linearity.

4.1 Preparation of Working Calibration Standar ds and Development of Calibration Curve

- Section 12 of the ASTM method under consideration WK23240 explains the procedure in

detail, and OSS will also provide a detailed SOP along with the ASTM method.

4.2 Quality Control (QC) Standard

- Spiking solution w ill be provided by OSS. This spiking solution will be used to perform

laboratory control samples and matrix spikes during testing.

4.3 Method Detection and Quantitation Sample Discussion

- Some samples will be purposefully chosen to be near the detection limit of the method. Thus,

some raw data may come back below the recommended reporting limit. Despite this, it is

imperative that the lab provide all of the raw, un

- rounded, un

- censored d ata.
4.4 Preparation of Collaborative Study Sample Matrices

- Preserved sample matrices will be provided by OSS. The laboratory will be required to

maintain preservation until the time of testing (pH 2, < 4 o C). The laboratory will need to verify

the co rrect pH (pH = 2) prior to testing. If pH is > 2 then the laboratory will be required to bring

the sample to pH = 2 prior to testing.

5.0 Sample Analysis Protocol

- Calibration verification will be performed prior to any analysis by examining the CSD at o r near 40 mg/L. Six samples will be provided to the lab. Upon completion of determining the

concentration of each sample, each sample will then be spiked with the spiking solution

following the procedure outlined in the method. There will also be a labo ratory control sample

(LCS) performed each day of testing, and a duplicate LCS (LCSD) if necessary. A method blank

will also be performed each day of testing.18

6.0 Data Report Format

Report determined concentrations on the attached Data Report Forms. Repo rt all numerical

values. Do not replace numerical values with zeroes or ―less than‖ values.19 DATA REPORTING FORMS

ASTM Interlaboratory Collaborative Study

— Data Report Form



### Multi

- Laboratory Validation Study Plan for Determination of Oil and Grease by

AS TM WK23240

– Standard Test Method for Solvent

- Free Membrane Recoverable Oil and Grease by Infrared Determination

Laboratory Name:_________________________________ Lab Code:______________________

Calibration Standards, Response Linearity

Calibration on Day #_______

CSD # CSD conc. Absorbance

mg/L

1

2

3

4

5

6

7

Each CSD will be labeled with the concentration it corresponds to. That concentration and the

measured absorbance should be recorded at the time of calibration.

Calibration verification will be performed each day of testing using the CSD at or near 40 mg/L.

Day Abs Concentration20 ASTM Interlaboratory Collaborative Study

— Data Report Form



### Multi

- Laboratory Validation Study Plan for Determination of Oil and Grease by

ASTM WK23240

– Standard Test Method for Solvent

- Free Membrane Recoverable Oil and Grease by Infrared Determination

Laboratory Name:______________________________ Lab Code:__________________

Laboratory Control Samples

Day LCS concentration LCS Recovery %21 ASTM Interlaboratory Collaborative Study

— Data Report Form



### Multi

- Laboratory Validation Study Plan for Determination of Oil and Grease by

ASTM WK23240

– Standard Test Method for Solvent

- Free Membrane Recoverable Oil and Grease by Infrared Determination

L aboratory Name:________________________________ Lab Code:___________________

Initial Demonstration of Laboratory Capability

Method Detection Limit Study

Method: ASTM Standard WK23240 Solvent

- Free Membrane Recoverable Oil and Grease by Infrared Determina tion
Matrix: Reagent Water

Fortification Level:

Measured Result mg/L as Oil and Grease
Replicate

#1 Replicate

#2 Replicate

#3 Replicate

#4 Replicate

#5 Replicate

#6 Replicate

#7

Average of Seven Replicates:

Standard Deviation of Seven Replicates: MDL (Student‘s t 3.143 x Standard Deviation) =
ML ( MDL x 3.18) =

Initial Precision and Recovery

Method: ASTM Standard WK23240 Solvent

- Free Membrane Recoverable Oil and Grease by Infrared Determination
Matrix: Reagent Water

Fortification Level:

Measured Result mg L - 1 as Oil and Grease
Replicate #1 Replicate #2 Replicate #3 Replicate #4

Percent Recovery (%) as Oil and Grease

Average Recovery of Four Replicates:
Standard Deviation of Average Recovery:

ASTM Interlaboratory Collaborative Study

— Data Report Form22



### Multi

- Laboratory Validation Study Plan for Determination of Oil and Grease by

ASTM WK23240

– Standard Test Method for Solvent

- Free Membrane Recoverable Oil and Grease by Infrared Determination

Labora tory Name:_______________________________ Lab Code:___________________

Sample Test Data and Sample Spike Data

ASTM WK 23240
Study Laboratory

Sample ID Measured Result, mg/L MS or LCS, Recovery %

1A

1B

2A

2B

3A

3B

LCS

LCSD (if required)23

Operate your instrument according to the manufacturer‘s instructions, and this test method. Please

complete this questionnaire to document your specific operating conditions, any deviations you made, and

any observations.

Laboratory Name : _____________________________________Lab Code:__________________

Operator Name & Title:____________________________________________________________

Telephone: ____________________FAX#: _________________E

- mail:_____________________

Date Started: _________

__________Date Completed:_________ Date Mailed:________________

Instrument Used:__________________________________________________________________

Describe any difficulties you encountered using this method.

Were any numeric data censored? If so, why?

Your comments, observations, or suggested changes to the method.24 APPENDIX B

– Equipment/Apparatus25

Equipment for WK 23240 ILS

Extractor: Oil and Grease Extractor ( Orono Spectral Solutions, P/N 1018SPE (US Patent Application

12/324,688))

FTIR Spectrometer: MB3000, ABB

- Bomem

Spectrum Processing software: Horizon MB, ABB

- Bomem

Shaker: New Brunswick Excella E1 with bottle holder attachment ( Orono Sp ectral Solutions, P/N

0916BH )

Syringe Pump: Harvard Apparatus PHD

- 2000; Syringepump.com NE

- 1010

Drying System: Custom built of pneumatic tubing fittings and designed to direct compressed

air through the Extractor ( Orono Sp ectral Solutions, P/N 0627DS )26 APPENDIX C

– Raw Data27

Single Laboratory Validation Study Raw Data

Matrix→ Publicly Owned Treatment

Works Gunsmith

Rep↓ / Method → WK23240 EPA 1664 WK23240 EPA1664

1 31.3 36.1 76.1 77.4

2 29.2 30.2 73.3 80.9

3 24.7 29.7 75.3 78.6

4 30.7 31.3 78.3 79.3

5 34.1 35.2 74.5 78.1

6 29.7 29.9 91.2 80.0

7 27.4 32.8 89.4 78.1

Average Recovery (mg/L) 29.6 32.2 79.7 78.9

Standard Deviation (mg/L) 3.0 2.6 7.4 1.2

Matrix Spike Recovery (%) 102% 73% 69% 89%

Table C

- 1. Single Laboratory Study

- Raw data for real

- world matrices repeated seven times.

Matrix→ Auto Garage Dairy

Rep↓ / Method→ WK23240 EPA 1664 WK23240 EPA1664

1 21.0 22.4 89.5 108.1

2 19.9 21 91.4 110.8

3 18.4 20 103.8 107.5

Average Recovery (mg/L) 19.8 21.1 94.9 108.8

Standard Deviation (mg/L) 1.3 1.2 7. 8 1.7

Matrix Spike
Recovery (%) 101% 78% * *

Table C

- 2. Single Laboratory Study

- Raw data for real

- world matrices repeated three times.

*Matrix spike not performed on this matrix.28

Matrix→ Machine Shop Auto Salvage Yard

Rep↓ / Method→ WK23240 EPA 1664 WK23240 EPA1664

1 74.0 89.1 5.8 7.2

2 69.9 88.7 4.8 * 5.9

3 98.4 90.3 5.8 6.5

Average Recovery (mg/L) 80.8 89.4 5. 8 6.6

Standard Deviation (mg/L) 15.4 0.8 0 0 .7

Matrix Spike Recovery (%) 96 % 0%** 94% 84%

Table C

- 2 (cont‘d). Single Laboratory Study

- Raw data for real

- world matrices repeated

three times.

* Below recommended reporting limit, thus not included in summary statistics as directed by

ASTM D2777.

**This result was double

- checked and the technician was asked about this matrix spike and
this was the actual result obtained, and no explanation as to what may have happened.

Matrix→ Fish Processor

Rep↓ / Method→ WK23240 EPA 1664

1 50.5 55 .0

2 54.1 53 .0

3 50.9 54 .0

Average Recovery (mg/L) 51.8 54.0

Standard Deviation (mg/L) 2.0 1.0

Matrix Spike Recovery (%) 94% 61%

Table C

- 2 (cont‘d). Single Laboratory Study

- Raw data for real

- world matrices repeated

three times.29

Matrix→ Day 1 Day 2

Rep↓ / Method→ WK23240 EPA 1664 WK23240 EPA1664

1 22.6 17. 2 25.6 29.8

2 19.5 16. 3 29.9 29.7

3 23.7 17. 6 28.6 27.3

Average Recovery (mg/L) 21.9 17.0 28.0 28.9

Standard Deviation (mg/L) 2.2 0.7 2.2 1.4

Matrix Spike Recovery (%) 96% 85% 96% 79%

Table C

- 3. Single Laboratory Study

- Seven collections from the same POTW spread over a period of time.

Matrix→ Day 3 Day 4

Rep↓ / Method→ WK23240 EPA 1664 WK23240 EPA1664

1 21.7 20.3 23.2 23.2

2 14.8 21. 2 20.8 22.6

3 21.2 21. 3 25.1 22.7

Average Recovery (mg/L) 19.3 20.9 23.0 22.9

Standard Deviation (mg/L) 3.8 0.5 2.1 0.3

Matrix Spike Recovery (%) 88% 83% *** ***

Table C

- 3 (cont‘d). Single Laboratory Study

- Seven collections from the same POTW spread over a period of time

. ***Matrix spikes not performed on these matrices.30

Matrix→ Day 5 Day 6

Rep↓ / Method→ WK23240 EPA 1664 WK23240 EPA1664

1 19.6 30.9 28.8 28.1

2 19.5 27.4 25.7 30.1

3 25.9 31.6 22.6 31. 2

Average Recovery (mg/L) 21.7 30.0 25.7 29.8

Standard Deviation (mg/L) 3.7 2.2 3.1 1.5

Matrix Spike Recovery (%) *** *** *** ***

Table C

- 3 (cont‘d). Single Laboratory Study

- Seven collections from the same POTW spread over a period of time.
***Matrix spikes not performed on these matrices.

Matrix→ Day 7

Rep↓ / Method→ WK23240 EPA 1664

1 1.1 * 0. 9*

2 1.3* 1. 5*

3 0.9 * 1. 4*

Average Recovery (mg/L) N/A** N/A**

Standard Deviation (mg/L) N/A** N/A**

Matrix Spike Recovery (%) 110% 88%

Table C

- 3 (cont‘d). Single Laboratory Study

- Seven collections from the same POTW spread over a period of time.

* Below recommended reporting limit, thus not included in summary statistics as directed by

ASTM D2777.

** These recoveries and precision are not calculated because of data below the recommended reporting limit. ***Matrix spikes not performed on these matrices.31

Interlaboratory Validation Study Raw Data

***NOTE: 15 labs were asked to participate in the study, 12 of those 15 labs (LABS 1

- 10, LAB 14, LAB 15) accepted

– see labels in the following tables. ***

Parameter MDL

Amount Spiked (mg/L) 4

Lab 1 0.91

Lab 2 1.51

Lab 3 1.41

Lab 4 1.51

Lab 5 1.04

Lab 6 1.38

Lab 7 1.48

Lab 8 1.41

Lab 9 1.38

Lab 10 0.77

Lab 14 1.29

Lab 15 0.97

Average MDL (mg/L) 1.26

Standard Deviation of MDL (mg/L) 0.26

Pooled MDL (mg/L) 1.0

Table C

- 4. Interlaboratory Study

- Method Detection Limit data (see Appendix D for calculation of Pooled MDL).32 Parameter IPR
Average Recovery (%) RSD (%)

Amount Spiked (mg/L) 40

Lab 1 90.5 5.4

Lab 2 94.1 7.0

Lab 3 94.6 7.6

Lab 4 106.0 4.8

Lab 5 94.7 9.4

Lab 6 95.8 8.6

Lab 7 97.3 11.0

Lab 8 87.2 8.4

Lab 9 89.6 6.1

Lab 10 97.0 10.6

Lab 14 99.6 3.8

Lab 15 99.8 7.6

Average Recovery (%) 95.5

Standard Deviation of Recovery (%) 5. 1
Average RSD (%) 7.5
Standard Deviation of RSD (%) 2.2

Table C

- 5

. Interlaboratory Study

- Initial Precision and Recovery data33 Parameter LCS

Amount Spiked (mg/L) 4 0

Lab 1 112.5%

Lab 2 97.0%

Lab 3 102.5%

Lab 4 103.8%

Lab 5 101.3%

Lab 6 94.0%

Lab 7 101.8%

Lab 8 102.8%

Lab 9 97.5%

Lab 10 101.5%

Lab 14 102.8%

Lab 15 78.5%

Average Recovery (%) 99.6%

Standard Deviation of Recovery (%) 8.0%

Table C

- 6

. Interlaboratory Study

- Laboratory Control Sample data34

Parameter IPR 1 IPR 2 IPR 3 IPR 4 LCS

Lab 1 89.4% 98.1% 85.5% 88.7% 112.5%

Lab 2 100.2% 92.8% 84.8% 98.7% 97.0%

Lab 3 83.5% 98.0% 97.2% 100.2% 102.5%

Lab 4 103.9% 109.5% 110.3% 100.2% 103.8%

Lab 5 85.8% 107.9% 91.3% 93.8% 101.3%

Lab 6 86.2% 91.9% 98.8% 106.1% 94.0%

Lab 7 90.3% 99.7% 111.8% 87.3% 101.8%

Lab 8 90.3% 83.6% 97.3% 77.7% 102.8%

Lab 9 86.9% 90.5% 83.3% 97.7% 97.5%

Lab 10 90.7% 95.0% 112.5% 89.9% 101.5%

Lab 14 95.2% 98.2% 100.9% 104.1% 102.8%

Lab 15 89.4% 102.6% 107.3% 99.7% 78.5%

Average Recovery (%) 96.3%

Standard Deviation of Recovery (%) 8.4%

Table C

- 7

. Interlaboratory Study

- Combined Ini tial Precision and Recovery data and Laboratory Control Sample data used for determining QC acceptance limits for IPR and LCS as described in Appendix D. 40 ppm spikes of equal parts hexadecane and stearic acid

in lab water.35 Matrix POTW 1 POTW 2 Dairy 1 Dairy 2 Bilge 1 Bilge 2

True Value (mg/L) 10.8 9.3 58.8 51.6 91.8 86.6

Lab 1 20.3 * 12.4 60.6 54.1 99.5 88.5

Lab 2 8.7 8.3 69.2 57.0 87.1 76.7

Lab 3 10.1 7.4 63.8 62.8 107.1 95.3

Lab 4 13.6 10.9 51.7 80.6 * 105.1 86.3

Lab 5 8.3 9.2 60.4 56.8 88.7 67.3

Lab 6 10.9 9.6 59.3 47.3 95.4 61.4 ***

Lab 7 11.9 9.2 47.9 52.9 101.2 82.5

Lab 8 11.8 10.6 68.7 54.9 103.7 94.6

Lab 9 9.8 8.3 65.7 55.6 93.2 86.2

Lab 10 12.9 6.6 62.3 53.1 98.9 87.8

Lab 14 13.4 7.0 60.2 46.6 76.5 77.6

Lab 15 9.5 3.6 ** 49.7 43.1 81.6 47.2 ***

Average Recovery (mg/L) 11.0 9.1 60.0 53.1 94.8 84.3

Relative Overall Standard
Deviation,% 16.8% 19.6% 11.6% 10.5% 10.2% 10.2%

Relative Single Operator
Standard Deviation, % 16.5% 7.3% 5.2%

Table C

- 8

. Interlaboratory Study

- S ample test results. *Outlier discarded according to the Grubbs test per ASTM E178 **Value is below the lab‘s ML, and thus is discarded from further calculations according to

ASTM D2777 ***Outlier discarded according to the Grubbs single

- sided two

- outlier test per ASTM E17836

Matrix POTW

1 POTW

2 Dairy 1 Dairy 2 Bilge 1 Bilge 2 Lab
Avg Lab Std.

Dev. (s i )

Lab 1 95** 114 78 101 50 77 84 25

Lab 2 108 110 73 114 91 61 93 22

Lab 3 124 157 131*** 109 86 119 119 26

Lab 4 * * * * * * * *

Lab 5 100 89 77 92 65 45 78 20

Lab 6 108 108 112 104 88 120** 104 9

Lab 7 106 126 93 67  86 110 104 16

Lab 8 121 135 95 98 102 84 106 19

Lab 9 104 136 92 134 100 97 110 19

Lab 10 89 106 107 104 106 106 103 7

Lab 14 118 92 36** * 94 124 83 102 18

Lab 15 111 118** 125 125 86 86** 97 27

Average Spike Recovery ( % ) 109 117 91 108 87 87 99.6

Standard

Deviation ( % ) 10 21 13 13 21 24 11.6 ( s b )

Table C

- 9

. Interlaboratory Study

- M atrix spike results , in percent recovery of the spike

.
* Lab performed matrix spikes incorrectly according to written record in lab notebook, so

data is discarded . Technician left the lab and could not be contacted.

** Value discarded since the sample point the spike was based on was discarded from the sample data set.

*** Outliers discarded based on Tietjen and Moore criteria for doubl e

- sided two

- outlier test

per ASTM E178

**** Outlier discarded according to the Grubbs test per ASTM E17837 APPENDIX D

– Calculations38

Calculation of Method Comparability from Single Lab Study

RMSD and RMSDmax calculation s for method comparability are from the ―Preliminary

Report of EPA Efforts to Replace Freon for the Determination of Oil and Grease, Appendix C‖

and the current (October 2009) EPA‘s ―Guidance on Implementation and Use‖ for method

1664A. Readers are referred to those documents for details of the calculations. The general

procedure is:

- log transform results to remove the inherent heteroskedasticity of the data, and all subsequent

steps are performed on the log

- transformed data.

- triplicate means of the log transformed results are calculated.

- outliers in deviation between the methods are removed. Two Grubbs outliers were found: Day

1 of the seven day POTW study failed at the 97.5% significance level and subsequently Day 5

failed at the 95% significance level.

- The root mean square deviation (RMSD) between the methods is determined (RMSD = 0.091)

.

- the root mean square error (RMSE) is determined and corrected by the F critical value to find a

maximum allowed RMSD (i.e. values of RMSD higher than this value would indicate the

methods are different at the chosen significance level) (RMSDmax = 0.10)

- In this case RMSD < RMSDmax, so it is determined WK 23240 is not s ignificantly different

from EPA 1664.

Calculations Detection Limit and QC Acceptance Limits from ILS

Calculation of Method Detection Limit

The MDL for WK 23240 is based on the pooled MDL as in the ― Protocol for EPA

Approval of New Methods for Organic a nd Inorganic Analytes in Wastewater and Drinking

Water , March 1999‖ and found from the ILS data in Table C

- 4 and the equation:39

w here i is the lab number (1

- 12) and d i is one less than the number of replicates used by lab i to

derive the MDL (all d i = 6)

. This calculation results in a pooled MDL of 1.0 mg/L. Often, the

lower reporting limit, (i.e. minimum level , ML ) is determined as the MDL times 3.18, then

rounded to the nearest whole number, which would result in a 4.0 mg/L lower limit. However,

the lower reporting limit has been set at 5 mg/L to match existing the U S EPA oil and grease

method 1664

.

Calculation o f IPR acceptance limits

IPR recovery limits were determined from the pooled IPR and LCS data (pooling the data

as opposed to treating IPR and LCS data separately is described in ―Report of the Method 1664

Validation Studies, April 1995, with Addendum, Jan uary 1996 ) shown in Table C

- 7

, and the

equation (from a recent EPA method validation report ―Method 1668A Interlaboratory

Validation Study Report, November 2008‖) :

w here n is the number of points (n
= 60 ), t 95%, n

- 1 = 1.99 5 , and s is the standard deviation of

recoveries from Table C

- 7 (s = 0 .0844 )

.

The maximum RSD for IPR samples is calculated as

w here s is the standard deviation of all IPR and LCS recoveries from Table C

- 7 (s = 0.0844 ) and

F 95%, n

- 1, n

- 1 = 1.54

.40

Calculation of LCS acceptance limits

LCS recovery limits were determined from the pooled IPR and LCS data in Table C

- 7

and the equation (same references as for the IPR calculations above ) :

w here n is t he number of points (n
= 60 ), t 95%, n

- 1 = 1.995 , and s is the standard deviation of

recoveries from Table C

- 7 (s = 0.0844 ).

Calculation of MS acceptance limits

Matrix Spike acceptance limits were calculated by two different methods, then the results

were averaged to get final acceptance limits. The first methodology was taken from the way MS

limits were determined for EPA 1664 as described in ―Report of the Method 1664 Validation

Studies, April 1995, with Addendum, January 1996‖ . In this case, MS accept ance limits were

determined as being the same as the LCS acceptance limits determined above (79

- 113%) . The

second method is from ― Protocol for EPA Approval of New Methods for Organic and Inorganic

Analytes in Wastewater and Drinking Water , March 1999‖ and uses data in Table C

- 9. First, the

pooled within

- lab standard deviation ( s w ) is determined:

w here s i is the standard deviation of recover ies for lab i found in Table C

- 9

. Then the combined

standard deviation (s

c ) is determined:41

w here s b is standard deviation of average matrix spi ke recoveries found in Table C

- 9 . Then the

limits are found:

w here m is the number of labs (m = 12) and t 95,14 = 2.145. The result of this calculation is the

range 60

- 139%. By averaging the lower and upper bounds with those determined by the method

used to determine the limits for EPA 1664, the final QC acceptance limits for matrix spikes are

determined and listed in Table 9.42 APPENDIX E

– Method