# 3

This laboratory maintains A2LA accreditation to: AN SI/ISO/IEC 17025:2005 for the specific

tests/calibrations listed in A2LA Certificate # 205 2.01 and meets the principles of ISO 9001:2000. The test

results included in this report are covered by this accreditation following method ETS-8-045.1 

# Analytical Report

Determination of PFC Concentrations in Fish Collect ed from the Tennessee

River in the Fall of 2012



# Study Title

Analytical Study ISO12-01-01-04: Tennessee River Fi sh Sample Analysis, October

2012



# Project Coordinator(s)

Jai Kesari, P.E., DEE and Charles Young

Weston Solutions, Inc.

1400 Weston Way

Westchester, MA 19380



# Principal Analytical Investigator

Cleston C. Lange, Ph.D.

3M Environmental Laboratory



# Report Completion Date

Date of Final Signature



# Analytical Laboratory

3M Environmental Health and Safety Operations

Environmental Laboratory

3M Center, Bldg 260-05-N-17

St. Paul, MN 55144



# Project Identification

ISO12-01-01-04

Total Number of Pages,.

39

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

2 

# T ABLE OF 

# C ONTENTS

Table of Contents .................................

................................................... ................................................... .... 2

List of Tables ....................................

................................................... ................................................... ......... 3

1 Study Information .................................

................................................... ................................................ 4

2 Summary ...........................................

................................................... ................................................... 5

3 Introduction ......................................

................................................... .................................................. 15

4 Reference Substances...............................

................................................... ....................................... 16

5 Method Summary ....................................

................................................... .......................................... 19

5.1 Methods ...........................................

................................................... ..................................... 19

5.2 Sample Receipt ....................................

................................................... ................................ 19

5.3 Sample Preparation/Extraction .....................

................................................... ....................... 20

5.4 LC/MS/MS Analysis .................................

................................................... ............................. 20

6 Results ...........................................

................................................... ................................................... . 24

6.1 Calibration .......................................

................................................... ...................................... 24

6.2 Limits of Quantitation (LOQs) .....................

................................................... ......................... 25

6.3 Continuing Calibration ............................

................................................... .............................. 25

6.4 Blanks ............................................

................................................... ........................................ 26

6.5 Laboratory Control Samples (LCSs) .................

................................................... ................... 27

6.6 Laboratory Matrix Spikes (LMSs) ...................

................................................... ..................... 34

6.7 NIST SRM 1947 Results .............................

................................................... ......................... 34

6.8 Fillet Equipment Aqueous Rinseate Results .........

................................................... .............. 34

7 Conclusion ........................................

................................................... ................................................. 3 6

8 Data/Sample Retention .............................

................................................... ........................................ 37

9 List of Attachments ...............................

................................................... ............................................. 37

10 Signatures ........................................

................................................... .................................................. 37

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

3 

# L IST OF 

# T

ABLES

Table 1. Concentration Results for Tennessee River Fish Tissue Samples .............................. ................ 6

Table 2. Laboratory Matrix Spike (LMS) Recoveries

................................................... ............................. 14

Table 3. Reference Substances: Target Analytes ...

................................................... .............................. 16

Table 4. Reference Substances: Stable Isotope Labe led Internal Standards (IS) ....................... .......... 17

Table 5. Reference Substances: Stable Isotope Labe led Surrogate Recovery Standards (SRS) ........ 19

Table 6. LC/MS/MS Instrument Information (ETS-8-045 ) ................................................. ........................ 21

Table 7. Gradient Liquid Chromatography Conditions (ETS-8-045); C 7 

# -C 12 

# PFCAs, PFSAs and

SRSs ..............................................

................................................... ................................................... . 21

Table 8. Gradient Liquid Chromatography Conditions (ETS-8-045); for C 4 

# -C 6 

# PFCAs .......................... 22

Table 9. Analytes, Surrogates, Internal Standards, and Transitions................................... ..................... 22

Table 10. Analytical Batches .....................

................................................... .............................................. 23

Table 11. LCS Results ............................

................................................... ................................................ 28

Table 12. Concentration Results for Fillet-Process Equipment Rinseate Blanks ........................ ........... 35

Table A1. Concentration Results for Bass & Catfish Fillet Control Materials (Right Side Fillets) .... ....... 38

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

4 

# 1 Study Information

Sponsor

3M Company



# Project Requestor

Gary Hohenstein

Manager, Environmental/ Regulatory Affairs

Special Projects, EHS Operations

Bldg 224-5W-03

St. Paul, MN 55144

Phone: (651) 737-3570

gahohenstein@mmm.com



# Project Coordinator(s)

Jai Kesari, P.E., DEE

Charles Young

Weston Solutions, Inc.

1400 Weston Way

Westchester, MA 19380

Jai phone: 610-701-3761

Charles phone: 610-701-3787

Jai.kesari@westonsolutions.com

Charles.young@westonsolutions.com



# Principal Analytical Investigator (PAI)

Cleston C. Lange, Ph.D.

3M Environmental Laboratory

3M Center, Bldg 260-5N-17

St. Paul, MN 55144

Phone: (651)-733-9860

clange@mmm.com



# Analytical Testing Facility

3M EHS Operations

3M Environmental Laboratory

Building 260-5N-17

St. Paul, MN 55144



# Study Personnel (3M Environmental Laboratory)

Marlene Heying, Analyst (Pace Analytical, Professio nal Services)

Jon Steege, Analyst (Pace Analytical, Professional Services)

Patrick Kenney, Analyst (Pace Analytical, Professio nal Services)

Kevin Eich, Analyst (Quality Associates, Inc., Prof essional Services)

Laura Harrington, Analyst (Pace Analytical, Profess ional Services)



# Study Dates

Study Initiation: October 12, 2012

Analytical Initiation: December 5, 2012

Analytical Completion: February 6, 2012

Report Completion: Date of Final Signature

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

5 

# 2 Summary

Reported herein are the determined concentrations o f certain perfluorinated chemicals (PFCs)

measured in fish collected from the Tennessee River in the Fall of 2012. These analyses were

conducted as 3M Environmental analytical project IS O12-01-01-04 and was performed in

support of the Weston Solutions, Inc. study plan, t itled “Revised Work Plan for Fall 2012 Fish

and Surface Water Sampling in the Tennessee River”, and dated October 2012.

During the Fall of 2012, Weston Solutions, Inc. per sonnel caught five largemouth bass and five

catfish from each of fifteen pre-defined fish sampl ing reaches located within the confines of the

Wilson and Wheeler Reservoirs of the Tennessee Rive r, located near Decatur, AL. The fish

were filleted (skin removed) in the field and provi ded blind to the laboratory in terms of specific

information on fish collections. A total of 175 f ish tissue samples were received and

quantitatively analyzed. The PFCs analyzed were per fluorobutanoate (PFBA),

perfluorohexanoate (PFHxA), perfluoroheptanoate (PF HpA), perfluorooctanoate (PFOA),

perfluorobutane sulfonate (PFBS), perfluorohexanesu lfonate (PFHS), perfluorooctanesulfonate

(PFOS) , perfluorobutanesulfonamide (FBSA), perfluo rooctanesulfonamide (FOSA),2-[Nmethyl-FOSA]acetic acid (MeFOSAA) and 2-[N-ethyl-FO SA]acetic acid (EtFOSAA) and results

reported in Table 1

.

Each sample was extracted in duplicate for determin ation of the average PFC concentrations,

and approximately every10th fish sample (21 in tota l; 12% of total fish) had two laboratory

matrix spike (LMS) samples prepared from a third an d fourth 0.5 gram aliquot and fortified with

known quantities of target analytes. The LMS recov ery results were used as a measure of the

sample measurement accuracy. The average recoverie s of LMSs for all analytes were within

100 + 10%, except FBSA which was 82.3%. The average rec overy for PFOS was 97.2% with

an RSD of 8.6%. The LMS recovery results are shown in Table 2 . Each sample preparation

also received two surrogate recovery standards (SRS s) 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS. The

recovery of 13 

# C 4 

# -PFOS was 88.0% (RSD = 3.8%) and for 13 

# C 4 

# -PFOA was 85.2% (RSD =

5.2%). The overall RSDs determined from the SRS rec overy results for samples were used as

a measure of the analytical method precision and in dicated excellent precision.

Laboratory control spikes (LCSs) were prepared at 3 levels, each level in triplicate, and

analyzed with each analytical batch. Average LCS r ecoveries were within 100 + 14% at all

levels, and overall, for each analyte. The LCS sta tistical results (for all LCS levels) were used

to determine the analytical method uncertainty (95% CI) at + 18% for PFBA, + 15% for PFHxA,

+ 21% for PFHpA, + 20% for PFOA, + 18% for PFBS, + 19% for PFHS, + 22% for total-PFOS,

+ 40% for FBSA, + 20% for FOSA, + 18% for MeFOSAA and + 19% for EtFOSAA.

Overall, the sample results showed that PFOS, FBSA and FOSA were detected with highest

frequencies in the Tennessee River fish fillet tiss ue samples, with PFOS detected in ~98% of

the samples received, and FBSA and FOSA detected in ~80-90% of the samples. PFOS was

measured at the highest concentration of all analyt es with a maximum value of 599 ng/g (wet

weight; ww) followed by FBSA with a maximum measure d value of 567 ng/g (ww) and FOSA

maximum measured value of 123 ng/g (ww). MeFOSAA and EtFOSAA were detected at

moderate frequency of ~50%, and lower maximum level s measured at 22.0 and 19.5 ng/g,

respectively. PFBS was detected at ~20% frequency in samples, with a maximum measured

concentration of 3.82 ng/g (ww). The remaining ana lytes PFBA, PFHxA, PFHpA, PFOA, and

PFHS were detected infrequently at less than 15% of samples and maximum measured values

were lower than PFBS maximum value of 3.82 ng/g (ww ).ISO12-01-01-04Tennessee River Fish StudyFall 20126Table 1.Concentration Results for Tennessee River Fish Tissue SamplesSAMPLE IDLIMS IDAverage Concentration (ng/g; wet wt.)Avg. SRS RecoveryPFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAA13C4-PFOA13C4-PFOSTN_11-0194-1_1 Sample 1Pre-StudyHomogenizationProcess controlISO12-01-01-04-0011.42<0.101<0.503<0.960<0.101<0.09600.731<0.250 [c]<0.253<0.500<0.500107%105%MSFOO1ISO12-01-01-04-002<0.250<0.0503<0.252<0.480<1.10<0.25025986.2 [b, c]2.371.451.1989.7%92.6%IPF002ISO12-01-01-04-003<0.250<0.0503<0.252<0.480<1.10<0.2508.4920.2 [c]0.947 [b]<0.500<0.50086.1%88.0%IPF003ISO12-01-01-04-004<0.250<0.0503<0.252<0.4803.82<0.25018.3164 [b, c]2.231.070.71484.4%87.1%MSF004ISO12-01-01-04-005<0.250<0.0503<0.252<0.480<1.10<0.25016758.2[c]3.051.371.1786.3%87.9%MSF005ISO12-01-01-04-006<0.250<0.0503<0.252<0.480<1.10<0.25016860.9[c]3.081.351.1385.1%87.1%MSF006ISO12-01-01-04-007<0.250<0.0503<0.252<0.480<1.10<0.25017379.5[c]2.260.9620.94384.4%87.7%IPF007ISO12-01-01-04-008<0.250<0.0503<0.252<0.480<1.10<0.2508.864.83[c]<0.505<0.500<0.50088.3%89.3%IPF009ISO12-01-01-04-009<0.250<0.0503<0.252<0.480<1.10<0.25013.45.29[c]0.487<0.500<0.50086.8%88.8%MSF010ISO12-01-01-04-010<0.250<0.0503<0.252<0.480<1.10<0.25071.068.6[c]1.42<0.500<0.50083.0%87.4%MSF011ISO12-01-01-04-011<0.250<0.0503<0.2520.576 [a,c]NR<0.25098.581.7 [c]2.03<0.500<0.50089.0%91.7%IPF012ISO12-01-01-04-012<0.250<0.0503<0.252<0.480<1.10<0.25012.196.3 [b, c]4.810.8290.51186.5%90.7%MSF013ISO12-01-01-04-013<0.250<0.0503<0.252<0.480<1.10<0.25068.984.1[c]1.370.546 [a]<0.50085.6%87.7%IPF014ISO12-01-01-04-014<0.250<0.0503<0.252<0.480<1.10<0.25014.352.4 [c]2.350.654 [a]<0.50084.4%87.5%MSF015ISO12-01-01-04-015<0.250<0.0503<0.252<0.480<1.10<0.25067.156.2 [c]1.240.684 [a]<0.50081.8%85.7%IPF016ISO12-01-01-04-016<0.250<0.0503<0.252<0.480<1.10<0.25014.655.5 [c]2.400.696 [a]<0.50086.8%89.1%MSF017ISO12-01-01-04-017<0.250<0.0503<0.252<0.480<1.10<0.25079.651.3 [c]1.09<0.500<0.50087.1%92.0%IPF018ISO12-01-01-04-018<0.250<0.0503<0.252<0.480<1.10<0.25011.728.6 [c]1.21<0.500<0.50085.0%87.9%ISO12-01-01-04Tennessee River Fish StudyFall 20127Table 1.Concentration Results for Tennessee River Fish Tissue SamplesSAMPLE IDLIMS IDAverage Concentration (ng/g; wet wt.)Avg. SRS RecoveryPFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAA13C4-PFOA13C4-PFOSMSF020ISO12-01-01-04-020<0.250<0.0503<0.252<0.480<1.10<0.25065.856.7 [c]1.11<0.500<0.50084.2%87.5%MSF021ISO12-01-01-04-021<0.250<0.0503<0.252<0.480<1.10<0.25098.223.3 [c]0.699<0.5000.520 [a]85.7%86.2%IPF022ISO12-01-01-04-022<0.250<0.0503<0.252<0.480<1.10<0.2505.1036.9 [c]<0.505<0.500<0.50087.6%88.6%MSF023ISO12-01-01-04-023<0.250<0.101<0.101<0.4800.184 [b]<0.25054.933.0 [c]0.800<0.5000.32684.7%85.8%IPF024ISO12-01-01-04-024<0.250<0.101<0.101<0.4802.668<0.25010.312.3 [c]1.07 [b]<0.5000.260 [a]85.4%86.1%MSF025ISO12-01-01-04-025<0.250<0.101<0.101<0.4800.294 [a]<0.25066.447.9 [b, c]0.887<0.5000.28285.7%89.2%IPF026ISO12-01-01-04-0262.34 [a]1.78 [a]1.79 [a]3.13 [a]0.887 [b]1.54 [a]45.5 [b]19.6 [c]6.382.40 [a]4.73 [a]83.4%84.3%MSF027ISO12-01-01-04-027<0.250<0.101<0.101<0.4800.176 [b]<0.25065.348.1 [c]0.988<0.5000.344 [b]92.7%92.9%MSF028ISO12-01-01-04-028<0.250<0.101<0.101<0.4800.859<0.25065.945.0 [c]0.970<0.5000.33787.5%90.2%IPF029ISO12-01-01-04-029<0.250<0.101<0.101<0.4800.460<0.25018.669.2 [c]4.201.940.84487.6%85.8%MSF030ISO12-01-01-04-030<0.250<0.1010.198 [a]<0.4800.590<0.25065.222.1 [c]0.571<0.5000.388 [b]97.3%100%IPF031ISO12-01-01-04-031<0.250<0.101<0.101<0.4801.31<0.25021.984.2 [c] 3.01 0.7290.28187.8%87.0%MSF033ISO12-01-01-04-032<0.250<0.101<0.101<0.4800.459<0.25019031.5 [c] 1.12 1.670.84585.2%86.4%IPF034ISO12-01-01-04-033<0.250<0.101<0.101<0.4801.862<0.2508.125.38 [c] 0.884 [b] <0.500<0.25085.0%85.2%MSF035ISO12-01-01-04-034<0.250<0.101<0.101<0.4800.219<0.25020741.3 [c] 1.28 0.5960.60289.0%90.2%IPF036ISO12-01-01-04-035<0.250<0.101<0.101<0.4800.371 [b]<0.25014.47.86 [c] 0.629 <0.500<0.25087.9%87.9%MSF037ISO12-01-01-04-037<0.250<0.101<0.101<0.4800.582 [b]<0.250101.963.6 [c] 0.716 <0.5000.42584.8%84.6%MSF038ISO12-01-01-04-038<0.250<0.101<0.101<0.4800.996 [b]<0.25010666.4 [c] 0.750 0.5230.46488.6%88.8%IPF039ISO12-01-01-04-039<0.250<0.101<0.101<0.4800.305 [b]<0.25015.753.7 [c] 3.58 0.7160.37388.0%89.6%MSF040ISO12-01-01-04-040<0.250<0.101<0.101<0.4800.259<0.25010435.6 [c] 1.26 0.7980.65284.7%87.6%IPF041ISO12-01-01-04-041<0.250<0.101<0.101<0.4800.405 [b]<0.25022.235.4 [c] 2.41 1.300.38185.5%86.0%MSF042ISO12-01-01-04-042<0.250<0.1010.120 [a]1.57 [a]0.131 [b]<0.250210 [b]52.8 [c] 1.25 2.61 [b]1.5282.5%82.5%MSF043ISO12-01-01-04-043NR<0.101<0.101NR0.138 [b]<0.25022455.5 [c] 1.32 2.861.6294.1%96.0%MSF044ISO12-01-01-04-044<0.100<0.1010.118 [a]<0.240<0.252<0.250116107 3.09 1.060.79185.2%90.7%MSF045ISO12-01-01-04-045<0.100<0.101<0.101<0.240<0.252<0.25022537.9 1.99 0.768 [b]0.77783.4%89.2%ISO12-01-01-04Tennessee River Fish StudyFall 20128Table 1.Concentration Results for Tennessee River Fish Tissue SamplesSAMPLE IDLIMS IDAverage Concentration (ng/g; wet wt.)Avg. SRS RecoveryPFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAA13C4-PFOA13C4-PFOSMSF046ISO12-01-01-04-046<0.100<0.101<0.101<0.240<0.252<0.25054.156.5 1.11 0.4350.31583.3%89.3%IPF047ISO12-01-01-04-047<0.100<0.101<0.101<0.2400.327<0.25014.363.9 2.71 0.2930.116 [a]85.3%90.8%MSF048ISO12-01-01-04-048<0.100<0.101<0.101<0.2400.269 [a]<0.25088.9129 2.40 1.000.79583.5%90.7%IPF049ISO12-01-01-04-049<0.100<0.101<0.101<0.240<0.252<0.2505.5920.6 1.29 <0.2500.141 [a]85.5%92.6%MSF050ISO12-01-01-04-050<0.100<0.101<0.101<0.240<0.252<0.25081.6116 2.11 0.8830.73187.0%89.7%IPF051ISO12-01-01-04-051<0.100<0.101<0.101<0.2400.337<0.2508.0128.0 1.71 0.307<0.10081.4%87.2%MSF052ISO12-01-01-04-052<0.100<0.101<0.101<0.240<0.252<0.25093.5 [b]71.1 2.11 0.448 [b]0.45884.2%87.9%IPF053ISO12-01-01-04-053<0.100<0.101<0.101<0.2401.54 [c]<0.2506.6815.9 1.31 <0.250<0.10085.0%89.6%IPF055ISO12-01-01-04-055<0.100<0.101<0.101<0.240<0.252<0.25011.031.5 1.37 [b] 0.333 [b]0.113 [b]84.9%87.3%MSF056ISO12-01-01-04-056<0.100<0.101<0.101<0.240<0.252<0.25054.819.3 0.530 <0.2500.226 [b]82.1%87.9%IPF057ISO12-01-01-04-057<0.100<0.101<0.101<0.240<0.252<0.2507.3322.8 1.58 <0.2500.102 [a]80.9%84.6%MSF058ISO12-01-01-04-058<0.100<0.101<0.101<0.240<0.252<0.25075.369.7 1.59 0.4040.38481.6%86.4%IPF059ISO12-01-01-04-059<0.100<0.101<0.101<0.2400.327<0.2507.5626.1 1.69 <0.250<0.10081.5%85.4%MSF060ISO12-01-01-04-060<0.100<0.101<0.101<0.240<0.252<0.25064.774.2 2.48 0.365 [b]0.44777.4%84.4%MSF061ISO12-01-01-04-061<0.100<0.101<0.101<0.2400.289<0.25075.585.9 2.21 0.3930.54483.1%87.7%MSF062ISO12-01-01-04-062<0.100<0.101<0.101<0.240<0.252<0.25010392.6 2.42 0.4190.45781.4%88.1%IPF063ISO12-01-01-04-063<0.100<0.101<0.101<0.2400.854<0.2506.6716.0 1.43 0.3000.14781.5%82.3%MSF065ISO12-01-01-04-064<0.100<0.101<0.101<0.240<0.252<0.25086.946.8 1.19 0.3310.28480.8%83.2%IPF066ISO12-01-01-04-065<0.250<0.0503<0.101<0.2400.353 [b]<0.25010.5 [c]37.5 1.95 0.1740.34492.5%96.2%MSF067ISO12-01-01-04-066<0.250<0.05030.176 [a]<0.240<0.252<0.25094.638.6 1.98 0.3130.49381.2%85.7%IPF068ISO12-01-01-04-067<0.250<0.0503<0.101<0.240<0.252<0.2509.6040.8 1.48 0.1980.27991.2%91.7%IPF069ISO12-01-01-04-068<0.250<0.05030.138 [a]<0.2400.739<0.2505.6318.0 0.771 0.142<0.25091.3%93.0%IPF070ISO12-01-01-04-069<0.250<0.0503<0.101<0.240<0.252<0.2501.81<0.250 0.153 [b] <0.100<0.25084.7%85.4%MSF071ISO12-01-01-04-070<0.250<0.0503<0.101<0.240<0.252<0.25035.80.555 0.137 <0.100<0.25081.2%89.8%MSF073ISO12-01-01-04-072<0.250<0.0503<0.101<0.240<0.252<0.25036.30.529 0.161 <0.100<0.25084.2%86.6%ISO12-01-01-04Tennessee River Fish StudyFall 20129Table 1.Concentration Results for Tennessee River Fish Tissue SamplesSAMPLE IDLIMS IDAverage Concentration (ng/g; wet wt.)Avg. SRS RecoveryPFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAA13C4-PFOA13C4-PFOSMSF074ISO12-01-01-04-073<0.250<0.0503<0.101<0.240<0.252<0.25032.71.72 0.215 <0.100<0.25088.2%92.5%MSF075ISO12-01-01-04-074<0.250<0.0503<0.101<0.240<0.252<0.25027.90.648 0.122 <0.100<0.25083.8%88.7%IPF076ISO12-01-01-04-075<0.2500.139 [a]<0.101<0.240<0.252<0.2500.768<0.250 <0.101 <0.100<0.25087.7%87.2%IPF077ISO12-01-01-04-076<0.250<0.0503<0.101<0.240<0.252<0.2501.50<0.250 <0.101 <0.100<0.25085.0%89.6%IPF078ISO12-01-01-04-077<0.250<0.0503<0.101<0.240<0.252<0.2505.173.58 0.277 [b] 0.149<0.25085.0%89.0%IPF079ISO12-01-01-04-078<0.250<0.0503<0.101<0.240<0.252<0.2500.836<0.250 <0.101 <0.100<0.25080.9%82.3%MSF080ISO12-01-01-04-079<0.250<0.0503<0.101<0.240<0.252<0.25018.60.361 0.148 <0.100<0.25081.2%86.4%MSF081ISO12-01-01-04-080<0.250<0.0503<0.101<0.240<0.252<0.25037.80.802 0.135 [a] <0.100<0.25088.2%93.8%IPF082ISO12-01-01-04-081<0.250<0.0503<0.101<0.240<0.252<0.2502.920.295 0.196 [a] <0.100<0.25091.1%92.5%IPF083ISO12-01-01-04-082<0.2500.137 [a]<0.101<0.240<0.252<0.2503.41 [b]0.512 [b] 0.123 [a] <0.100<0.25082.9%88.3%MSF084ISO12-01-01-04-083<0.250<0.0503<0.101<0.240<0.252<0.25044.17.01 1.44 0.3980.530 [b]83.8%90.6%MSF085ISO12-01-01-04-084<0.250<0.05030.110 [a]<0.240<0.252<0.25032.62.72 0.580 0.122 [b]<0.25084.6%88.7%MSF086ISO12-01-01-04-085<0.250<0.0503<0.101<0.240<0.252<0.25049.56.87 0.127 [a] <0.100<0.25076.0%81.9%MSF087ISO12-01-01-04-086<0.250<0.101<0.503<0.240<0.252<0.25049.025.8 0.379 0.2840.31878.4%84.0%MSF089ISO12-01-01-04-087<0.2500.260 [a]<0.503<0.240<0.252<0.25040.814.8 1.22 [b] 0.288 [b]0.322 [a]81.1%88.2%MSF090ISO12-01-01-04-088<0.2500.151 [a]<0.503<0.240<0.252<0.25010428.4 1.71 0.2890.350 [a]85.3%89.8%MSF091ISO12-01-01-04-090<0.2500.174 [a]<0.503<0.240<0.252<0.25015920.4 2.24 0.4780.46483.8%90.1%MSF092ISO12-01-01-04-091<0.250<0.101<0.503<0.240<0.252<0.2504348.55 4.69 0.688 [b]0.70482.9%87.4%MSF094ISO12-01-01-04-0920.860 [a]0.132<0.503<0.240<0.252<0.25069.028.1 0.672 0.446<0.25082.7%85.8%IPF95ISO12-01-01-04-093<0.2500.0965 [a]<0.503<0.240<0.252<0.2504.371.16 0.258 <0.100<0.25082.6%86.5%MSF096ISO12-01-01-04-094<0.2500.144<0.503<0.240<0.252<0.25046.92.95 0.396 0.171<0.25082.9%87.0%IPF97ISO12-01-01-04-095<0.2500.137 [a]<0.503<0.240<0.252<0.2504.371.90 0.413 0.121<0.25080.8%87.7%IPF98ISO12-01-01-04-096<0.250<0.101<0.503<0.240<0.252<0.2502.220.867 0.299 [a] <0.100<0.25084.4%89.2%MSF099ISO12-01-01-04-097<0.250<0.101<0.503<0.240<0.252<0.2501246.10 1.60 0.6480.478 [b]81.3%86.6%IPF100ISO12-01-01-04-098<0.250<0.101<0.503<0.240<0.252<0.2504.690.575 0.656 0.122 [a]<0.25081.6%86.8%ISO12-01-01-04Tennessee River Fish StudyFall 201210Table 1.Concentration Results for Tennessee River Fish Tissue SamplesSAMPLE IDLIMS IDAverage Concentration (ng/g; wet wt.)Avg. SRS RecoveryPFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAA13C4-PFOA13C4-PFOSIPF101ISO12-01-01-04-099<0.250<0.101<0.503<0.240<0.252<0.25050.93.37 6.68 0.236 [b]<0.25079.9%89.2%MSF102ISO12-01-01-04-100<0.250<0.101<0.503<0.240<0.252<0.25021822.4 1.37 [b] 1.391.2782.2%91.2%IPF103ISO12-01-01-04-101<0.250<0.101<0.503<0.240<0.252<0.25042.03.56 6.26 0.243<0.25084.9%91.3%IPF104ISO12-01-01-04-102<0.250<0.101<0.503<0.240<0.252<0.2504.151.16 <0.253 0.111 [a]<0.25078.2%85.0%IPF105ISO12-01-01-04-103<0.250<0.101<0.503<0.240<0.252<0.2503.410.633 0.255 [a] <0.100<0.25085.9%87.7%IPF106ISO12-01-01-04-104<0.250<0.101<0.503<0.240<0.252<0.2502.420.609 0.271 [a] 0.107 [a]<0.25082.1%88.2%IPF107ISO12-01-01-04-105<0.250<0.101<0.503<0.240<0.252<0.2507.851.39 1.28 <0.100<0.25078.7%85.8%IPF108ISO12-01-01-04-106<0.250<0.101<0.503<0.240<0.252<0.25029.11.29 1.07 0.246 [b]<0.25082.6%88.0%TN_11-0194-1_1Sample 2Mid-StudyHomogenizationProcess controlISO12-01-01-04-1081.420.126 [a]<0.503<0.240<0.252<0.2500.523 [a]<0.250 <0.253 <0.100<0.25082.9%92.4%MSF109ISO12-01-01-04-109<1.00<2.52<0.252<0.480<0.252<0.10021.81.03 <0.101 <0.100<0.50083.8%86.6%MSF111ISO12-01-01-04-110<1.00<2.52<0.252<0.480<0.252<0.10025.71.57 0.235 [b] <0.100<0.50084.9%88.2%IPF112ISO12-01-01-04-111<1.00<2.52<0.252<0.480<0.252<0.1001.78<0.500 <0.101 <0.100<0.50085.8%88.6%IPF113ISO12-01-01-04-112<1.00<2.52<0.252<0.480<0.252<0.1001.30<0.500 <0.101 <0.100<0.50088.9%92.0%IPF114ISO12-01-01-04-113<1.00<2.52<0.252<0.480<0.252<0.1001.24<0.500 <0.101 <0.100<0.50084.1%88.0%MSF115ISO12-01-01-04-114<1.00<2.52<0.252<0.480<0.252<0.10031.24.28 <0.101 <0.100<0.50077.0%84.1%IPF116ISO12-01-01-04-115<1.00<2.52<0.252<0.480<0.252<0.1002.65<0.500 <0.101 <0.100<0.50079.6%85.2%IPF117ISO12-01-01-04-116<1.00<2.52<0.252<0.480<0.252<0.1000.897<0.500 <0.101 <0.100<0.50086.7%89.4%IPF118ISO12-01-01-04-117<1.00<2.52<0.252<0.480<0.252<0.1002.01<0.500 <0.101 <0.100<0.50080.6%85.2%MSF119ISO12-01-01-04-118<1.00<2.52<0.252<0.480<0.252<0.10023.92.26 0.162 <0.100<0.50082.1%91.8%IPF120ISO12-01-01-04-119<1.00<2.52<0.252<0.480<0.252<0.1000.961<0.500 0.300 <0.100<0.50082.0%84.1%MSF121ISO12-01-01-04-120<1.00<2.52<0.252<0.480<0.252<0.10019.92.06 0.141 <0.100<0.50081.6%89.6%IPF122ISO12-01-01-04-121<1.00<2.52<0.252<0.480<0.252<0.1004.45<0.500 <0.101 <0.100<0.50080.6%83.8%ISO12-01-01-04Tennessee River Fish StudyFall 201211Table 1.Concentration Results for Tennessee River Fish Tissue SamplesSAMPLE IDLIMS IDAverage Concentration (ng/g; wet wt.)Avg. SRS RecoveryPFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAA13C4-PFOA13C4-PFOSIPF123ISO12-01-01-04-122<1.00<2.52<0.252<0.480<0.252<0.1002.11<0.500 <0.101 <0.100<0.50083.4%85.4%MSF124ISO12-01-01-04-123<1.00<2.52<0.252<0.480<0.252<0.10073.26.47 0.258 [b] <0.100<0.50080.7%87.7%MSF125ISO12-01-01-04-124<1.00<2.52<0.252<0.480<0.252<0.10040.8 [d]6.23 0.200 [d] <0.100 [d]<0.500 [d]78.8%89.4% [d]MSF127ISO12-01-01-04-126<1.00<2.52<0.252<0.4800.4440.416408182 4.19 2.221.7383.6%86.6%MSF128ISO12-01-01-04-127<1.00<2.52<0.252<0.480<0.252<0.100262110 4.00 1.181.0875.5%82.7%MSF129ISO12-01-01-04-128<1.00<2.52<0.252<0.480<0.2520.123250137 2.46 1.651.1977.6%83.7%MSF130ISO12-01-01-04-129<1.00<2.52<0.252<0.480<0.252<0.100333119 8.18 5.072.4278.7%87.5%MSF131ISO12-01-01-04-130<0.250 [c]<0.101<0.252<0.240<0.252<0.250178128 [c] 2.12 1.691.4382.7%85.4%MSF132ISO12-01-01-04-131<0.2500.0992 [a]<0.252<0.240<0.252<0.250324143 [c] 7.21 5.542.8790.8%85.8%IPF133ISO12-01-01-04-132<0.2500.417 [a]<0.252<0.240<0.252<0.25022.68.87 [c] 0.384 <0.250<0.25096.5%95.7%IPF134ISO12-01-01-04-133<0.250<0.101<0.252<0.240<0.252<0.2503.98 [b]1.80 [c] <0.253 <0.250<0.25087.7%84.9%IPF135ISO12-01-01-04-134<0.250<0.101<0.252<0.240<0.252<0.2504.89 [b]3.67 [c] <0.253 <0.250<0.25091.2%85.9%IPF137ISO12-01-01-04-135<0.250<0.101<0.252<0.240<0.252<0.2501.902.50 [c] <0.253 <0.250<0.25085.2%87.4%IPF138ISO12-01-01-04-136<0.250<0.101<0.252<0.240<0.252<0.2508.553.97 [c] <0.253 <0.250<0.25093.1%90.0%MSF139ISO12-01-01-04-137<0.2500.0942 [a]<0.252<0.240<0.252<0.25027972.9 [c] 1.73 1.431.4589.4%87.3%MSF140ISO12-01-01-04-138<0.250<0.101<0.252<0.240<0.252<0.250281133 [c] 3.75 1.851.7689.3%84.5%MSF141ISO12-01-01-04-139<0.2500.136<0.252<0.240<0.252<0.250134114 [c] 1.75 1.060.70386.8%89.3%MSF142ISO12-01-01-04-140<0.250<0.101<0.252<0.240<0.252<0.25045.015.4 [c] 0.441 [b] <0.250<0.25089.7%90.5%MSF143ISO12-01-01-04-141<0.250<0.101<0.252<0.240<0.252<0.250143111 [c] 1.83 1.020.75392.9%92.8%MSF144ISO12-01-01-04-142<0.2500.122 [a]<0.252<0.240<0.252<0.25076.911.4 [c] 0.243 [a] <0.250<0.25091.5%92.6%TN_11-0194-1_1 Sample 3Post-StudyHomogenizationISO12-01-01-04-1441.420.170 [a]<0.252<0.240<0.252<0.250<0.184 [a]<0.250 [c] <0.253 <0.250<0.25087.3%84.8%IPF181ISO12-01-01-04-145<0.250<0.101<0.252<0.240<0.252<0.2505.00<0.250 [c] <0.253 <0.250<0.25084.5%82.8%MSF145ISO12-01-01-04-146<0.250<0.101<0.252<0.240<0.252<0.25017515.2 [c] 0.348 1.160.89688.2%93.6%ISO12-01-01-04Tennessee River Fish StudyFall 201212Table 1.Concentration Results for Tennessee River Fish Tissue SamplesSAMPLE IDLIMS IDAverage Concentration (ng/g; wet wt.)Avg. SRS RecoveryPFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAA13C4-PFOA13C4-PFOSMSF146ISO12-01-01-04-147<0.250<0.101<0.252<0.240<0.252<0.25080.317.7 [c] 0.465 0.336<0.25086.5%87.2%IPF147ISO12-01-01-04-148<0.250<0.101<0.252<0.240<0.252<0.25026.972.8 [c] 7.23 0.806<0.25089.8%90.0%IPF148ISO12-01-01-04-149<0.250<0.101<0.252<0.240<0.252<0.25069.665.9 [c] 3.64 0.351<0.25091.2%91.4%IPF149ISO12-01-01-04-150<0.250<0.101<0.252<0.240<0.252<0.25064.245.9 [c] 2.45 0.299<0.25093.0%91.5%IPF151ISO12-01-01-04-151<0.250<0.252<0.252<0.480<0.503<0.25011.749.5 1.81 <0.500<0.50087.7%83.1%IPF152ISO12-01-01-04-152<0.250<0.252<0.252<0.480<0.503<0.25029.2106 4.92 0.731<0.50085.8%87.1%IPF153ISO12-01-01-04-153<0.250<0.252<0.252<0.480<0.503<0.25016.310.1 0.816 <0.500<0.50085.2%84.6%IPF155ISO12-01-01-04-154<0.250<0.252<0.252<0.480<0.503<0.25011.938.6 1.83 <0.500<0.50077.2%85.6%MSF157ISO12-01-01-04-155<0.250<0.252<0.252<0.480<0.503<0.25011574.7 1.20 0.7860.83591.7%93.2%MSF158ISO12-01-01-04-156<0.2500.317 [a]<0.252<0.480<0.503<0.25014567.3 2.28 0.9740.84785.9%87.9%IPF159ISO12-01-01-04-157<0.250<0.252<0.252<0.480<0.503<0.25011.220.4 0.620 <0.500<0.50084.1%85.7%IPF160ISO12-01-01-04-158<0.250<0.252<0.252<0.480<0.503<0.2504.878.47 0.490 <0.500<0.50083.1%85.8%IPF161ISO12-01-01-04-159<0.250<0.252<0.252<0.480<0.503<0.25085.669.2 34.1 3.422.6687.3%89.7%IPF162ISO12-01-01-04-160<0.250<0.252<0.252<0.480<0.503<0.25079.365.8 31.4 3.092.5079.3%80.9%IPF163ISO12-01-01-04-162<0.250<0.252<0.252<0.480<0.503<0.25083.444.8 25.6 1.851.1681.4%82.1%IPF164ISO12-01-01-04-163<0.250<0.252<0.252<0.480<0.503<0.25028337.5 63.5 [b] 3.511.0081.3%89.4%IPF165ISO12-01-01-04-164<0.250<0.252<0.252<0.480<0.503<0.25054234.1 123 13.24.3088.6%89.6%IPF166ISO12-01-01-04-165<0.250<0.252<0.252<0.480<0.503<0.25028633.3 88.1 7.963.3285.1%87.5%MSF167ISO12-01-01-04-166<0.250<0.252<0.252<0.480<0.503<0.25050.612.1 1.28 <0.500<0.50088.1%90.6%MSF168ISO12-01-01-04-167<0.250<0.252<0.252<0.480<0.503<0.25012020.2 4.22 1.211.5681.2%81.8%MSF169ISO12-01-01-04-168<0.250<0.252<0.252<0.480<0.503<0.25043567.2 [b] 7.44 3.553.0177.9%86.0%MSF170ISO12-01-01-04-169<0.250<0.252<0.2520.6432.210.783373567 59.9 22.519.086.0%91.6%MSF172ISO12-01-01-04-170<0.250<0.252<0.252<0.480<0.5030.39196.445.8 2.92 0.5450.63979.7%80.6%MSF173ISO12-01-01-04-171<0.250<0.252<0.252<0.480<0.503<0.250599135 9.75 4.673.4689.5%89.1%MSF174ISO12-01-01-04-172<0.2500.294 [a]<0.252<0.240<0.252<0.50052195.0 5.20 1.621.4385.7%87.7%ISO12-01-01-04Tennessee River Fish StudyFall 201213Table 1.Concentration Results for Tennessee River Fish Tissue SamplesSAMPLE IDLIMS IDAverage Concentration (ng/g; wet wt.)Avg. SRS RecoveryPFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAA13C4-PFOA13C4-PFOSIPF175ISO12-01-01-04-173<0.250<0.252<0.252<0.240<0.252<0.5008.5736.2 1.36 <0.500<0.50088.2%87.9%IPF176ISO12-01-01-04-174<0.250<0.252<0.252<0.240<0.252<0.50011.828.6 1.54 <0.500<0.50086.2%84.9%IPF177ISO12-01-01-04-175<0.2500.421<0.252<0.240<0.252<0.50012.729.9 1.60 <0.500<0.50090.7%90.5%IPF178ISO12-01-01-04-176<0.250<0.252<0.252<0.2400.322<0.50010.551.2 1.23 <0.500<0.50085.3%88.4%IPF179ISO12-01-01-04-177<0.250<0.252<0.252<0.2400.506<0.50010.735.7 0.670 <0.500<0.50088.1%86.5%IPF180ISO12-01-01-04-178<0.2500.277<0.252<0.240<0.252<0.5007.6927.8 1.43 0.539<0.50093.8%89.8%DAL-MSF-LS10001ISO12-01-01-04-180<0.250<0.252<0.252<0.240<0.252<0.500<0.366<0.250 <0.253 <0.500<0.50092.1%91.1%DAL-IPF-LS10002ISO12-01-01-04-181<0.250<0.252<0.252<0.240<0.252<0.5003.95<0.250 <0.253 <0.500<0.50084.3%86.1%DAL-MSF-LS10003ISO12-01-01-04-182<0.250<0.252<0.252<0.240<0.252<0.5003.67<0.250 <0.253 <0.500<0.50094.7%91.4%DAL- IPF-LS10004ISO12-01-01-04-183<0.250<0.252<0.252<0.240<0.252<0.500<0.366<0.250 <0.253 <0.500<0.50087.3%85.8%DAL- IPF-LS10005ISO12-01-01-04-184<0.250<0.252<0.252<0.240<0.252<0.500<0.366<0.250 <0.253 <0.500<0.50087.1%84.3%DAL-MSF-LS10006ISO12-01-01-04-185<0.250<0.252<0.252<0.240<0.252<0.5004.31<0.250 <0.253 <0.500<0.50080.4%85.7%**Frequency of Detection (n = 175)** **2.9% 12%** **4.0% 2.3%** **19% 2.9%** **98% 87% 82% 49%** **45% 100%** **100% Average Recovery** **NA NA** **NA NA** **NA NA** **NA NA NA NA** **NA 85.2%** **88.1% Standard Deviation** **NA NA** **NA NA** **NA NA** **NA NA NA NA** **NA 4.4%** **3.4% RSD** **NA NA** **NA NA** **NA NA** **NA NA NA NA** **NA 5.2%** **3.8%**Analytical method uncertainty (95% CI) at +18% for PFBA, +15% for PFHxA, +21% for PFHpA, +20% for PFOA, +18% for PFBS, +19% for PFHS, +22% for total-PFOS, +40% for FBSA, +20% for FOSA, +18% for MeFOSAA and +19% for EtFOSAANR; Sample had LMS prepared and recovery was outside of 100 +50% and result not reported*; quantified from a linear-PFOS (L-PFOS) calibration for total-PFOS (sum of linear and branched isomer peaks) in samples, LMSs and LCSs.[a] Value reported for single replicate results, duplicate result was <LLOQ[b]RPD > 20%[c] Sample had LMS prepared and LMS recovery resultwas outside of 100 + 30%, but within 100 + 50% andwere reported with an expanded uncertainty of +50%[d] Value reported for single result, duplicate sample analysis had peak shift out of retention time window and no result determinedISO12-01-01-04Tennessee River Fish StudyFall 201214Table 2.Laboratory Matrix Spike (LMS) RecoveriesLIMS ID Study IDLMS Analyte RecoveriesPFBA PFHxA PFHpA PFOA PFBS PFHS PFOS FBSA FOSA MeFOSAA EtFOSAA13C4-PFOA13C4-PFOSISO12-01-01-04-001TN11-0194-1/1Sample 1 PrestudyHomogenizationProcess Control107% 109% 101% 111% 109% 102% 111% 139% [a] 92.1% 107% 105% 102% 101%ISO12-01-01-04-002 MSFOO1 109% 101% 124% 114% 111%106% 113% [b] 53.5%[a, b] 95.3% 104% 98.1% 87.1% 93.2%ISO12-01-01-04-011 MSF011 112% 102% 128% 116% NR 115% 97.5% [b] 55.4%[a, b] 96.6% 111% 111% 83.0% 88.6%ISO12-01-01-04-022 IPF022 108% 96.8% 126% 109% 95.6% 100% 103% 86.9% [b] 97.6% 107% 97.8% 88.9% 93.2%ISO12-01-01-04-023 MSF023 112% 106% 99.4% 115% 115% 98.6% 96.9 [b]% 52.2%[a, b] 97.7% 115% 97.7% 89.3% 86.3%ISO12-01-01-04-032 MSF033 107% 100% 91.8% 109% 104% 91.8% 105% [b] 69.7%[a, b] 94.6% 101% 88.1% 84.9%93.0%ISO12-01-01-04-043 MSF043 NR 122% 101% NR NR 102% 102% [b] 53.1%[a, b] 106% 111% 107% 90.3% 88.0%ISO12-01-01-04-044 MSF044 112% 100% 92.0% 94.0% 107% 140%[a] 94.7% [b] 85.9% [b] 109% 102% 93.6% 82.9%89.6%ISO12-01-01-04-053 IPF053 106% 96.2% 91.8% 90.8% 89.7% 91.6% 86.4% 90.5% [b] 122% 99.0% 88.5% 83.4% 86.3%ISO12-01-01-04-064MSF065100% 97.0% 85.0% 91.7% 87.6% 92.5% 95.1% [b] 89.1%[b] 108% 88.0% 88.7% 84.4% 89.4%ISO12-01-01-04-065IPF066106% 89.6% 89.0% 90.8% 93.3% 97.2% 80.4% 94.6% [b]114% 100% 85.7% 89.9% 91.2%ISO12-01-01-04-076 IPF077 114% 102% 86.2% 91.7% 90.0% 95.6% 88.7% 104% 115% 98.0% 82.4% 86.1% 87.0%ISO12-01-01-04-086 MSF087 112% 101% 92.0% 95.4% 94.8% 96.0% 95.7% [b] 93.8% [b] 97.2% 106% 93.8% 87.4%87.4%ISO12-01-01-04-096 IPF098 104% 95.6% 88.4% 90.6% 92.8% 86.6% 99.6% 92.2% 97.3% 104% 86.0% 85.6% 91.1%ISO12-01-01-04-109 MSF109 86.0% 78.2% 82.6% 84.4% 86.2% 84.0% 97.0% [b] 93.1% 108% 96.6% 81.2% 82.9% 90.5%ISO12-01-01-04-118 MSF11984.2% 86.2% 84.0% 91.9% 89.8% 85.4% 82.5% [b] 79.8%113% 103% 82.8% 81.7% 79.4%ISO12-01-01-04-130 MSF131135%[a] 103% 106% 108% 113% 103% 97.4%[b] 50.7%[a,b] 104% 105% 98.9% 91.9% 89.4%ISO12-01-01-04-139 MSF141125% 107% 104% 97.5% 107% 101% 96.9% [b] 64.8% [a,b] 98.8% 97.8% 88.9% 94.2% 89.0%ISO12-01-01-04-151 IPF151104% 98.4% 103% 107% 112% 97.2% 106% [b] 111% [b] 104% 105% 102% 85.2% 87.4%ISO12-01-01-04-159 IPF161104% 93.6% 93.6% 108% 96.0% 100% 90.7% [b] 84.0% [b] 77.0% [b] 90.1% 99.5% 87.6% 91.1%ISO12-01-01-04-172 MSF174117% 89.3% 98.6% 97.9% 98.4% 103% 101% [b] 85.7% [b] 99.4% 109% 98.4% 83.0% 86.6%**Average Recovery 108% 98.8% 98.4% 101% 99.6% 99.4% 97.2% 82.3% 102%** **103% 94.1% 87.2% 89.5% Standard Deviation** **11% 9.0% 13% 10% 9.6% 12% 8.4% 22% 10% 6.6% 8.5% 4. 8% 4.1%** **RSD 10% 9.1% 14% 10% 9.7% 12% 8.6% 27% 10% 6.4% 9.0% 5.** **5% 4.5% n** **20 21 21 20 19 21 21 21 21 21 21 21 21**Values shown are those determined for the Low-LMS (fortified at nominal 5 ng/g), unless otherwise noted for those shown for high LMS (fortified at nominal 125 ng/g)NR; value was outside of 100 +50% and result not reported for that sample and LMS recovery excluded from summary statistics[a] Recovery outside of 100+30%, but within 100 +50% and included in summary statistics[b] Value shown was obtained from high-level LMS (fortified at nominal 250 ng/g); accurate concentrations of LMSs were used for recovery calculations.

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

15 

# 3 Introduction

A scientific investigation to determine the concent rations of select perfluorinated compounds

(PFC) fish and surface water from the Tennessee Riv er was conducted in the Fall of 2012, per

the study work plan titled: “Revised Work Plan for Fall 2012 Fish and Surface Water Sampling

in the Tennessee River” Weston Solutions, Inc. (Wes tchester, MA), dated October 2012. The

overall purpose of the study was to provide additio nal site-specific information on PFCs in fish

tissues and surface water from within the confines of the Wilson and Wheeler Reservoirs,

Tennessee River, located near Decatur, Alabama. L aboratory blinded fish tissue samples

were provided for analysis by Weston Solutions, Inc . Weston Solutions, Inc. personnel were

responsible for coordinating all field aspects of t he study such as site parameters, fish and

water collection procedures, handling and storage o f collected specimens, documentation of

individual fish information, shipment of whole fish for processing, filleting, homogenization at a

contracted laboratory (TestAmerica-Burlington, VT) and the shipment of frozen fillets to

TestAmerica, and then coordinating shipment of tiss ue homogenates to the 3M environmental

Laboratory and Axys Laboratories for analysis of PF Cs. TestAmerica personnel were

responsible for homogenizing fillets and sending th e tissue homogenates to the analytical

laboratories for quantitative analysis of PFCs, und er the direction of Weston Solutions, Inc.

personnel.

Prior to the commencement of the Tennessee River Fa ll 2012 fish and water sampling

campaign, whole largemouth bass and catfish were pu rchased by the 3M Environmental

Laboratory from Osage Catfisheries (Osage Beach, MO ) as control materials. The right side

fillet from each of three bass and three catfish we re removed in the laboratory, the skin was

removed from each fillet, and then control fillet t issue homogenate was prepared and kept at

the laboratory. The remaining three bass and three catfish with only left side fillets intact were

sent to the field, to have left side fillets remove d as filleting process controls. The control fill et

tissues kept at the laboratory were analyzed prior to the study to establish baseline levels for the

field process control materials. The results from t he extraction and LC/MS/MS analysis of the

control tissues maintained at the laboratory are pr ovided in Table A1 of Attachment A.

With the exception of the homogenization process co ntrols, this was a laboratory-blinded study

and the true identities of the fish tissues were no t provided and were only identifiable by the

anonymous IDs assigned to the samples by Weston Sol utions, Inc. personnel. Additionally,

Weston Solutions, Inc. personnel are responsible fo r associating the determined PFC

concentrations with the fish collection locations, fish species, age, weight, etc. and overall

evaluation of control materials. This report focus es solely on analytical aspects controlled by

the laboratory during the analysis of those supplie d fish tissues, and describes the extraction

procedures and quantitative LC/MS/MS analysis perfo rmed at the 3M Environmental

Laboratory for measuring the target analyte concent rations.

Additionally included in this report are the assess ment of the precision and accuracy of the data

based on several quality control (QC) parameters, a s follows: 1) recovery of fortified target

analytes in a control bluegill fish fillet homogena te matrix as laboratory control samples (LCSs)

was assessed as a measure of intra-batch and inter- batch precision and accuracy; 2) recovery

of fortified target analytes into fish sample matri x for ~10% of the samples (i.e. prepared for

every 10 th 

# fish sample) as laboratory matrix spikes (LMSs), a nd providing a measure of

sample-specific target analyte measurement accuracy ; and 3) recovery of target analytes from

each sample was indirectly determined from the reco very of the two stable isotope labeled

surrogate recovery standards (SRSs), with 13 

# C 4 

# -PFOS representative of the perfluorinated

sulfonates (PFSAS) and MeFOSAA, EtFOSAA, FOSA and F BSA and 13 

# C 4 

# -PFOA

representative of the perfluorinated carboxylates ( PFCAs). The SRSs were fortified into each

sample, LMS and LCS sample aliquot prior to extract ion.

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

16 

# 4 Reference Substances

The reference substances used during the analyses a re listed in Table 3

, Table 4 and Table 5

. Most were purchased from commercial vendors and exi st as predominantly linear isomers. Four of

the reference substances were 3M-supplied materials , and were as follows: PFBS, FBSA, FOSA and

technical PFHS; technical grade materials each cont ain pre-determined percentages of linear and

branched isomers. All standard solutions were pre pared from neat materials and were corrected for

purity during solution preparations. Several refer ence standards are provided as salts, and additiona l

correction factors for salt cations (Na + 

# , K + 

# , NH 4 + 

# ) and protons (H + 

# ) were also applied, when

applicable. All analyte concentrations were determ ined and reported for the anion form of each.

Table 3. Reference Substances: Target Analytes

Reference Substance

PFBA PFHxA PFHpA Technical PFOA

(linear + branched)

Chemical Name Perfluorobutyric acid Perfluorohexanoic acid Perfluoroheptanoic acid Technical Ammonium

Perfluorooctanoate

Chemical Formula C 3 

# F 7 

# COOH C 5 

# F 11 

# COOH C 6 

# F 13 

# COOH C 8 

# HF 15 

# O 2



# Identifier CAS # 375-22-4 CAS # 307-24-4 CAS # 375-85-9 CAS # 95328-99-7

Source Sigma Oakwood Aldrich Wellington Laboratories

Expiration Date 4/20/2017 8/18/2018 12/24/2018 3/17/2014

Storage Conditions Frozen Frozen Frozen Frozen

Chemical Lot Number 11921CH C150099B1 PU/07219EU TPFOA0311

TCR Number TCR07-0011 TCR-673 TCR-268 TCR12-0027

Physical Description Colorless Liquid Colorless Liquid Clear Crystals Liquid

Purity 97.6% 98.2% 98.0% >97% *

Reference Substance PFBS PFHS Br-PFOS (linear +

branched isomers) FBSA

Chemical Name Potassium

perfluorobutane sulfonate Sodium perfluoro-1-

hexanesulfonate Potassium perfluorooctane

sulfonate Nonafluoro-1-

butanesulfonamide

Chemical Formula C 4 

# F 9 

# SO 3 - 

# K + 

# C 6 

# F 13 

# SO 3 - 

# Na + 

# C 8 

# F 17 

# SO 3 - 

# K + 

# C 4 

# F 9 

# SO 2 

# NH 2



# Identifier CAS # 29420-49-3 N/A CAS # 2795-39-3 N/A

Source 3M Wellington Laboratories Wellington Laboratories 3M

Expiration Date 1/10/2017 3/25/2015 12/1/2014 1/31/2021

Storage Conditions Frozen Frozen Frozen Frozen

Chemical Lot Number 101 LPFHxSAM08 br-PFOSK1111 F-11954

TCR Number TCR-121 TCR12-0021 TCR12-0026 TCR-314

Physical Description White powder Powder Liquid Fine White Crystals

Purity

96.7% >98% * >98% purity **; 78.8%

composition is linear

isomer, 21.2% is branched

isomers 99.3%

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

17 Table 3. Reference Substances: Target Analytes

Reference Substance

FOSA MeFOSAA EtFOSAA

Chemical Name Perfluorooctane

sulfonamide N-methylperfluoro-1-

octanesulfonamidoacetic

acid N-ethylperfluoro-1-

octanesulfonamidoacetic

acid

Chemical Formula C 8 

# F 17 

# SO 2 

# NH 2 

# C 11 

# H 6 

# F 17 

# NO 4 

# S C 12 

# H 8 

# F 17 

# NO 4 

# S

Identifier CAS # 754-91-6 N/A N/A

Source

3M Wellington Laboratories Wellington Laboratories

Expiration Date

3/27/2018 8/26/2013 3/14/2015

Storage Conditions

Frozen Frozen Frozen

Chemical Lot Number NB#120067-108 NMeFOSAA0308 NEtFOSAA0312

TCR Number TCR-280 TCR12-0044 TCR12-0042

Physical Description White powder Liquid Liquid

Purity 98.3% ***; 22.2%

branched isomers >98% * >98% *

* ; 100% Purity was assumed to calculate concentrat ions used in this study and solution concentrations were based on the concentration of

the standard material provided by the commercial ve ndor and assume the vendor adjusted for purity. ** ; 78.8% Purity (78.8% linear isomer)

was used to calculate concentrations of calibration standards, L-PFOS LCS results and CCVs. 100% puri ty was used to calculated LMS

recoveries.

***; Calculated concentrations used in the study us ing 98.3% purity



# Table 4. Reference Substances: Stable Isotope Labe led Internal Standards (IS)

Reference Substance MPFBA MPFHxA MPFHpA M8PFOA

Chemical Name Perfluoro-n-[1,2,3,4-

13 

# C 4 

# ]-butanoic acid Perfluoro-n-[1,2- 13 

# C 2 

# ]-

hexanoic acid Perfluoro-n-[ 13 

# C 4 

# ]-

heptanoic acid Perfluoro-n-[ 13 

# C 8 

# ]-octanoic

acid

Chemical Formula 13 

# C 4 

# HF 7 

# O 2 13 

# C 2 12 

# C 4 

# HF 11 

# O 2 13 

# C 4 12 

# C 3 

# HF 13 

# O 2 13 

# C 8 

# HF 15 

# O 2



# Identifier IS for PFBA IS for PFHxA IS for PFHpA IS for PFOA and MPFOA

Source Wellington Laboratories Wellington Laboratories Wellington Laboratories Wellington Laboratories

Expiration Date 10/3/2015 10/3/2015 10/3/2015 10/3/2015

Storage Conditions Frozen Frozen Frozen Frozen

Chemical Lot Number MPFC-C-100212 MPFC-C-100212 MPFC-C-100212 MPFC-C-100212

TCR Number TCR12-0038 TCR12-0038 TCR12-0039 TCR12-0038

Physical Description Liquid Liquid Liquid Liquid

Purity >98% * >98% * >98% * >98% *

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

18 Reference Substance 18 

# O

2 

# -PFBS M3PFHxS M8PFOS M8FOSA

Chemical Name [18O2]-Ammonium

Perfluorobutanesulfonate Sodium perfluoro-1-

[ 13 

# C 3 

# ]hexanesulfonate Sodium perfluoro-1-

[ 13 

# C 8 

# ]octanesulfonate Perfluoro-1-[ 13 

# C 8 

# ]-

octanesulfonamide

Chemical Formula C 4 

# F 9 

# S 18 

# O 2 

# O

- 

# NH4 + 13 

# C 3 12 

# C 3 

# F 13 

# SO 3 - 

# Na + 13 

# C 8 

# F 17 

# SO 3 - 

# Na + 13 

# C 8 

# F 17 

# SO 2 

# NH 2



# Identifier IS for PFBS and FBSA IS for PFHS IS for PFOS and MPFOS IS for FOSA

Source RTI International Wellington Laboratories Wellington Laboratories Wellington Laboratories

Expiration Date 3/9/2015 10/3/2015 10/3/2015 10/3/2015

Storage Conditions Frozen Frozen Frozen Frozen

Chemical Lot Number 11546-107-2 MPFC-C-100212 MPFC-C-100212 MPFC-C-100212

TCR Number TCR-964, TCR-965 TCR12-0038 TCR12-0038 TCR12-0038

Physical Description Liquid Liquid Liquid Liquid

Purity N/A >98% * >98% * >98% *

Reference Substance d3-MeFOSAA d5-EtFOSAA

Chemical Name N-methyl-d3-perfluoro-1-

octanesulfonamidoacetic

acid N-ethyl-d5-perfluoro-1-

octanesulfonamidoacetic

acid

Chemical Formula C 11 

# D 3 

# H 3 

# F 17 

# NO 4 

# S C 12 

# D 5 

# H 3 

# F 17 

# NO 4 

# S

Identifier IS for N-MeFOSAA IS for N-EtFOSAA

Source Wellington Laboratories Wellington Laboratories

Expiration Date 6/14/2015 6/14/2015

Storage Conditions Frozen Frozen

Chemical Lot Number d3NMeFOSAA0408 d5NEtFOSAA0408

TCR Number TCR12-0045 TCR12-0043

Physical Description Liquid Liquid

Purity >98% * >98% *

* ; 100% Purity was assumed to calculate concentrat ions used in this study and solution concentrations were based on the concentration of

the standard material provided by the commercial ve ndor and assume the vendor adjusted for purity.

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

19 Table 5. Reference Substances: Stable Isotope Labe led Surrogate Recovery Standards (SRS)

Reference Substance 13 

# C 4 

# -PFOA 13 

# C 4 

# -PFOS

Chemical Name Perfluoro-n-[1,2,3,4- 13 

# C 4 

# ]-octanoic acid Sodium perfluoro-1-[1,2,3,4- 13 

# C 4 

# ]-

octanesulfonate

Chemical Formula 13 

# C 4 12 

# C 4 

# HF 15 

# O 2 13 

# C 4 12 

# C 4 

# F 17 

# SO 3 - 

# Na +



# Purpose/Use Surrogate for PFCAs Surrogate for PFSAs, MeFOSAA, EtFOSAA,

FOSA and FBSA

Source Wellington Laboratories Wellington Laboratories

Expiration Date 12/7/2013 12/21/2014

Storage Conditions Frozen Frozen

Chemical Lot Number MPFOA1210 MPFOS1211

TCR Number TCR11-0001 TCR12-0003

Physical Description Liquid Liquid

Purity >98% * >98% *

*; 100% purity was used to calculate concentrations used in this study and based on the concentration of the standard material provided

by the commercial vendor.



# 5 Method Summary

5.1 Methods

The re-homogenization of fish tissue samples, and t he sample extractions and quantitative

LC/MS/MS analyses were performed following validate d 3M Environmental Laboratory method

ETS-8-045.1 [Malinsky et. al. 2011, Analytica Chemica Acta 683 :pp248-257]. The bulk of the

fish tissue sample analyses occurred from December 27, 2012 through February 6, 2013, with

the exception of the original analysis for the fill et process controls and the first homogenization

process controls which occurred in November, 2012. The identities of the instruments used for

collection of the LC/MS/MS data are listed in Table 6 . The analytical batches used for reporting

the results of the fish analyses are listed in Table 10

.



# 5.2 Sample Receipt

All samples were received frozen on dry ice and wer e shipped for overnight delivery from

TestAmerica (Burlington, VT) to 3M by FedEx. Upon receipt at 3M, that sample was entered

into the 3M Environmental Laboratory LIMS system as sample ID ISO12-01-01-04-001

A single homogenization process control fish tissue sample was received from TestAmerica on

November 22, 2011 with appropriate chain of custody , logged into LIMS as ISO12-01-01-04-

001 and analyzed on December 6, 2012.

The remainder of control fish tissues (9), Tennesse e River fish fillet tissues (165) and aqueous

fillet equipment rinseates (10) were received from TestAmerica on two dates: December 27,

2012 and January 3, 2013 and were logged into the s ame LIMS project as ISO12-01-01-04-

002 through -185.

Copies of all chains of custody for the samples rec eived were retained within the study folder

and electronically scanned into the 3M Environmenta l Laboratory LIMS system. Data for this

study is associated with study ISO12-01-01-04.

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

20 

# 5.3 Sample Preparation/Extraction

For the frozen fish tissue homogenates received for this study, each was thawed ~30 minutes

and then removed from its sample bag and re-homogen ized with dry ice using a Robot-Coupe

model RSI 2Y-1 stainless steel tissue homogenizer, then transferred back into the same

sample baggie and left overnight in a freezer to al low the dry ice to dissipate. For preparative

extraction of each analytical sample, a nominal 0.5 gram aliquot of re-homogenized fish tissue

sample was accurately weighed into a centrifuge tub e. Each fish sample was typically

aliquoted in duplicate with one being the primary a nalytical sample and the second being a

duplicate analytical sample. A stable isotope lab eled PFC internal standard (IS) and stable

isotope labeled surrogate recovery standard (SRS) m ixture was added to each sample aliquot,

after which 5.0 mL of acetonitrile was added. For LMSs, a target analyte mixture was also

added. The sample was mixed and then placed into a freezer for greater than 1 hr to facilitate

precipitation of proteins and fats. Samples were th en centrifuged at greater than 10,000 x g to

pellet the solids. Then, a 1.00 mL aliquot of supe rnatant extract was transferred to an

autosampler vial with 0.010 mL formic acid to make the final analyzed extract.

Typically, approximately twenty fish samples were p repared and extracted, each sample in

duplicate, and analyzed as part of each analytical batch. Each analytical batch was also

prepared with the following QC elements: (1) each sample and LMS aliquot received two

SRSs ( 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS) and ISs prior to extraction, for evaluation of overall data

precision for PFC carboxylates and PFC sulfonates/s ulfonamides, respectively; (2) for

approximately every tenth fish sample extracted, tw o additional aliquots were prepared as

LMSs and fortified with known quantities of target analytes, ISs and SRSs; (3) a set of sixteen

solvent calibration standards were prepared in acet onitrile; (4) a set of LCSs was prepared by

fortifying known quantities of target analytes, ISs and SRSs into a control bluegill fillet tissue

homogenate at three levels, each level in triplicat e; (5) matrix blanks (method blanks) were

nominal 0.5 g of control bluegill fillet matrix and prepared with and without IS and SRS and

used to evaluate endogenous levels of analyte in th e LCS matrix; and (6) solvent blanks were

prepared with the acetonitrile used to make calibra tion standards and prepared with and without

ISs and SRSs.



# 5.4 LC/MS/MS Analysis

Analyses were performed following a previously vali dated and published LC/MS/MS method

[Malinsky et. al. 2011, Analytica Chemica Acta 683 :pp248-257], described in 3M Environmental

Laboratory method ETS-8-045.1. The relative percen t difference (RPD) of the duplicate

analytical sample results was used as a measure of the sample-specific analytical precision.

The determined recoveries of fortified target analy tes from LMS samples were used as a

measure of sample-specific and sample matrix analyt ical accuracy. Recovery of the SRSs ( 13 

# C 4 

# - PFOS and 13 

# C 4 

# -PFOA) from each analytical sample was additionally used as a secondary

measure of the target analyte accuracy for each sam ple.

Details of the specific instrument parameters, the liquid chromatography gradient program, and

the specific mass transitions analyzed are detailed in the raw data, and are briefly described

below in Table 6

, Table 7

, Table 8 and Table 9 . Additionally, the analytical batches conducted

for collection of LC/MS/MS data associated with thi s study are listed in Table10

.

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

21 Table 6. LC/MS/MS Instrument Information (ETS-8-045 )

Instrument Name(s) Beethoven (Be), Ginger (g), Jona s (j) and MaryAnn (m),

Analytical Method ID ETS-8-045.1

Liquid Chromatograph Agilent 1200 or 1290 HPLC Syst em

Guard column (instrument cleanup) Placed between Purge Valve and Autosampler; Prism R P

(2.1 x 50 mm, 5 μ m)

Injection Volume 20 μ L , 40 μ L or 50 μ L

Mass Spectrometer API 5000 Triple Quadrapole or API 4500 Triple Quad.

Ion Source TurboIon Spray (Electrospray)

Polarity Negative Ion

Software Analyst 1.6.1

C 7 

# and C 8 

# PFCAs, PFSAs and SRSs

Extraction column Waters Corp. Oasis HLB Online Column (3 x 20 mm,

25 μ m); 30°C

Analytical column Betasil C18 (2.1 x 100 mm, 5 μ m); 30°C

Column Switching Valve 0 to 5 min (waste); 5 minutes to 21 minutes to anal ytical

column

C 4 

# and C 6 

# PFCAs

Analytical column Prism RP (2.1 x 50 mm, 5 μ m); 30°C

Table 7. Gradient Liquid Chromatography Conditions (ETS-8-045); C 7 

# -C 12 

# PFCAs, PFSAs and SRSs

Step

Number Total Time

[min] Flow Rate

[mL/min] Percent Mobile Phase A

[2 mm Aqueous Ammonium

Acetate] Percent Mobile Phase B

[ Acetonitrile]

0 0.00 0.400 97.0 3.0

1 3.00 0.400 97.0 3.0

2 3.50 0.400 70.0 30.0

3 13.5 0.400 40.0 60.0

4 15.5 0.400 40.0 60.0

5 16.0 0.400 10.0 90.0

6 18.0 0.400 10.0 90.0

7 18.3 0.400 97.0 3.0

8 21.0 0.400 97.0 3.0

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

22 Table 8. Gradient Liquid Chromatography Conditions (ETS-8-045); for C 4 

# -C 6 

# PFCAs

Step

Number Total Time

[min] Flow Rate

[mL/min] Percent Mobile Phase A

[5 mm Aqueous Ammonium Acetate

with 0.1% Acetic Acid] Percent Mobile Phase B

[ Methanol]



# 0 0.00 0.300 90.0 10.0

1 3.00 0.300 90.0 10.0

2 3.50 0.300 30.0 70.0

3 9.00 0.300 5.0 95.0

4 15.0 0.300 5.0 95.0

5 15.1 0.300 90.0 10.0

6 19.0 0.300 90.0 10.0

Table 9. Analytes, Surrogates, Internal Standards, and Transitions

Compound Analyte Description MRM Transition(s)

PFBA (C4 Acid) Target 213>169

PFHxA (C6 Acid) Target 313>269

313>119

PFHpA (C7 Acid) Target 363>319

363>169

PFOA (C8 Acid) Target 413>369

413>219

413>169

PFBS (C4 Sulfonate) Target 299>80

299>99

PFHS (C6 Sulfonate) Target 399>80

399>99

PFOS (C8 Sulfonate) Target 499>80

499>99

499>130

FBSA Target 298>78

FOSA Target 498>78

MeFOSAA Target 570>83

570>169

570>219

EtFOSAA Target 584>83

584>169

584>219

Internal Standards (IS)

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

23 Table 9. Analytes, Surrogates, Internal Standards, and Transitions

Compound Analyte Description MRM Transition(s)

[1,2,3,4 - 13 

# C]-PFBA IS for PFBA 217>172

[1,2 - 13 

# C 2 

# ]-PFHxA IS for PFHxA and PFHpA 315>270

[1,2,3,4,5,6,7,8- 13 

# C 8 

# ]-PFOA IS for PFOA and [1,2,3,4- 13 

# C 4 

# ]-PFOA 421>376

[ 18 

# O 2 

# ]-PFBS IS for PFBS and FBSA 303>84

[ 13 

# C 3 

# ]-PFHS IS for PFHS 402>80

[1,2,3,4,5,6,7,8- 13 

# C 8 

# ]-PFOS IS for PFOS and [1,2,3,4- 13 

# C 4 

# ]-PFOS 507>80

[1,2,3,4,5,6,7,8- 13 

# C 8 

# ]-FOSA IS for FOSA 506>78

d 3 

# -MeFOSAA IS for MeFOSAA 573>83

573>169

573>219

d 5 

# -EtFOSAA IS for Et FOSAA 589>83

589>169

589>219

Surrogate Recovery Standards (SRS)

[1,2,3,4- 13 

# C 4 

# ]-PFOA Surrogate for PFCAs 417>372

[1,2,3,4- 13 

# C 4 

# ]-PFOS Surrogate for PFSAs, EtFOSAA,

MeFOSAA, FOSA & FBSA 503>80

Multiple MS/MS ion transitions are summed when more than one transition is listed for the analyte.

Table 10. Analytical Batches

Analytical

Batch # Analytical

Batch ID Analytes Reported [a] 

# Samples Analyzed

1 j121206a PFBA, PFHxA ISO12-01-01-04-001

2 j130118a PFBA, PFHxA ISO12-01-01-04-002 to -022

3 j130121a PFBA, PFHxA ISO12-01-01-04-023 to -043

4 j130123a PFBA, PFHxA ISO12-01-01-04-044 to -064

5 g130124a PFBA, PFHxA ISO12-01-01-04-065 to -085

6 j130125a PFBA, PFHxA ISO12-01-01-04-086 to -108

7 j130128a PFBA, PFHxA ISO12-01-01-04-109 to -129

8 g130128a PFBA, PFHxA ISO12-01-01-04-130 to -150

9 m130204a PFBA, PFHxA ISO12-01-01-04-151 to -171

10 g130204a PFBA, PFHxA ISO12-01-01-04-172 to -185

11 m121206a PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-001

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

24 Table 10. Analytical Batches

Analytical

Batch # Analytical

Batch ID Analytes Reported [a] 

# Samples Analyzed

12 m130122b PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-002 to -022

13 m130129b PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-023 to -043

14 Be130121b PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-044 to -064

15 Be130123b PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-065 to -085

16 Be130128a PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-086 to -108

17 Be130130a PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-109 to -129

18 j130201a PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-130 to -150

19 g130130b PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-151 to -171

20 g130201a PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA,

MeFOSAA, EtFOSAA, 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS ISO12-01-01-04-172 to -185

[a] Each extract was analyzed in one analytical run for short chain acids (PFBA and PFHxA) and then re -analyzed in a

second analytical run for PFHpA, PFOA, PFBS, PFHS, PFOS, FBSA, FOSA, MeFOSAA and EtFOSAA



# 6 Results

All reported concentration results and recovery val ues are rounded to three significant figures.

Statistical values (RSDs, RPDs, etc.) are rounded t o two significant figures. All results were

calculated using full precision raw data during dat a calculations in Microsoft ExcelTM, and as a result

some values may vary slightly when the rounded valu es reported herein are carried through the

same calculations.



# 6.1 Calibration

Calibration standards were prepared by spiking vary ing known quantities of target analytes,

SRSs and ISs into acetonitrile. A set of 16 aceton itrile solvent calibration standards were

prepared with each analytical batch, ranging from n ominal 0.00250 ng/mL to 250 ng/mL for

target analytes and SRSs; equivalent to nominal 0.0 250 ng/g to 2500 ng/g in fish tissue after

adjustment for dilution during extraction. The sa me amount of ISs were added as were added

to each fish sample during extraction. Following t he analysis of calibration standards, the

nominal concentration of each standard was plotted versus the measured analyte peak area/IS

peak area response ratio. A quadratic equation wit h 1/x weighting was used to fit the calibration

response data. Calibration curves were not forced through zero. Calculating the determined

standard concentration using the resultant calibrat ion curve and comparing to the known

concentration confirmed accuracy of each curve poin t within 100 ± 25% (+ 30% at the LLOQ).

The correlation coefficient (r) was greater than 0. 999, and coefficient of determination (r 2 

# ) was

greater than 0.990. High and/or low calibration sta ndards were excluded as needed to meet

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

25 LLOQ requirements and to focus quantitation at high end or low end of the curve, as needed

based on sample concentrations in the batch.

For PFOS, the calibration standards were prepared w ith brPFOS, which is composed of a well

characterized isomer mixture composed of 78.8% (w/w ) linear PFOS isomer and 21.2% (w/w)

of branched isomers. Because fish samples showed a predominance of linear isomer, PFOS

quantitation was based on the chromatographically d istinct L-PFOS peak in calibration

standards and those results used to construct a L-P FOS calibration curve. Samples, LMSs

and LCSs were quantified by integration of total PF OS (linear + branched isomers) using the LPFOS calibration. For LCSs, both L-PFOS and total- PFOS (branched + linear isomers)

recoveries were determined and are reported in Table 11 of section 6.5.



# 6.2 Limits of Quantitation (LOQs)

The lower limit of quantitation (LLOQ) was typical ly the lowest non-zero calibration standard in

the curve that met linearity and accuracy requireme nts (100 + 30%) and for which the peak

area counts were at least 2X that of the average an alyte peak area counts determined from the

acetonitrile solvent blanks. The LLOQ for all anal ytes in this study was the typically nominal

0.250 ng/g fish concentration, equivalent to the 0. 0250 ng/mL standard, with some exceptions

as noted in the raw data. Results in Table 1 indicate the nominal LLOQ values when results

were below the LLOQ. The ULOQ was typically the n ominal 250 ng/mL standard (equivalent

to 2500 ng/g in fish tissue), with some exceptions as noted in the raw data.



# 6.3 Continuing Calibration

During the course of analysis of each analytical ba tch, continuing calibration verifications

(CCVs) were performed by regular injection of mid-l evel calibration standards throughout the

analysis. The back calculated concentration result s of CCVs were used to verify that the

instrument response and the initial calibration cur ve were still in control. All CCVs for all

analytes were within 100+ 25% and met method criteria, with the following ex ceptions:

PFHpA:m130122b052 (993%), m130122b098 (NA, outside of curve range), m130122b110

(497%), m130122b122 (72.1%), m130122b128 (NA, outsi de of curve range).

PFOA: m130122b052 (559%), m130122b110 (383%), m1301 22b122 (134%) and

m130122b128 (NA, outside of curve range).

PFBS: m130122b052 (425%), m130122b110 (126%), m1301 22b122 (2.02%) and

m130131a005 (130%).

PFHS:m130122b052 (NA, outside of curve range) and m 130122b128 (57.6%)

PFOSm130122b052 (529%), m130122b110 (4050%), j13020 1a074 (74.3%) and j130201a112

(71.4%)

FBSA: m130122b052 (402%), m130122b110 (304%), m1301 22b128 (61.6%), m130129b052

(70.5%), m130131a005 (70.5%), j130201a041 (134%), j 130201a074 (128%) and j130201a112

(126%).

FOSA: m130122b052 (392%) and m130122b110 (31.7%).

MeFOSAA: m130122b052 (160%) and m130122b110 (48.5%)

EtFOSAA: m130122b052 (38.8%) and m130122b110 (18.5% )

13 

# C 4 

# -PFOA (SRS): m130122b052 (32.3%), m130122b098 (4.06 %), m130122b110 (41.6%)

and m130122b128 (11.4%)

13 

# C 4 

# -PFOS (SRS): m130122b052 (16.2%), m130122b098 (4.06 %), m130122b110 (37.0%)

and m130122b128 (4.07%)

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

26 When CCVs failed to meet criteria, the analytical r un was evaluated more closely for LMS

recoveries and LCS results, and a potential cause o f CCV recovery failures was evaluated and

documented with the batch paperwork. For example, in analytical run m130122b the standard

solution used for several CCV injections ran low an d were not effectively injected, resulting in

CCV values well outside of the 100 + 25% criterion. In all instances, LMS results and LCS

results were additionally evaluated to provide just ification for reporting the results from those

batches. All CCV failures were documented into a m ethod deviation and included in the raw

data.



# 6.4 Blanks

Three types of blanks were analyzed with fish sampl es: (1) acetonitrile solvent blanks were

typically analyzed before or after calibration stan dards and CCVs for evaluating and abating any

potential carry over and at times are used in conju nction with method blanks to help assign

LLOQs; (2) fish matrix blanks were prepared with no minal 0.5 g of a control bluegill fillet

homogenate TN11-0194-1/1 and were spiked with and w ithout ISs and SRSs and used to

provide endogenous background levels for the fish m atrix used to prepare LCSs; and (3)

process control blanks used to evaluate the homogen ization process at TestAmerica (control

sunfish tissue TN11-0194-1/1) and the process contr ols for the field filleting process

(Bass,TN12-0365-1/13, -2/13 and -3/13 and Catfish T N12-0365-1/13, -2/13 and -3/13).

The fillet process controls were 3 largemouth whole bass (TN12-0364-1/13, -2/13 and -3/13)

and 3 whole catfish (TN12-0365-1/13, -2/13 and -3/1 3) for which the right side fillets were

removed at the 3M Environmental Laboratory, homogen ized and analyzed prior to fish sample

receipt for the study. Those fish, with right side fillets removed, were sent to the field and left

side fillets removed in the field during fish fille ting process. Those process control samples

were returned blinded to the laboratory regarding t heir true identities. Homogenization process

controls were returned and were identifiable to the laboratory based on identities placed on

them by TestAmerica. Final results and fish identi ties for process controls will be verified in the

Weston Solutions, Inc. final study report.

All blank results were carefully evaluated for issu es such as background instrument

interferences, analytical carry over during the ana lysis to provide a background level with which

to use for assigning LLOQs, and to determine endoge nous values of each analyte in the fish

matrix used for preparation of LCSs. All of the bl ank sample results were considered

acceptable upon evaluation of the raw data based on the intended use of the different types of

blanks.

The sunfish control matrix TN11-0194-1/1 was the ma trix used for LCS preparations, and was

the same matrix used for the independent evaluation of the homogenization process

(homogenization process controls) at TestAmerica. That matrix has low measurable levels of

endogenous total PFOS, and other analytes, and thos e values subtracted from LCS results

during recovery calculations. The results of all s amples, including homogenization and filleting

process controls are included in Table 1

.

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

27 

# 6.5 Laboratory Control Samples (LCSs)

Laboratory control samples (LCSs) were prepared for each analytical batch by fortifying known

quantities of the target analytes, ISs and SRSs int o accurately weighed (nominal 0.5 gram)

aliquots of a control bluegill fillet tissue prior to extraction then carrying the LCS through the

extraction and analysis procedure. The LCS sets we re prepared at three levels of nominal 1.0,

10 and 100 ng/g (ww), and each level was prepared i n triplicate. The PFOS used to fortify

LCSs was brPFOS purchased from Wellington Laborator ies and with a known 78.8% (w/w)

linear isomer composition and 22.2 %(w/w) branched isomer composition. Because calibration

was performed using only the chromatographically se parated L-PFOS isomer peak, both LPFOS and total-PFOS recoveries from LCSs were deter mined. Total-PFOS was always

determined from samples, LMS s and matrix blanks.

The LCS results were used to determine the analytic al method accuracy (average recovery)

and precision (RSD). The LCS accuracy and precisio n results, and the analytical method

uncertainties at the 95% confidence interval (95% C I; 2-times standard deviation), determined

from the statistical evaluation of LCS results are shown in Table 11 . The method acceptance

criteria of 100 ± 30% was typically met, with some exceptions as noted in the Table. The

average low-LCS, mid-LCSs and high-LCSs recoveries over 10 analytical batches, and overall

for all LCSs levels, was within 100 + 14% for all analytes and SRSs.

The LCS recoveries were calculated using the follow ing equation:



# 100% *

ion Concentrat Spike ) Conc. Matrix Endogenous

- LCS of ion Concentrat d (Determine Recovery LCS

=ISO12-01-01-04Tennessee River Fish StudyFall 201228Table 11. LCS Results

**Analytical Batch ID** **LCS ID Analyte Recovery (%)** **PFBA PFHx** **A PFHpA PFOA PFBS PFHS** **Total- PFOS** **[b] L-PFOS** **[b] FBSA FOSA MeFOSAA EtFOSAA** **13 C** **4 -PFOA** **13 C** **4 -PFOS** 

j121206a (PFBA, PFPHxA) and m121206aLCS Low-1115% 108% 84.4% 74.7% 108% 106% 118% 105% 123% 94.6% 119% 115%112% 109%LCS Low-2101% 102% 69.9% 63.9% 99.2% 97.1% 111% 96.6% 117% 90.6% 102% 100%100% 101%LCS Low-3105% 100% 72.5% 66.3% 104% 98.9% 117% 105% 120% 91.1% 103% 101%102% 101%LCS Mid-1111% 110% 105% 110% 109% 105% 114% 111% 133% 93.4%110% 104%106% 104%LCS Mid-2111% 109% 107% 115% 110% 105% 116% 112% 135% 94.7%110% 108%110% 106%LCS Mid-3106% 108% 106% 111% 107% 102% 113% 110% 132% 91.3%103% 104%108% 104%LCS High-1109% 108% 87.5% 102% NA 103% 115% 108% 126% 96.1% 108% 107%104% 102%LCS High-2103% 104% 89.6% 100% NA 98.6% 110% 104% 117% 91.2%105% 103%104% 101%LCS High-3108% 106% 88.7% 101% NA 101% 112% 106% 123% 95.7% 108% 104%102% 100%

j130118a (PFBA, PFPHxA) and m130122bLCS Low-1106% 96.5% 126% 109% 104% 99.3% 110% 108% 90.3% 90.6% 98.4% 92.1%87.4% 88.6%LCS Low-2107% 96.5% 125% 116% 104% 102% 117% 115% 91.8% 95.8% 103% 97.6%89.6% 90.8%LCS Low-3103% 93.8% 119% 110% 98.6% 96.8% 110% 108% 87.6% 89.5% 98.7% 92.7%86.9% 88.2%LCS Mid-1105% 104% 106% 104% 97.9% 99.0% 111% 105% 78.1% 96.2% 99.7% 91.8%85.5% 89.6%LCS Mid-2111% 105% 112% 106% 101% 102% 116% 111% 80.3% 104%108% 100%87.2% 88.4%LCS Mid-3103% 98.8% 105% 101% 96.8% 96.2% 110% 105% 76.6% 99.2% 99.0% 93.8%87.7% 93.3%LCS High-1NA NA NA NA 83.6% 85.9% NA 67.5%[a]67.0% NA 87.1% 83.3%83.3% 88.0%LCS High-2NA NA NA NA 70.0% 78.0% NA 67.7%[a]51.7% NA 86.1% 79.0%82.8% 88.9%LCS High-3NA NA NA NA 66.8%[a]79.1% NA 69.3%[a]49.1% NA 89.1% 84.2%87.2% 92.7%ISO12-01-01-04Tennessee River Fish StudyFall 201229Table 11. LCS Results

**Analytical Batch ID** **LCS ID Analyte Recovery (%)** **PFBA PFHx** **A PFHpA PFOA PFBS PFHS** **Total- PFOS** **[b] L-PFOS** **[b] FBSA FOSA MeFOSAA EtFOSAA** **13 C** **4 -PFOA** **13 C** **4 -PFOS** 

j130121a (PFBA, PFPHxA) and m130129bLCS Low-1106% 97.5% 92.3% 106% 105% 85.0% 101% 99.2% 75.4% 93.4% 99.3% 93.4%86.8% 89.8%LCS Low-2102% 94.7% 87.9% 104% 101% 81.2% 95.6% 94.0% 72.5%90.2% 93.9% 90.8%84.2% 82.9%LCS Low-3105% 97.4% 92.5% 107% 104% 83.9% 102% 99.8% 71.3% 93.6% 103% 92.2%87.3% 89.2%LCS Mid-1105% 101% 84.3% 104% 107% 90.2% 100% 97.4%68.8%[a]95.9% 97.6% 90.1%87.9% 85.7%LCS Mid-2103% 100% 82.7% 99.7% 107% 90.2% 97.4% 94.3%67.4%[a]94.8% 95.5% 86.3%86.5% 86.0%LCS Mid-3101% 99.8% 84.9% 98.8% 104% 87.3% 97.8% 94.9%68.4%[a]95.0% 96.8% 87.4%105% 104%LCS High-1NA NA NA 100% 90.3% 80.7% 115% 76.0% 75.4% NA 89.5%82.0%84.7% 89.7%LCS High-2NA NA NA 98.1% 94.7% 71.6% 104% 70.6%61.6%[a]NA 86.1% 80.4%86.0% 88.3%LCS High-3NA NA NA 97.5% 89.7% 69.4%[a]103% 69.2%[a]51.1%[a]NA 84.2% 81.8%86.4% 86.5%

j130123a (PFBA, PFPHxA) and Be130121bLCS Low-1105% 96.0% 82.3% 95.1% 87.3% 93.6% 89.6% 89.2% 128%104% 97.0% 88.6%86.4% 88.0%LCS Low-2105% 95.4% 88.4% 93.6% 90.4% 89.2% 87.1% 86.8% 134%[a]106% 98.6% 93.7%87.8% 90.5%LCS Low-3103% 95.4% 84.4% 95.3% 91.2% 91.4% 88.0% 88.0% 129%107% 99.6% 89.9%85.6% 84.1%LCS Mid-1109% 107% 97.7% 101% 93.8% 103% 98.1% 98.1% 119% 108% 101% 97.2%92.8% 97.5%LCS Mid-2109% 104% 95.9% 97.1% 96.7% 102% 94.3% 94.2% 119% 113% 101% 92.7%86.9% 89.3%LCS Mid-398.2% 94.5% 83.5% 88.5% 85.2% 91.4% 84.9% 85.3% 110% 104% 93.5% 85.5%81.7% 85.8%LCS High-1NA NA 86.1% 83.9% 87.6% 83.7% 97.6% 90.0%65.7%[a]86.5% 90.3% 79.5%85.1% 89.1%LCS High-2NA NA 83.2% 79.3% 87.4% 81.3% 97.6% 89.0%66.6%[a]94.0% 82.8% 78.2%80.8% 87.5%LCS High-3NA NA 87.7% 83.8% 90.5% 83.7% 101% 92.3%67.6%[a]93.6% 88.8% 78.8%87.0% 91.7%ISO12-01-01-04Tennessee River Fish StudyFall 201230Table 11. LCS Results

**Analytical Batch ID** **LCS ID Analyte Recovery (%)** **PFBA PFHx** **A PFHpA PFOA PFBS PFHS** **Total- PFOS** **[b] L-PFOS** **[b] FBSA FOSA MeFOSAA EtFOSAA** **13 C** **4 -PFOA** **13 C** **4 -PFOS** 

g130124a (PFBA, PFPHxA) and Be130123bLCS Low-1122% 104% 89.7% 95.0% 93.4% 106% 95.2% 94.8% 105% 116% 109% 87.2%85.3% 91.1%LCS Low-2116% 100% 87.6% 93.5% 91.2% 104% 94.6% 94.1% 102% 113% 103% 85.1%88.8% 91.8%LCS Low-3117% 96.8% 86.5% 95.2% 93.7% 103% 94.8% 93.9% 104%108% 101% 88.1%85.7% 88.4%LCS Mid-1106% 89.5% 84.3% 101% 83.5% 87.0% 84.5% 83.9% 94.0%97.7% 91.3% 78.8%81.6% 80.5%LCS Mid-2118% 90.8% 87.8% 97.1% 85.7% 96.4% 94.3% 93.5% 97.2% 110% 101% 83.3%93.5% 101%LCS Mid-3116% 91.1% 90.6% 88.5% 88.1% 95.2% 92.1% 92.5% 101%101% 99.0% 84.1%90.2% 97.5%LCS High-1NA NA 87.0% 83.9% 92.3% 69.2%[a]105% 93.8% 91.2% 84.2% 89.6% 84.9%84.4% 90.3%LCS High-2NA NA 85.7% 79.3% 84.4% 68.5%[a]102% 91.6% 87.4% 82.8% 82.9% 81.5%83.0% 92.5%LCS High-3NA NA 86.0% 83.9% 87.0% 68.1%[a]103% 91.6% 85.9% 85.0% 84.8% 82.4%81.4% 86.8%

j130125a(PFBA, PFPHxA) and Be130128aLCS Low-1110% 100% 89.7% 96.0% 94.7% 97.4% 101% 99.4% 100% 98.1% 94.7% 93.0%84.3% 94.0%LCS Low-2105% 97.1% 91.4% 92.1% 91.4% 92.0% 91.7% 90.8% 99.5% 98.1% 96.1% 87.0%80.9% 85.3%LCS Low-3107% 99.6% 89.0% 96.7% 96.3% 96.5% 93.7% 92.6% 105%103% 97.7% 87.7%88.1% 88.8%LCS Mid-198.6% 95.4% 85.5% 92.1% 90.5% 89.2% 87.5% 86.0% 94.4% 104% 93.7% 83.2%86.2% 88.0%LCS Mid-297.2% 94.3% 80.8% 91.1% 89.5% 87.4% 89.2% 88.2% 91.1% 99.3% 96.5% 82.2%77.8% 84.4%LCS Mid-388.8% 85.8% 75.1% 82.1% 80.9% 81.1% 78.9% 78.0% 84.9% 87.2% 80.4% 76.5%74.9% 79.1%LCS High-1NA NA 86.9% 85.9% 85.4% 84.1% 105% 92.2% 80.2% 108%87.7% 81.7%85.0% 88.9%LCS High-2NA NA 85.2% 83.5% 81.8% 80.1% 102% 90.7% 79.0% 95.8% 82.4% 81.2%77.8% 85.7%LCS High-3NA NA 82.8% 84.1% 83.2% 79.1% 101% 89.2% 76.8% 90.6% 86.3% 77.9%85.6% 91.5%ISO12-01-01-04Tennessee River Fish StudyFall 201231Table 11. LCS Results

**Analytical Batch ID** **LCS ID Analyte Recovery (%)** **PFBA PFHx** **A PFHpA PFOA PFBS PFHS** **Total- PFOS** **[b] L-PFOS** **[b] FBSA FOSA MeFOSAA EtFOSAA** **13 C** **4 -PFOA** **13 C** **4 -PFOS** 

j130128a (PFBA, PFPHxA) and Be130130aLCS Low-196.7% 87.9% 82.1% 90.3% 87.2% 85.2% 86.3% 85.4% 93.3% 114% 93.1% 84.2%80.4% 86.4%LCS Low-2104% 95.1% 89.6% 97.6% 95.2% 91.3% 91.3% 91.3% 96.6% 117% 96.0% 93.6%85.2% 86.5%LCS Low-397.7% 90.0% 84.9% 92.4% 87.0% 88.3% 90.6% 89.4% 96.8% 97.3% 100% 87.7%85.1% 86.3%LCS Mid-198.7% 93.7% 82.0% 87.8% 85.9% 82.2% 85.8% 86.1% 87.8% 99.7% 89.6% 82.6%88.1% 91.0%LCS Mid-2NA NA 84.8% 93.1% 88.6% 88.3% 93.5% 93.3% 93.2% 102% 96.9% 87.7%82.8% 88.6%LCS Mid-3NA NA 74.0% 80.9% 78.9% 78.4% 80.7% 80.1% 80.4% 95.2% 83.5% 79.2%74.9% 79.3%LCS High-1NA NA 78.7% 83.2% 79.6% 80.4% 94.9% 83.8% 76.8% 88.4% 84.8% 77.8%76.4% 79.6%LCS High-2NA NA 84.5% 90.7% 87.3% 84.7% 101% 89.3% 81.2% 105%85.2% 83.4%84.7% 86.3%LCS High-3NA NA 83.9% 89.7% 85.1% 85.7% 101% 90.0% 79.5% 86.4% 88.4% 80.1%83.7% 88.5%

g130128a (PFBA, PFPHxA) and j130201aLCS Low-1101% 80.2% 88.8% 92.0% 89.6% 89.3% 97.7% 89.6% 125%88.8% 87.1% 81.3%80.3% 81.3%LCS Low-2102% 92.6% 87.7% 91.3% 86.5% 85.4% 91.2% 82.9% 116%84.1% 85.6% 78.2%75.7% 74.2%LCS Low-396.2% 83.2% 82.9% 91.9% 81.0% 85.1% 94.1% 85.7% 106% 84.8% 87.4% 78.1%81.8% 79.6%LCS Mid-196.6% 83.8% 79.5% 83.4% 76.6% 78.5% 98.1% 89.2% 86.8% 81.3% 81.2% 75.0%80.5% 81.2%LCS Mid-2120% 98.4% 103% 102% 92.1% 94.4% 116% 106% 100% 96.7% 94.4% 92.7%88.5% 91.1%LCS Mid-3100% 86.7% 84.3% 87.3% 80.0% 82.2% 96.8% 87.9% 93.8% 83.3% 81.6% 77.6%77.9% 75.6%LCS High-1110% 87.9% NA NA NA NA 130% 75.6% NA 75.1% 77.9% 76.4%83.6% 83.1%LCS High-2111% 93.0% NA NA NA NA 148% 79.0% NA 77.7% 76.9% 76.1%80.6% 86.5%LCS High-3105% 85.6% NA NA NA NA 105% 67.4% NA 71.0% 74.3% 72.3%74.8% 78.8%ISO12-01-01-04Tennessee River Fish StudyFall 201232Table 11. LCS Results

**Analytical Batch ID** **LCS ID Analyte Recovery (%)** **PFBA PFHx** **A PFHpA PFOA PFBS PFHS** **Total- PFOS** **[b] L-PFOS** **[b] FBSA FOSA MeFOSAA EtFOSAA** **13 C** **4 -PFOA** **13 C** **4 -PFOS** 

m130204a (PFBA, PFPHxA) and j130201aLCS Low-187.5% 84.1% 85.0% 90.8% 83.6% 85.9% 102% 101% 80.5%84.8% 90.8% 80.4%77.5% 81.5%LCS Low-288.1% 87.2% 84.7% 95.1% 91.2% 85.7% 107% 104% 82.2%81.9% 95.8% 79.9%88.7% 94.7%LCS Low-396.4% 93.6% 95.9% 99.5% 96.5% 93.5% 112% 108% 99.7%94.5% 100% 85.8%92.9% 93.1%LCS Mid-189.4% 87.1% 89.8% 98.6% 86.2% 86.1% 99.4% 95.4% 93.8% 90.6% 82.3% 81.3%90.1% 85.3%LCS Mid-295.2% 91.4% 89.8% 99.9% 99.0% 94.5% 105% 101% 103%93.7% 92.3% 82.6%98.3% 104%LCS Mid-395.2% 92.4% 94.4% 100% 88.5% 94.2% 105% 100% 93.5%94.4% 89.5% 86.8%88.8% 93.7%LCS High-180.9% 93.4% 75.9% 78.0% NA 73.5% 100% 75.9% 85.5% 92.5% 74.5% 65.3%[a]71.6% 76.9%LCS High-281.4% NA 80.9% 76.8% NA 71.3% 88.7% 70.0% 85.6% 89.6% 75.9% 63.2%[a]70.5% 76.1%LCS High-388.1% NA NA 93.4% NA 88.5% 104% 80.8% 103% 96.6% 78.1% 73.5%78.7% 83.8%

g130204a (PFBA, PFPHxA) and g130201aLCS Low-1110% 95.7% 90.6% 93.0% 92.7% 96.3% 101% 96.8% 93.4%90.4% 91.0% 90.2%78.6% 88.6%LCS Low-2120% 94.8% 93.0% 104% 100% 100% 104% 99.5% 99.5% 94.8% 103% 92.9%92.1% 87.4%LCS Low-3110% 93.6% 93.1% 102% 96.7% 95.1% 98.4% 92.5% 105%94.1% 96.7% 90.9%90.0% 87.9%LCS Mid-1113% 89.4% 94.6% 99.1% 90.9% 94.7% 97.8% 93.1% 95.5% 94.2% 92.5% 88.1%80.7% 81.5%LCS Mid-299.1% 78.8% 85.0% 83.8% 83.8% 81.6% 85.9% 81.9% 88.2% 81.4% 79.0% 74.9%77.5% 80.0%LCS Mid-3110% 83.4% 91.5% 98.0% 93.0% 96.6% 99.9% 95.2% 104%93.9% 93.5% 89.1%83.0% 83.6%LCS High-1104% NA 87.2% 87.1% NA 82.8% 107% 77.8% 85% 109% 86.0% 71.0%81.7% 86.2%LCS High-2124% NA NA 100% NA 93.4% 115% 86.0% 103% 126% 94.3%80.3%86.8% 95.1%LCS High-3112% NA NA 90.3% NA 84.4% 98.3% 75.6% 99.80% 118% 81.4% 79.4%85.7% 86.4%ISO12-01-01-04Tennessee River Fish StudyFall 201233Table 11. LCS Results

**Analytical Batch ID** **LCS ID Analyte Recovery (%)** **PFBA PFHx** **A PFHpA PFOA PFBS PFHS** **Total- PFOS** **[b] L-PFOS** **[b] FBSA FOSA MeFOSAA EtFOSAA** **13 C** **4 -PFOA** **13 C** **4 -PFOS** Low LCS Average105% 95.0% 90.6% 95.0% 94.8% 93.5% 99.5% 96.2% 102%97.0% 98.1% 89.9%87.2% 89.0%Low-LCS Std. Dev.8.1% 6.0% 12% 11% 6.9% 7.1% 9.4% 7.9% 17% 9.8% 6.6%7.5%7.4% 6.7%Low LCS RSD7.7% 6.3% 14% 12% 7.3% 7.6% 9.4% 8.3% 17% 10% 6.8%8.4%8.4% 7.5%Low LCS (n)30 30 30 30 30 30 30 30 30 30 30 3030 30Mid-LCS Average104% 95.5% 90.9% 96.8% 92.5% 92.0% 98.1% 94.9% 95.7% 96.5% 94.5% 87.5%88.0% 89.9%Mid-LCS Std. Dev.8.1% 8.5% 10% 8.8% 9.5% 7.9% 11% 9.5% 18% 7.6% 8.5%8.7%9.4% 8.8%Mid-LCS RSD7.8% 8.9% 11% 9.1% 10% 8.6% 11% 10% 19% 7.8% 9.0% 9.9%11% 9.7%Mid-LCS RSD (n)28 28 30 30 30 30 30 30 30 30 30 3030 30High-LCS Average103% 96.8% 84.9% 89.0% 84.8% 81.8% 106% 83.7% 82.3%93.3% 86.5% 81.0%84.3% 88.3%High-LCS Std. Dev.13% 9.1% 3.5% 8.2% 7.1% 9.5% 12% 11.7% 20% 13% 8.5%9.5%7.8% 6.3%High LCS RSD13% 9.4% 4.2% 9.2% 8.3% 12% 11% 14% 25% 14% 9.8% 12%9.2% 7.1%High LCS (n)12 7 18 24 18 27 27 30 27 24 30 3030 30**Overall Study Average 104% 95.4% 89.4% 93.9% 91.6% 89.4% 101% 91.6% 93.6% 95.8% 93.0% 86.2% 86.5% 89.1** **% Standard Deviation 9.0% 7.4% 10% 10% 8.9% 9.6% 11%** **11% 20% 10% 9.2% 9.3% 8.3% 7.3% RSD 8.6% 7.8% 12% 11% 9.7% 11% 11% 12% 21% 11% 9.9** **% 11% 9.6% 8.2% n (Total Number of LCS** **Results) 70 65 78 84 78 87 87 90 87 84 90 90 90 90** **Analytical Method Uncertainty (95%CI)** **+ 18% +** **15% + 21% +** **20% + 18% +** **19% + 22% +** **23% + 40% +** **20% + 18% +** **19% + 17% +** **15%**[a] Recovery was outside of 100 +30%, but data used in summary statistics.[b] PFOS quantified with L-PFOS calibration, LCS recoveries determined for both L-PFOS and total-PFOS(linear + branched isomers); all QCs and samples quantified for total-PFOSNA; No value determined for the LCS either due to instrument error or value outside of the calibrationcurve range for that batch

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

34 

# 6.6 Laboratory Matrix Spikes (LMSs)

As additional quality control for the study, labora tory matrix spike (LMS) samples were prepared

for ~10% of the study samples. Approximately every 10 th 

# fish received in the series was

prepared with four replicate weighings. The first two replicates were prepared as analytical

sample and sample duplicate, the third replicate wa s fortified with nominal 5 ng/g (ww) of each

target analyte prior to extraction as a low-LMS and the fourth replicate fortified with nominal 250

ng/g (ww) of each target analyte as a high-LMS. Al l four replicates were then fortified with ISs,

SRSs and carried through the extraction and analysi s procedure. In total, 21 of the 175 fish

samples received in the study (12%) had a set of LM Ss prepared and analyzed. The LMS

recovery results are summarized in Table 2 . The PFOS fortified into LMSs was a mixture of

linear and branched isomers (br-PFOS from Wellingto n Laboratories), and were quantified for

total PFOS using L-PFOS calibration, as described e arlier.

For sample analytes PFBA, PFHpA, PFOA, PFBS and PFH S and most samples for EtFOSAA

and MeFOSAA, the results for the nominal 5 ng/g (ww ) low-LMS were appropriate for

evaluating recovery of the target analytes based on the low level of endogenous target analyte

concentration in the equivalently non-fortified sam ples (i.e. appropriate fortification is considered

that between 0.5-times to 20-times the endogenous l evel). For many of the LMS sample

results for PFOS and FBSA the high- LMS level was t he more appropriate fortification level

relative to the endogenous analyte level in the non -fortified samples for determining the LMS

recoveries.

The overall average LMS analyte recoveries for all target analytes was typically within 100 +

10%, except for FBSA which was 82.3%. The RSDs of LMS recoveries were also < 15%,

except FBSA which had an RSD of 27%. LMSs for FBSA in runs m130122b, m130129b,

be130121b and j130130a were outside of 100 + 30%, but within 100 + 50% and reported with

expanded uncertainties of + 50% for those affected results in Table 1

.

The LMS recoveries were calculated using the follow ing equation:



# 100% *

ion Concentrat Spike Sample) Field of ion Concentrat Determined LMS of ion Concentrat d (Determine Recovery LMS −

=



# 6.7 NIST SRM 1947 Results

As added QC for each analytical batch, two accurate ly weighed (nominal 0.5 gram) aliquots of

NIST standard reference material (SRM) 1947 were ex tracted and analyzed the same as

samples. The results of the analysis of the NIST S RM 1947 showed overall average +

standard deviation (n = 10 batches, 20 results) for PFOS at 5.19 + 0.58 ng/g (ww). This PFOS

result was in acceptable agreement with the NIST va lue and accurate to 88.0% + 10% with

respect to the NIST SRM 1947 reference value of 5.9 + 0.39 ng/g.



# 6.8 Fillet Equipment Aqueous Rinseate Results

As part of the filleting process, the equipment was rinsed and at times and the rinseates

collected for analysis. Ten aqueous equipment rins eate blanks were collected and shipped

with the fish samples for analysis of the target an alytes. Those samples were analyzed by 3M

Environmental Laboratory method ETS-8-044.1 and sho wed rinseates were generally less than

LLOQ except 1.4 ng/L PFOS measured in sample EBLK11 and PFBA measured at 5.69 and

5.09 ng/L in samples EBLK03 and EBLK005. The result s for the aqueous rinseates are

provided in Table 12 . Overall evaluation of rinseate blank results will be reserved for Weston

Solutions, Inc. in their final overall study report

.ISO12-01-01-04Tennessee River Fish StudyFall 201235Table 12.Concentration Results for Fillet-Process Equipment Rinseate BlanksSAMPLE IDLIMS IDConcentration (ng/L; parts per trillion)PFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAAEBLK01ISO12-01-01-04-019<5.00<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK02ISO12-01-01-04-036<5.00<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK03ISO12-01-01-04-054**5.69**<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK04ISO12-01-01-04-071<5.00<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK005ISO12-01-01-04-089**5.09**<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK006ISO12-01-01-04-107<5.00<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK07ISO12-01-01-04-125<5.00<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK08ISO12-01-01-04-143<5.00<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK09ISO12-01-01-04-161<5.00<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK10ISO12-01-01-04-179<5.00<2.00<2.00<4.80<5.00<5.00<0.927<2.00<2.00<9.98<15.0EBLK11ISO12-01-01-04-186<5.00<2.00<2.00<4.80<5.00<5.00**1.40**<2.00<2.00<9.98<15.0*Results of LCSs prepared in water during the analysis indicated an average +RSD accuracy for PFOS of 96.9% +3.5% and with an analytical method uncertainty (95% CI) of +17%; average +RSD accuracyfor PFBA was 100% +0.58% with an analytical method uncertainty (95% CI) of +16%.

# ISO12-01-01-04

Tennessee River Fish Study

Fall 2012

36 

# 7 Conclusion

The determined concentrations of perfluorinated che micals (PFCs) measured in fish collected

from the Tennessee River in the Fall of 2012 were r eported. These analyses were conducted

as 3M Environmental analytical project ISO12-01-01- 04 and were performed in support of the

Weston Solutions, Inc. study plan, titled “Revised Work Plant for Fall 2012 Fish and Surface

Water Sampling in the Tennessee River”, and dated O ctober 2012.

During the Fall of 2012, Weston Solutions, Inc. per sonnel caught five largemouth bass and five

catfish from each of fifteen pre-defined fish sampl ing reaches located within the confines of the

Wilson and Wheeler Reservoirs of the Tennessee Rive r, located near Decatur, AL. The fish

were filleted (skin removed) in the field. A total of 150 left side fillets and 15 randomly selected

right side fillets (as field duplicates) were remov ed and then shipped to TestAmerica

(Burlington, VT) for homogenization of the fillets. Frozen fillet homogenates were then shipped

on dry ice to the 3M Environmental Laboratory for e xtraction and quantitative analysis of PFCs.

An additional six process controls for evaluating t he filleting process, three process controls for

evaluating the homogenization process, and one NIST fish tissue standard reference material

(SRM 1947) were also received. A total of 175 fish tissue samples were received and

quantitatively analyzed. The PFCs analyzed for were perfluorobutanoate (PFBA),

perfluorohexanoate (PFHxA), perfluoroheptanoate (PF HpA), perfluorooctanoate (PFOA),

perfluorobutane sulfonate (PFBS), perfluorohexanesu lfonate (PFHS), perfluorooctanesulfonate

(PFOS) , perfluorobutanesulfonamide (FBSA), perfluo rooctanesulfonamide (FOSA),2-[Nmethyl-FOSA]acetic acid (MeFOSAA) and 2-[N-ethyl-FO SA]acetic acid (EtFOSAA).

Each sample was extracted in duplicate for determin ation of the average PFC concentrations,

and approximately every10th fish sample (21 in tota l; 12% of total fish) had two laboratory

matrix spike (LMS) samples prepared from a third an d fourth 0.5 gram aliquot and fortified with

known quantities of target analytes. The LMS recov ery results are shown in Table 2 and

average + standard deviation recovery results were within 100 + 10% for all analytes, with RSDs

less than 15%, except FBSA which had a recovery at 82.3% + 22% (RSD = 27%). Based on

the LMS recovery results, one PFBS result for sampl e MSF011 (ISO12-01-01-04-011) and

PFOA and PFBS results for fish MSF043 (ISO12-01-01- 04-043) were not reported (NR) due to

LMS recoveries outside of 100 + 50%.

Laboratory control spikes (LCSs) were prepared in c ontrol bluegill fillet tissue at 3 levels, each

level in triplicate, and analyzed with each analyti cal batch. Average LCS recoveries were within

100 + 14% at all LCS levels, and overall, for each analy te. The LCS statistical results (for all

LCS levels) were used to determine the analytical m ethod uncertainty (95% CI) for the dataset,

and were + 18% for PFBA, + 15% for PFHxA, + 21% for PFHpA, + 20% for PFOA, + 18% for

PFBS, + 19% for PFHS, + 22% for total-PFOS (linear + branched isomers), + 40% for FBSA,

+ 20% for FOSA, + 18% for MeFOSAA and + 19% for EtFOSAA.

The NIST standard reference material SRM 1947 (Lake Michigan fish tissue) was analyzed in

duplicate as part of each analytical batch. The ov erall average PFOS value + standard

deviation was determined at 5.19 + 0.58 ng/g (ww) over ten analytical batches (n = 20 results).

This result was accurate to 88.0% and in acceptable agreement with the SRM 1947 PFOS

reference value of 5.9 + 0.39 ng/g (ww).

Two stable-isotope labeled surrogate recovery stand ards (SRSs) 13 

# C 4 

# -PFOA and 13 

# C 4 

# -PFOS

were added to each sample aliquot prior to extracti on. Average SRS recoveries for all the fish

tissue samples analyzed (n = 175) were 85.3% for 13 

# C 4 

# -PFOA and 88.1% for 13 

# C 4 

# -PFOS. The

relative standard deviation (RSD) for the average S RS recoveries showed excellent method

precision with RSDs of 5.2% for 13 

# C 4 

# -PFOA and 3.8% for 13 

# C 4 

# -PFOS.

Overall, the sample results in Table 1 showed that PFOS and FBSA were detected with

highest frequency in Tennessee River fish samples a nd with the highest concentrations

measured. PFOS concentrations ranged from <0.366 n g/g (ww) to 599 ng/g (ww). FBSAISO12-01-01-04Tennessee River Fish StudyFall 201238Attachment APFC Results from Analysis of Bass & Catfish ControlMaterials Used for Fillet-Process ControlsTable A1.Concentration Results for Bass & Catfish Fillet Control Materials (Right Side Fillets)SAMPLE IDAverage Concentration (ng/g; wet wt.)PFBAPFHxAPFHpAPFOAPFBSPFHSPFOS*FBSAFOSAMeFOSAAEtFOSAATN12-0364-1/13-LM Bass-Right SideFillet<0.250<1.00<0.0500<0.100<0.250<0.1004.27<0.100<0.250<0.250<0.350TN12-0364-1/13-LM Bass-Right SideFillet<0.250<1.00<0.0500<0.100<0.250<0.1004.12<0.100<0.250<0.250<0.350TN12-0364-1/13-LM Bass-Right SideFillet<0.250<1.00<0.0500<0.100<0.250<0.1003.85<0.100<0.250<0.250<0.350TN12-0365-1/13-Catfish-Right SideFillet<0.250<1.00<0.0500<0.100<0.250<0.100<0.100<0.100<0.250<0.250<0.350TN12-0365-1/13-Catfish-Right SideFillet<0.250<1.00<0.0500<0.100<0.250<0.100<0.100<0.100<0.250<0.250<0.350TN12-0365-1/13-Catfish-Right SideFillet<0.250<1.00<0.0500<0.100<0.250<0.100<0.100<0.100<0.250<0.250<0.350[a] LMSs fortified with nominal 10ng/g of each target analytes were prepared with the samples and recoveries were all within 100 +20%LLOQs shown are nominal for a nominal 0.5 g tissuesample extracted and analyzedResults are shown for right side fillets only; removed at the laboratory and kept at the laboratory for analysis. Intact left side fillets attached to fish bodies sent to the field for fillet-process controls.ISO12-01-01-04Tennessee River Fish StudyFall 201239Attachment BPreparation Forms, Raw Data and Chromatograms(Available Upon Request)