3 

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

tests/calibrations listed in A2LA Certificate # 2052.01 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 Collected from the Tennessee 

River in the Fall of 2012

Study Title

Analytical Study ISO12-01-01-04: Tennessee River Fish 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 

TABLE OF CONTENTS

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 ............................................................................................................................................ 36

8 Data/Sample Retention ........................................................................................................................ 37

9 List of Attachments ............................................................................................................................... 37

10 Signatures ............................................................................................................................................. 37
ISO12-01-01-04 

Tennessee River Fish Study 

 Fall 2012 

3 

LIST OF TABLES

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 Labeled Internal Standards (IS) ................................. 17

Table 5. Reference Substances: Stable Isotope Labeled 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); C7-C12 PFCAs, PFSAs and 

SRSs ..................................................................................................................................................... 21

Table 8. Gradient Liquid Chromatography Conditions (ETS-8-045); for C4-C6 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, Professional Services) 

Jon Steege, Analyst (Pace Analytical, Professional Services) 

Patrick Kenney, Analyst (Pace Analytical, Professional Services) 

Kevin Eich, Analyst (Quality Associates, Inc., Professional Services) 

Laura Harrington, Analyst (Pace Analytical, Professional 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 of 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 ISO12-01-01-04 and was performed in 

support of the Weston Solutions, Inc. study plan, titled “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. personnel caught five largemouth bass and five 

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

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

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

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

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

perfluorohexanoate (PFHxA), perfluoroheptanoate (PFHpA), perfluorooctanoate (PFOA), 

perfluorobutane sulfonate (PFBS), perfluorohexanesulfonate (PFHS), perfluorooctanesulfonate 

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

reported in Table 1. 

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

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

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

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

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

100 +10%, except FBSA which was 82.3%. The average recovery 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 (SRSs) 13C4-PFOA and 13C4-PFOS. The 

recovery of 13C4-PFOS was 88.0% (RSD = 3.8%) and for 13C4-PFOA was 85.2% (RSD = 

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

a measure of the analytical method precision and indicated excellent precision.

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

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

levels, and overall, for each analyte. The LCS statistical 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 tissue 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 analytes with a maximum value of 599 ng/g (wet 

weight; ww) followed by FBSA with a maximum measured 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 levels 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 analytes 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). 
T

6 

Table 1. Concentration Results for Tennessee River Fish Tissue Samples 

SAMPLE ID LIMS ID 

Average Concentration (ng/g; wet wt.) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA MeFO

TN_11-0194-1_1 Sample 1 Pre-Study Homogenization Process control 

ISO12-01-01-04-001 1.42 <0.101 <0.503 <0.960 <0.101 <0.0960 0.731 <0.250 [c] <0.253 <0.5

MSFOO1 ISO12-01-01-04-002 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 259 86.2 [b, c] 2.37 1.4IPF002 ISO12-01-01-04-003 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 8.49 20.2 [c] 0.947 [b] <0.5IPF003 ISO12-01-01-04-004 <0.250 <0.0503 <0.252 <0.480 3.82 <0.250 18.3 164 [b, c] 2.23 1.0MSF004 ISO12-01-01-04-005 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 167 58.2[c] 3.05 1.3MSF005 ISO12-01-01-04-006 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 168 60.9[c] 3.08 1.3MSF006 ISO12-01-01-04-007 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 173 79.5[c] 2.26 0.9IPF007 ISO12-01-01-04-008 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 8.86 4.83[c] <0.505 <0.5IPF009 ISO12-01-01-04-009 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 13.4 5.29[c] 0.487 <0.5MSF010 ISO12-01-01-04-010 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 71.0 68.6[c] 1.42 <0.5MSF011 ISO12-01-01-04-011 <0.250 <0.0503 <0.252 0.576 [a,c] NR <0.250 98.5 81.7 [c] 2.03 <0.5IPF012 ISO12-01-01-04-012 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 12.1 96.3 [b, c] 4.81 0.8MSF013 ISO12-01-01-04-013 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 68.9 84.1[c] 1.37 0.546 IPF014 ISO12-01-01-04-014 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 14.3 52.4 [c] 2.35 0.654 MSF015 ISO12-01-01-04-015 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 67.1 56.2 [c] 1.24 0.684 IPF016 ISO12-01-01-04-016 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 14.6 55.5 [c] 2.40 0.696 MSF017 ISO12-01-01-04-017 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 79.6 51.3 [c] 1.09 <0.5IPF018 ISO12-01-01-04-018 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 11.7 28.6 [c] 1.21 <0.5
T

7 

Table 1. Concentration Results for Tennessee River Fish Tissue Samples 

SAMPLE ID LIMS ID 

Average Concentration (ng/g; wet wt.) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA MeFO

MSF020 ISO12-01-01-04-020 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 65.8 56.7 [c] 1.11 <0.5MSF021 ISO12-01-01-04-021 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 98.2 23.3 [c] 0.699 <0.5IPF022 ISO12-01-01-04-022 <0.250 <0.0503 <0.252 <0.480 <1.10 <0.250 5.10 36.9 [c] <0.505 <0.5MSF023 ISO12-01-01-04-023 <0.250 <0.101 <0.101 <0.480 0.184 [b] <0.250 54.9 33.0 [c] 0.800 <0.5IPF024 ISO12-01-01-04-024 <0.250 <0.101 <0.101 <0.480 2.668 <0.250 10.3 12.3 [c] 1.07 [b] <0.5MSF025 ISO12-01-01-04-025 <0.250 <0.101 <0.101 <0.480 0.294 [a] <0.250 66.4 47.9 [b, c] 0.887 <0.5IPF026 ISO12-01-01-04-026 2.34 [a] 1.78 [a] 1.79 [a] 3.13 [a] 0.887 [b] 1.54 [a] 45.5 [b] 19.6 [c] 6.38 2.40 MSF027 ISO12-01-01-04-027 <0.250 <0.101 <0.101 <0.480 0.176 [b] <0.250 65.3 48.1 [c] 0.988 <0.5MSF028 ISO12-01-01-04-028 <0.250 <0.101 <0.101 <0.480 0.859 <0.250 65.9 45.0 [c] 0.970 <0.5IPF029 ISO12-01-01-04-029 <0.250 <0.101 <0.101 <0.480 0.460 <0.250 18.6 69.2 [c] 4.20 1.9MSF030 ISO12-01-01-04-030 <0.250 <0.101 0.198 [a] <0.480 0.590 <0.250 65.2 22.1 [c] 0.571 <0.5IPF031 ISO12-01-01-04-031 <0.250 <0.101 <0.101 <0.480 1.31 <0.250 21.9 84.2 [c] 3.01 0.7MSF033 ISO12-01-01-04-032 <0.250 <0.101 <0.101 <0.480 0.459 <0.250 190 31.5 [c] 1.12 1.6IPF034 ISO12-01-01-04-033 <0.250 <0.101 <0.101 <0.480 1.862 <0.250 8.12 5.38 [c] 0.884 [b] <0.5MSF035 ISO12-01-01-04-034 <0.250 <0.101 <0.101 <0.480 0.219 <0.250 207 41.3 [c] 1.28 0.5IPF036 ISO12-01-01-04-035 <0.250 <0.101 <0.101 <0.480 0.371 [b] <0.250 14.4 7.86 [c] 0.629 <0.5MSF037 ISO12-01-01-04-037 <0.250 <0.101 <0.101 <0.480 0.582 [b] <0.250 101.9 63.6 [c] 0.716 <0.5MSF038 ISO12-01-01-04-038 <0.250 <0.101 <0.101 <0.480 0.996 [b] <0.250 106 66.4 [c] 0.750 0.5IPF039 ISO12-01-01-04-039 <0.250 <0.101 <0.101 <0.480 0.305 [b] <0.250 15.7 53.7 [c] 3.58 0.7MSF040 ISO12-01-01-04-040 <0.250 <0.101 <0.101 <0.480 0.259 <0.250 104 35.6 [c] 1.26 0.7IPF041 ISO12-01-01-04-041 <0.250 <0.101 <0.101 <0.480 0.405 [b] <0.250 22.2 35.4 [c] 2.41 1.3MSF042 ISO12-01-01-04-042 <0.250 <0.101 0.120 [a] 1.57 [a] 0.131 [b] <0.250 210 [b] 52.8 [c] 1.25 2.61 MSF043 ISO12-01-01-04-043 NR <0.101 <0.101 NR 0.138 [b] <0.250 224 55.5 [c] 1.32 2.8MSF044 ISO12-01-01-04-044 <0.100 <0.101 0.118 [a] <0.240 <0.252 <0.250 116 107 3.09 1.0MSF045 ISO12-01-01-04-045 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 225 37.9 1.99 0.768 
T

8 

Table 1. Concentration Results for Tennessee River Fish Tissue Samples 

SAMPLE ID LIMS ID 

Average Concentration (ng/g; wet wt.) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA MeFO

MSF046 ISO12-01-01-04-046 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 54.1 56.5 1.11 0.4IPF047 ISO12-01-01-04-047 <0.100 <0.101 <0.101 <0.240 0.327 <0.250 14.3 63.9 2.71 0.2MSF048 ISO12-01-01-04-048 <0.100 <0.101 <0.101 <0.240 0.269 [a] <0.250 88.9 129 2.40 1.0IPF049 ISO12-01-01-04-049 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 5.59 20.6 1.29 <0.2MSF050 ISO12-01-01-04-050 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 81.6 116 2.11 0.8IPF051 ISO12-01-01-04-051 <0.100 <0.101 <0.101 <0.240 0.337 <0.250 8.01 28.0 1.71 0.3MSF052 ISO12-01-01-04-052 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 93.5 [b] 71.1 2.11 0.448 IPF053 ISO12-01-01-04-053 <0.100 <0.101 <0.101 <0.240 1.54 [c] <0.250 6.68 15.9 1.31 <0.2IPF055 ISO12-01-01-04-055 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 11.0 31.5 1.37 [b] 0.333 MSF056 ISO12-01-01-04-056 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 54.8 19.3 0.530 <0.2IPF057 ISO12-01-01-04-057 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 7.33 22.8 1.58 <0.2MSF058 ISO12-01-01-04-058 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 75.3 69.7 1.59 0.4IPF059 ISO12-01-01-04-059 <0.100 <0.101 <0.101 <0.240 0.327 <0.250 7.56 26.1 1.69 <0.2MSF060 ISO12-01-01-04-060 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 64.7 74.2 2.48 0.365 MSF061 ISO12-01-01-04-061 <0.100 <0.101 <0.101 <0.240 0.289 <0.250 75.5 85.9 2.21 0.3MSF062 ISO12-01-01-04-062 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 103 92.6 2.42 0.4IPF063 ISO12-01-01-04-063 <0.100 <0.101 <0.101 <0.240 0.854 <0.250 6.67 16.0 1.43 0.3MSF065 ISO12-01-01-04-064 <0.100 <0.101 <0.101 <0.240 <0.252 <0.250 86.9 46.8 1.19 0.3IPF066 ISO12-01-01-04-065 <0.250 <0.0503 <0.101 <0.240 0.353 [b] <0.250 10.5 [c] 37.5 1.95 0.1MSF067 ISO12-01-01-04-066 <0.250 <0.0503 0.176 [a] <0.240 <0.252 <0.250 94.6 38.6 1.98 0.3IPF068 ISO12-01-01-04-067 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 9.60 40.8 1.48 0.1IPF069 ISO12-01-01-04-068 <0.250 <0.0503 0.138 [a] <0.240 0.739 <0.250 5.63 18.0 0.771 0.1IPF070 ISO12-01-01-04-069 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 1.81 <0.250 0.153 [b] <0.1MSF071 ISO12-01-01-04-070 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 35.8 0.555 0.137 <0.1MSF073 ISO12-01-01-04-072 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 36.3 0.529 0.161 <0.1
T

9 

Table 1. Concentration Results for Tennessee River Fish Tissue Samples 

SAMPLE ID LIMS ID 

Average Concentration (ng/g; wet wt.) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA MeFO

MSF074 ISO12-01-01-04-073 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 32.7 1.72 0.215 <0.1MSF075 ISO12-01-01-04-074 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 27.9 0.648 0.122 <0.1IPF076 ISO12-01-01-04-075 <0.250 0.139 [a] <0.101 <0.240 <0.252 <0.250 0.768 <0.250 <0.101 <0.1IPF077 ISO12-01-01-04-076 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 1.50 <0.250 <0.101 <0.1IPF078 ISO12-01-01-04-077 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 5.17 3.58 0.277 [b] 0.1IPF079 ISO12-01-01-04-078 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 0.836 <0.250 <0.101 <0.1MSF080 ISO12-01-01-04-079 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 18.6 0.361 0.148 <0.1MSF081 ISO12-01-01-04-080 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 37.8 0.802 0.135 [a] <0.1IPF082 ISO12-01-01-04-081 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 2.92 0.295 0.196 [a] <0.1IPF083 ISO12-01-01-04-082 <0.250 0.137 [a] <0.101 <0.240 <0.252 <0.250 3.41 [b] 0.512 [b] 0.123 [a] <0.1MSF084 ISO12-01-01-04-083 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 44.1 7.01 1.44 0.3MSF085 ISO12-01-01-04-084 <0.250 <0.0503 0.110 [a] <0.240 <0.252 <0.250 32.6 2.72 0.580 0.122 MSF086 ISO12-01-01-04-085 <0.250 <0.0503 <0.101 <0.240 <0.252 <0.250 49.5 6.87 0.127 [a] <0.1MSF087 ISO12-01-01-04-086 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 49.0 25.8 0.379 0.2MSF089 ISO12-01-01-04-087 <0.250 0.260 [a] <0.503 <0.240 <0.252 <0.250 40.8 14.8 1.22 [b] 0.288 MSF090 ISO12-01-01-04-088 <0.250 0.151 [a] <0.503 <0.240 <0.252 <0.250 104 28.4 1.71 0.2MSF091 ISO12-01-01-04-090 <0.250 0.174 [a] <0.503 <0.240 <0.252 <0.250 159 20.4 2.24 0.4MSF092 ISO12-01-01-04-091 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 434 8.55 4.69 0.688 MSF094 ISO12-01-01-04-092 0.860 [a] 0.132 <0.503 <0.240 <0.252 <0.250 69.0 28.1 0.672 0.4IPF95 ISO12-01-01-04-093 <0.250 0.0965 [a] <0.503 <0.240 <0.252 <0.250 4.37 1.16 0.258 <0.1MSF096 ISO12-01-01-04-094 <0.250 0.144 <0.503 <0.240 <0.252 <0.250 46.9 2.95 0.396 0.1IPF97 ISO12-01-01-04-095 <0.250 0.137 [a] <0.503 <0.240 <0.252 <0.250 4.37 1.90 0.413 0.1IPF98 ISO12-01-01-04-096 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 2.22 0.867 0.299 [a] <0.1MSF099 ISO12-01-01-04-097 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 124 6.10 1.60 0.6IPF100 ISO12-01-01-04-098 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 4.69 0.575 0.656 0.122 
T

10 

Table 1. Concentration Results for Tennessee River Fish Tissue Samples 

SAMPLE ID LIMS ID 

Average Concentration (ng/g; wet wt.) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA MeFO

IPF101 ISO12-01-01-04-099 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 50.9 3.37 6.68 0.236 MSF102 ISO12-01-01-04-100 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 218 22.4 1.37 [b] 1.3IPF103 ISO12-01-01-04-101 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 42.0 3.56 6.26 0.2IPF104 ISO12-01-01-04-102 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 4.15 1.16 <0.253 0.11IPF105 ISO12-01-01-04-103 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 3.41 0.633 0.255 [a] <0.1IPF106 ISO12-01-01-04-104 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 2.42 0.609 0.271 [a] 0.107 IPF107 ISO12-01-01-04-105 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 7.85 1.39 1.28 <0.1IPF108 ISO12-01-01-04-106 <0.250 <0.101 <0.503 <0.240 <0.252 <0.250 29.1 1.29 1.07 0.246 TN_11-0194-1_1Sample 2 Mid-Study Homogenization Process control 

ISO12-01-01-04-108 1.42 0.126 [a] <0.503 <0.240 <0.252 <0.250 0.523 [a] <0.250 <0.253 <0.1

MSF109 ISO12-01-01-04-109 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 21.8 1.03 <0.101 <0.1MSF111 ISO12-01-01-04-110 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 25.7 1.57 0.235 [b] <0.1IPF112 ISO12-01-01-04-111 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 1.78 <0.500 <0.101 <0.1IPF113 ISO12-01-01-04-112 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 1.30 <0.500 <0.101 <0.1IPF114 ISO12-01-01-04-113 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 1.24 <0.500 <0.101 <0.1MSF115 ISO12-01-01-04-114 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 31.2 4.28 <0.101 <0.1IPF116 ISO12-01-01-04-115 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 2.65 <0.500 <0.101 <0.1IPF117 ISO12-01-01-04-116 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 0.897 <0.500 <0.101 <0.1IPF118 ISO12-01-01-04-117 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 2.01 <0.500 <0.101 <0.1MSF119 ISO12-01-01-04-118 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 23.9 2.26 0.162 <0.1IPF120 ISO12-01-01-04-119 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 0.961 <0.500 0.300 <0.1MSF121 ISO12-01-01-04-120 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 19.9 2.06 0.141 <0.1IPF122 ISO12-01-01-04-121 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 4.45 <0.500 <0.101 <0.1
T

11 

Table 1. Concentration Results for Tennessee River Fish Tissue Samples 

SAMPLE ID LIMS ID 

Average Concentration (ng/g; wet wt.) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA MeFO

IPF123 ISO12-01-01-04-122 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 2.11 <0.500 <0.101 <0.1MSF124 ISO12-01-01-04-123 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 73.2 6.47 0.258 [b] <0.1MSF125 ISO12-01-01-04-124 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 40.8 [d] 6.23 0.200 [d] <0.10MSF127 ISO12-01-01-04-126 <1.00 <2.52 <0.252 <0.480 0.444 0.416 408 182 4.19 2.2MSF128 ISO12-01-01-04-127 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 262 110 4.00 1.1MSF129 ISO12-01-01-04-128 <1.00 <2.52 <0.252 <0.480 <0.252 0.123 250 137 2.46 1.6MSF130 ISO12-01-01-04-129 <1.00 <2.52 <0.252 <0.480 <0.252 <0.100 333 119 8.18 5.0MSF131 ISO12-01-01-04-130 <0.250 [c] <0.101 <0.252 <0.240 <0.252 <0.250 178 128 [c] 2.12 1.6MSF132 ISO12-01-01-04-131 <0.250 0.0992 [a] <0.252 <0.240 <0.252 <0.250 324 143 [c] 7.21 5.5IPF133 ISO12-01-01-04-132 <0.250 0.417 [a] <0.252 <0.240 <0.252 <0.250 22.6 8.87 [c] 0.384 <0.2IPF134 ISO12-01-01-04-133 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 3.98 [b] 1.80 [c] <0.253 <0.2IPF135 ISO12-01-01-04-134 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 4.89 [b] 3.67 [c] <0.253 <0.2IPF137 ISO12-01-01-04-135 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 1.90 2.50 [c] <0.253 <0.2IPF138 ISO12-01-01-04-136 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 8.55 3.97 [c] <0.253 <0.2MSF139 ISO12-01-01-04-137 <0.250 0.0942 [a] <0.252 <0.240 <0.252 <0.250 279 72.9 [c] 1.73 1.4MSF140 ISO12-01-01-04-138 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 281 133 [c] 3.75 1.8MSF141 ISO12-01-01-04-139 <0.250 0.136 <0.252 <0.240 <0.252 <0.250 134 114 [c] 1.75 1.0MSF142 ISO12-01-01-04-140 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 45.0 15.4 [c] 0.441 [b] <0.2MSF143 ISO12-01-01-04-141 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 143 111 [c] 1.83 1.0MSF144 ISO12-01-01-04-142 <0.250 0.122 [a] <0.252 <0.240 <0.252 <0.250 76.9 11.4 [c] 0.243 [a] <0.2TN_11-0194-1_1 Sample 3 Post-Study Homogenization 

ISO12-01-01-04-144 1.42 0.170 [a] <0.252 <0.240 <0.252 <0.250 <0.184 [a] <0.250 [c] <0.253 <0.2

IPF181 ISO12-01-01-04-145 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 5.00 <0.250 [c] <0.253 <0.2MSF145 ISO12-01-01-04-146 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 175 15.2 [c] 0.348 1.1
T

12 

Table 1. Concentration Results for Tennessee River Fish Tissue Samples 

SAMPLE ID LIMS ID 

Average Concentration (ng/g; wet wt.) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA MeFO

MSF146 ISO12-01-01-04-147 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 80.3 17.7 [c] 0.465 0.3IPF147 ISO12-01-01-04-148 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 26.9 72.8 [c] 7.23 0.8IPF148 ISO12-01-01-04-149 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 69.6 65.9 [c] 3.64 0.3IPF149 ISO12-01-01-04-150 <0.250 <0.101 <0.252 <0.240 <0.252 <0.250 64.2 45.9 [c] 2.45 0.2IPF151 ISO12-01-01-04-151 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 11.7 49.5 1.81 <0.5IPF152 ISO12-01-01-04-152 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 29.2 106 4.92 0.7IPF153 ISO12-01-01-04-153 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 16.3 10.1 0.816 <0.5IPF155 ISO12-01-01-04-154 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 11.9 38.6 1.83 <0.5MSF157 ISO12-01-01-04-155 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 115 74.7 1.20 0.7MSF158 ISO12-01-01-04-156 <0.250 0.317 [a] <0.252 <0.480 <0.503 <0.250 145 67.3 2.28 0.9IPF159 ISO12-01-01-04-157 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 11.2 20.4 0.620 <0.5IPF160 ISO12-01-01-04-158 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 4.87 8.47 0.490 <0.5IPF161 ISO12-01-01-04-159 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 85.6 69.2 34.1 3.4IPF162 ISO12-01-01-04-160 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 79.3 65.8 31.4 3.0IPF163 ISO12-01-01-04-162 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 83.4 44.8 25.6 1.8IPF164 ISO12-01-01-04-163 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 283 37.5 63.5 [b] 3.5IPF165 ISO12-01-01-04-164 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 542 34.1 123 13IPF166 ISO12-01-01-04-165 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 286 33.3 88.1 7.9MSF167 ISO12-01-01-04-166 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 50.6 12.1 1.28 <0.5MSF168 ISO12-01-01-04-167 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 120 20.2 4.22 1.2MSF169 ISO12-01-01-04-168 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 435 67.2 [b] 7.44 3.5MSF170 ISO12-01-01-04-169 <0.250 <0.252 <0.252 0.643 2.21 0.783 373 567 59.9 22MSF172 ISO12-01-01-04-170 <0.250 <0.252 <0.252 <0.480 <0.503 0.391 96.4 45.8 2.92 0.5MSF173 ISO12-01-01-04-171 <0.250 <0.252 <0.252 <0.480 <0.503 <0.250 599 135 9.75 4.6MSF174 ISO12-01-01-04-172 <0.250 0.294 [a] <0.252 <0.240 <0.252 <0.500 521 95.0 5.20 1.6
T

13 

Table 1. Concentration Results for Tennessee River Fish Tissue Samples 

SAMPLE ID LIMS ID 

Average Concentration (ng/g; wet wt.) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA MeFO

IPF175 ISO12-01-01-04-173 <0.250 <0.252 <0.252 <0.240 <0.252 <0.500 8.57 36.2 1.36 <0.5IPF176 ISO12-01-01-04-174 <0.250 <0.252 <0.252 <0.240 <0.252 <0.500 11.8 28.6 1.54 <0.5IPF177 ISO12-01-01-04-175 <0.250 0.421 <0.252 <0.240 <0.252 <0.500 12.7 29.9 1.60 <0.5IPF178 ISO12-01-01-04-176 <0.250 <0.252 <0.252 <0.240 0.322 <0.500 10.5 51.2 1.23 <0.5IPF179 ISO12-01-01-04-177 <0.250 <0.252 <0.252 <0.240 0.506 <0.500 10.7 35.7 0.670 <0.5IPF180 ISO12-01-01-04-178 <0.250 0.277 <0.252 <0.240 <0.252 <0.500 7.69 27.8 1.43 0.5DAL-MSF-LS10001 ISO12-01-01-04-180 <0.250 <0.252 <0.252 <0.240 <0.252 <0.500 <0.366 <0.250 <0.253 <0.5DAL-IPF-LS10002 ISO12-01-01-04-181 <0.250 <0.252 <0.252 <0.240 <0.252 <0.500 3.95 <0.250 <0.253 <0.5DAL-MSF-LS10003 ISO12-01-01-04-182 <0.250 <0.252 <0.252 <0.240 <0.252 <0.500 3.67 <0.250 <0.253 <0.5DAL- IPF-LS10004 ISO12-01-01-04-183 <0.250 <0.252 <0.252 <0.240 <0.252 <0.500 <0.366 <0.250 <0.253 <0.5DAL- IPF-LS10005 ISO12-01-01-04-184 <0.250 <0.252 <0.252 <0.240 <0.252 <0.500 <0.366 <0.250 <0.253 <0.5DAL-MSF-LS10006 ISO12-01-01-04-185 <0.250 <0.252 <0.252 <0.240 <0.252 <0.500 4.31 <0.250 <0.253 <0.5Frequency of Detection (n = 175) 2.9% 12% 4.0% 2.3% 19% 2.9% 98% 87% 82% 49Average Recovery NA NA NA NA NA NA NA NA NA NA Standard Deviation NA NA NA NA NA NA NA NA NA NA RSD NA NA NA NA NA NA NA NA NA NA 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 FBS19% for EtFOSAA NR; 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 result was outside of 100 + 30%, but within 100 + 50% and were 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 determined 
T

14 

Table 2. Laboratory Matrix Spike (LMS) Recoveries

LIMS ID Study ID 

LMS Analyte Recoveries 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS FBSA FOSA MeFOSAA 

ISO12-01-01-04-001 

TN11-0194-1/1 Sample 1 Prestudy Homogenization Process Control 

107% 109% 101% 111% 109% 102% 111% 139% [a] 92.1% 107% 

ISO12-01-01-04-002 MSFOO1 109% 101% 124% 114% 111% 106% 113% [b] 53.5%[a, b] 95.3% 104% ISO12-01-01-04-011 MSF011 112% 102% 128% 116% NR 115% 97.5% [b] 55.4%[a, b] 96.6% 111% ISO12-01-01-04-022 IPF022 108% 96.8% 126% 109% 95.6% 100% 103% 86.9% [b] 97.6% 107% ISO12-01-01-04-023 MSF023 112% 106% 99.4% 115% 115% 98.6% 96.9 [b]% 52.2%[a, b] 97.7% 115% ISO12-01-01-04-032 MSF033 107% 100% 91.8% 109% 104% 91.8% 105% [b] 69.7%[a, b] 94.6% 101% ISO12-01-01-04-043 MSF043 NR 122% 101% NR NR 102% 102% [b] 53.1%[a, b] 106% 111% ISO12-01-01-04-044 MSF044 112% 100% 92.0% 94.0% 107% 140%[a] 94.7% [b] 85.9% [b] 109% 102% 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% ISO12-01-01-04-064 MSF065 100% 97.0% 85.0% 91.7% 87.6% 92.5% 95.1% [b] 89.1% [b] 108% 88.0% ISO12-01-01-04-065 IPF066 106% 89.6% 89.0% 90.8% 93.3% 97.2% 80.4% 94.6% [b] 114% 100% ISO12-01-01-04-076 IPF077 114% 102% 86.2% 91.7% 90.0% 95.6% 88.7% 104% 115% 98.0% 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% ISO12-01-01-04-096 IPF098 104% 95.6% 88.4% 90.6% 92.8% 86.6% 99.6% 92.2% 97.3% 104% 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% ISO12-01-01-04-118 MSF119 84.2% 86.2% 84.0% 91.9% 89.8% 85.4% 82.5% [b] 79.8% 113% 103% ISO12-01-01-04-130 MSF131 135%[a] 103% 106% 108% 113% 103% 97.4%[b] 50.7%[a, b] 104% 105% ISO12-01-01-04-139 MSF141 125% 107% 104% 97.5% 107% 101% 96.9% [b] 64.8% [a, b] 98.8% 97.8% ISO12-01-01-04-151 IPF151 104% 98.4% 103% 107% 112% 97.2% 106% [b] 111% [b] 104% 105% ISO12-01-01-04-159 IPF161 104% 93.6% 93.6% 108% 96.0% 100% 90.7% [b] 84.0% [b] 77.0% [b] 90.1% ISO12-01-01-04-172 MSF174 117% 89.3% 98.6% 97.9% 98.4% 103% 101% [b] 85.7% [b] 99.4% 109% 

Average Recovery 108% 98.8% 98.4% 101% 99.6% 99.4% 97.2% 82.3% 102% 103% 

Standard Deviation 11% 9.0% 13% 10% 9.6% 12% 8.4% 22% 10% 6.6% 

RSD 10% 9.1% 14% 10% 9.7% 12% 8.6% 27% 10% 6.4% 

n 20 21 21 20 19 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 concentrations of select perfluorinated compounds 

(PFC) fish and surface water from the Tennessee River 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. (Westchester, MA), dated October 2012. The 

overall purpose of the study was to provide additional 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. Laboratory blinded fish tissue samples 

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

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

water collection procedures, handling and storage of 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 tissue homogenates to the 3M environmental 

Laboratory and Axys Laboratories for analysis of PFCs. TestAmerica personnel were 

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

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

personnel. 

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

campaign, whole largemouth bass and catfish were purchased 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 were removed in the laboratory, the skin was 

removed from each fillet, and then control fillet tissue 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 removed as filleting process controls. The control fillet 

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

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

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

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

and the true identities of the fish tissues were not provided and were only identifiable by the 

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

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

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

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

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

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

Laboratory for measuring the target analyte concentrations. 

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

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

analytes in a control bluegill fish fillet homogenate 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 matrix for ~10% of the samples (i.e. prepared for 

every 10th fish sample) as laboratory matrix spikes (LMSs), and providing a measure of 

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

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

surrogate recovery standards (SRSs), with 13C4-PFOS representative of the perfluorinated 

sulfonates (PFSAS) and MeFOSAA, EtFOSAA, FOSA and FBSA and 13C4-PFOA 

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

sample, LMS and LCS sample aliquot prior to extraction. 
ISO12-01-01-04 

Tennessee River Fish Study 

 Fall 2012 

16 

4 Reference Substances 

The reference substances used during the analyses are listed in Table 3, Table 4 and Table 5. 

Most were purchased from commercial vendors and exist 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 contain pre-determined percentages of linear and 

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

purity during solution preparations. Several reference standards are provided as salts, and additional 

correction factors for salt cations (Na+, K+, NH4

+

) and protons (H+) were also applied, when 

applicable. All analyte concentrations were determined 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 C3F7COOH C5F11COOH C6F13COOH C8HF15O2

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 C4F9SO3

-K+ C6F13SO3-Na+ C8F17SO3-K+ C4F9SO2NH2

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 C8F17SO2NH2 C11H6F17NO4S C12H8F17NO4S 

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 concentrations used in this study and solution concentrations were based on the concentration of 

the standard material provided by the commercial vendor 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% purity was used to calculated LMS 

recoveries. 

***; Calculated concentrations used in the study using 98.3% purity 

Table 4. Reference Substances: Stable Isotope Labeled Internal Standards (IS) 

Reference Substance MPFBA MPFHxA MPFHpA M8PFOA 

Chemical Name 

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

13C4]-butanoic acid 

Perfluoro-n-[1,2-13C2]-

hexanoic acid 

Perfluoro-n-[13C4]-

heptanoic acid 

Perfluoro-n-[13C8]-octanoic 

acid 

Chemical Formula 13C4HF7O2

13C212C4HF11O213C412C3HF13O213C8HF15O2

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 

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Reference Substance 18O2-PFBS M3PFHxS M8PFOS M8FOSA 

Chemical Name 

[18O2]-Ammonium 

Perfluorobutanesulfonate 

Sodium perfluoro-1-

[

13C3]hexanesulfonate 

Sodium perfluoro-1-

[

13C8]octanesulfonate 

Perfluoro-1-[13C8]-

octanesulfonamide 

Chemical Formula C4F9S

18O2O-NH4+ 13C312C3F13SO3-Na+ 13C8F17SO3-Na+ 13C8F17SO2NH2

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 C11D3H3F17NO4S C12D5H3F17NO4S 

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 concentrations used in this study and solution concentrations were based on the concentration of 

the standard material provided by the commercial vendor and assume the vendor adjusted for purity. 
ISO12-01-01-04 

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Table 5. Reference Substances: Stable Isotope Labeled Surrogate Recovery Standards (SRS) 

Reference Substance 13C4-PFOA 13C4-PFOS 

Chemical Name Perfluoro-n-[1,2,3,4-13C4]-octanoic acid Sodium perfluoro-1-[1,2,3,4-13C4]-

octanesulfonate 

Chemical Formula 13C4

12C4HF15O2

13C4

12C4F17SO3-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 the sample extractions and quantitative 

LC/MS/MS analyses were performed following validated 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 fillet 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 were 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), Tennessee 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 same LIMS project as ISO12-01-01-04-

002 through -185. 

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

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

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

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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-homogenized 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 allow 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 tube. Each fish sample was typically 

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

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

isotope labeled surrogate recovery standard (SRS) mixture 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 then centrifuged at greater than 10,000 x g to 

pellet the solids. Then, a 1.00 mL aliquot of supernatant 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 prepared 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 (13C4-PFOA and 13C4-PFOS) and ISs prior to extraction, for evaluation of overall data 

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

approximately every tenth fish sample extracted, two 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 acetonitrile; (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 triplicate; (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 the LCS matrix; and (6) solvent blanks were 

prepared with the acetonitrile used to make calibration standards and prepared with and without 

ISs and SRSs. 

5.4 LC/MS/MS Analysis 

Analyses were performed following a previously validated 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 percent 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 analytes from LMS samples were used as a 

measure of sample-specific and sample matrix analytical accuracy. Recovery of the SRSs (13C4-

PFOS and 13C4-PFOA) from each analytical sample was additionally used as a secondary 

measure of the target analyte accuracy for each sample. 

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 this study are listed in Table10. 
ISO12-01-01-04 

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

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

Analytical Method ID ETS-8-045.1 

Liquid Chromatograph Agilent 1200 or 1290 HPLC System 

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

(2.1 x 50 mm, 5 μm) 

Injection Volume 20 μL , 40 μL or 50 μL 

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

Ion Source TurboIon Spray (Electrospray) 

Polarity Negative Ion 

Software Analyst 1.6.1 

C7 and C8 PFCAs, PFSAs and SRSs 

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

25μm); 30°C 

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

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

column 

C4 and C6 PFCAs

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

Table 7. Gradient Liquid Chromatography Conditions (ETS-8-045); C7-C12 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 

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Table 8. Gradient Liquid Chromatography Conditions (ETS-8-045); for C4-C6 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 

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

Compound Analyte Description MRM Transition(s) 

[1,2,3,4 -13C]-PFBA IS for PFBA 217>172 

[1,2 -13C2]-PFHxA IS for PFHxA and PFHpA 315>270 

[1,2,3,4,5,6,7,8-13C8]-PFOA IS for PFOA and [1,2,3,4-13C4]-PFOA 421>376 

[

18O2]-PFBS IS for PFBS and FBSA 303>84 

[

13C3]-PFHS IS for PFHS 402>80 

[1,2,3,4,5,6,7,8-13C8]-PFOS IS for PFOS and [1,2,3,4-13C4]-PFOS 507>80 

[1,2,3,4,5,6,7,8-13C8]-FOSA IS for FOSA 506>78 

d3-MeFOSAA IS for MeFOSAA 

573>83 

573>169 

573>219 

d5-EtFOSAA IS for Et FOSAA 

589>83 

589>169 

589>219 

Surrogate Recovery Standards (SRS) 

[1,2,3,4-13C4]-PFOA Surrogate for PFCAs 417>372 

[1,2,3,4-13C4]-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, 13C4-PFOA and 13C4-PFOS 

ISO12-01-01-04-001 
ISO12-01-01-04 

Tennessee River Fish Study 

 Fall 2012 

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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, 13C4-PFOA and 13C4-PFOS 

ISO12-01-01-04-002 to -022 

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

MeFOSAA, EtFOSAA, 13C4-PFOA and 13C4-PFOS 

ISO12-01-01-04-023 to -043 

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

MeFOSAA, EtFOSAA, 13C4-PFOA and 13C4-PFOS 

ISO12-01-01-04-044 to -064 

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

MeFOSAA, EtFOSAA, 13C4-PFOA and 13C4-PFOS 

ISO12-01-01-04-065 to -085 

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

MeFOSAA, EtFOSAA, 13C4-PFOA and 13C4-PFOS 

ISO12-01-01-04-086 to -108 

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

MeFOSAA, EtFOSAA, 13C4-PFOA and 13C4-PFOS 

ISO12-01-01-04-109 to -129 

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

MeFOSAA, EtFOSAA, 13C4-PFOA and 13C4-PFOS 

ISO12-01-01-04-130 to -150 

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

MeFOSAA, EtFOSAA, 13C4-PFOA and 13C4-PFOS 

ISO12-01-01-04-151 to -171 

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

MeFOSAA, EtFOSAA, 13C4-PFOA and 13C4-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 values are rounded to three significant figures. 

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

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

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

same calculations.

6.1 Calibration 

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

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

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

target analytes and SRSs; equivalent to nominal 0.0250 ng/g to 2500 ng/g in fish tissue after 

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

to each fish sample during extraction. Following the 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 with 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 calibration curve and comparing to the known 

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

The correlation coefficient (r) was greater than 0.999, and coefficient of determination (r2) was 

greater than 0.990. High and/or low calibration standards were excluded as needed to meet 
ISO12-01-01-04 

Tennessee River Fish Study 

 Fall 2012 

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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 with 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 distinct L-PFOS peak in calibration 

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

and LCSs were quantified by integration of total PFOS (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 typically the lowest non-zero calibration standard in 

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

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

acetonitrile solvent blanks. The LLOQ for all analytes 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 nominal 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 batch, continuing calibration verifications 

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

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

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

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

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

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

PFOA: m130122b052 (559%), m130122b110 (383%), m130122b122 (134%) and 

m130122b128 (NA, outside of curve range). 

PFBS: m130122b052 (425%), m130122b110 (126%), m130122b122 (2.02%) and 

m130131a005 (130%). 

PFHS:m130122b052 (NA, outside of curve range) and m130122b128 (57.6%) 

PFOSm130122b052 (529%), m130122b110 (4050%), j130201a074 (74.3%) and j130201a112 

(71.4%) 

FBSA: m130122b052 (402%), m130122b110 (304%), m130122b128 (61.6%), m130129b052 

(70.5%), m130131a005 (70.5%), j130201a041 (134%), j130201a074 (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%) 

13C4-PFOA (SRS): m130122b052 (32.3%), m130122b098 (4.06%), m130122b110 (41.6%) 

and m130122b128 (11.4%) 

13C4-PFOS (SRS): m130122b052 (16.2%), m130122b098 (4.06%), m130122b110 (37.0%) 

and m130122b128 (4.07%) 
ISO12-01-01-04 

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When CCVs failed to meet criteria, the analytical run was evaluated more closely for LMS 

recoveries and LCS results, and a potential cause of 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 and 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 justification for reporting the results from those 

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

data. 

6.4 Blanks 

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

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

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

LLOQs; (2) fish matrix blanks were prepared with nominal 0.5 g of a control bluegill fillet 

homogenate TN11-0194-1/1 and were spiked with and without ISs and SRSs and used to 

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

process control blanks used to evaluate the homogenization process at TestAmerica (control 

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

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

removed at the 3M Environmental Laboratory, homogenized 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 filleting process. Those process control samples 

were returned blinded to the laboratory regarding their 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 identities for process controls will be verified in the 

Weston Solutions, Inc. final study report. 

All blank results were carefully evaluated for issues such as background instrument 

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

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

matrix used for preparation of LCSs. All of the blank 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 matrix 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 those values subtracted from LCS results 

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

process controls are included in Table 1. 
ISO12-01-01-04 

Tennessee River Fish Study 

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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 into 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 were prepared at three levels of nominal 1.0, 

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

LCSs was brPFOS purchased from Wellington Laboratories 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 separated L-PFOS isomer peak, both LPFOS and total-PFOS recoveries from LCSs were determined. Total-PFOS was always 

determined from samples, LMS s and matrix blanks. 

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

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

uncertainties at the 95% confidence interval (95% CI; 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 following equation: 

*100%

Spike Concentration

(Determine d Concentration of LCS - Endogenous Matrix Conc.) 

LCSRecovery =
T

28 

Table 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 EtFO

j121206a (PFBA, PFPHxA) and m121206a 

LCS Low-1 115% 108% 84.4% 74.7% 108% 106% 118% 105% 123% 94.6% 119% 11LCS Low-2 101% 102% 69.9% 63.9% 99.2% 97.1% 111% 96.6% 117% 90.6% 102% 10LCS Low-3 105% 100% 72.5% 66.3% 104% 98.9% 117% 105% 120% 91.1% 103% 10LCS Mid-1 111% 110% 105% 110% 109% 105% 114% 111% 133% 93.4% 110% 10LCS Mid-2 111% 109% 107% 115% 110% 105% 116% 112% 135% 94.7% 110% 10LCS Mid-3 106% 108% 106% 111% 107% 102% 113% 110% 132% 91.3% 103% 10LCS High-1 109% 108% 87.5% 102% NA 103% 115% 108% 126% 96.1% 108% 10LCS High-2 103% 104% 89.6% 100% NA 98.6% 110% 104% 117% 91.2% 105% 10LCS High-3 108% 106% 88.7% 101% NA 101% 112% 106% 123% 95.7% 108% 10

j130118a (PFBA, PFPHxA) and m130122b 

LCS Low-1 106% 96.5% 126% 109% 104% 99.3% 110% 108% 90.3% 90.6% 98.4% 92LCS Low-2 107% 96.5% 125% 116% 104% 102% 117% 115% 91.8% 95.8% 103% 97LCS Low-3 103% 93.8% 119% 110% 98.6% 96.8% 110% 108% 87.6% 89.5% 98.7% 92LCS Mid-1 105% 104% 106% 104% 97.9% 99.0% 111% 105% 78.1% 96.2% 99.7% 91LCS Mid-2 111% 105% 112% 106% 101% 102% 116% 111% 80.3% 104% 108% 10LCS Mid-3 103% 98.8% 105% 101% 96.8% 96.2% 110% 105% 76.6% 99.2% 99.0% 93LCS High-1 NA NA NA NA 83.6% 85.9% NA 67.5% [a] 67.0% NA 87.1% 83LCS High-2 

NA NA NA NA 70.0% 78.0% NA 67.7% [a] 51.7% NA 86.1% 79

LCS High-3 NA NA NA NA 66.8% [a] 79.1% NA 69.3% [a] 49.1% NA 89.1% 84
T

29 

Table 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 EtFO

j130121a (PFBA, PFPHxA) and m130129b 

LCS Low-1 106% 97.5% 92.3% 106% 105% 85.0% 101% 99.2% 75.4% 93.4% 99.3% 93LCS Low-2 102% 94.7% 87.9% 104% 101% 81.2% 95.6% 94.0% 72.5% 90.2% 93.9% 90LCS Low-3 105% 97.4% 92.5% 107% 104% 83.9% 102% 99.8% 71.3% 93.6% 103% 92LCS Mid-1 105% 101% 84.3% 104% 107% 90.2% 100% 97.4% 68.8% [a] 95.9% 97.6% 90LCS Mid-2 103% 100% 82.7% 99.7% 107% 90.2% 97.4% 94.3% 67.4% [a] 94.8% 95.5% 86LCS Mid-3 101% 99.8% 84.9% 98.8% 104% 87.3% 97.8% 94.9% 68.4% [a] 95.0% 96.8% 87LCS High-1 NA NA NA 100% 90.3% 80.7% 115% 76.0% 75.4% NA 89.5% 82LCS High-2 NA NA NA 98.1% 94.7% 71.6% 104% 70.6% 61.6% [a] NA 86.1% 80LCS High-3 NA NA NA 97.5% 89.7% 69.4% [a] 103% 69.2% [a] 51.1% [a] NA 84.2% 81

j130123a (PFBA, PFPHxA) and Be130121b 

LCS Low-1 105% 96.0% 82.3% 95.1% 87.3% 93.6% 89.6% 89.2% 128% 104% 97.0% 88LCS Low-2 105% 95.4% 88.4% 93.6% 90.4% 89.2% 87.1% 86.8% 134% [a] 106% 98.6% 93LCS Low-3 103% 95.4% 84.4% 95.3% 91.2% 91.4% 88.0% 88.0% 129% 107% 99.6% 89LCS Mid-1 109% 107% 97.7% 101% 93.8% 103% 98.1% 98.1% 119% 108% 101% 97LCS Mid-2 109% 104% 95.9% 97.1% 96.7% 102% 94.3% 94.2% 119% 113% 101% 92LCS Mid-3 98.2% 94.5% 83.5% 88.5% 85.2% 91.4% 84.9% 85.3% 110% 104% 93.5% 85LCS High-1 NA NA 86.1% 83.9% 87.6% 83.7% 97.6% 90.0% 65.7% [a] 86.5% 90.3% 79LCS High-2 NA NA 83.2% 79.3% 87.4% 81.3% 97.6% 89.0% 66.6% [a] 94.0% 82.8% 78LCS High-3 NA NA 87.7% 83.8% 90.5% 83.7% 101% 92.3% 67.6% [a] 93.6% 88.8% 78
T

30 

Table 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 EtFO

g130124a (PFBA, PFPHxA) and Be130123b 

LCS Low-1 122% 104% 89.7% 95.0% 93.4% 106% 95.2% 94.8% 105% 116% 109% 87LCS Low-2 116% 100% 87.6% 93.5% 91.2% 104% 94.6% 94.1% 102% 113% 103% 85LCS Low-3 117% 96.8% 86.5% 95.2% 93.7% 103% 94.8% 93.9% 104% 108% 101% 88LCS Mid-1 106% 89.5% 84.3% 101% 83.5% 87.0% 84.5% 83.9% 94.0% 97.7% 91.3% 78LCS Mid-2 118% 90.8% 87.8% 97.1% 85.7% 96.4% 94.3% 93.5% 97.2% 110% 101% 83LCS Mid-3 116% 91.1% 90.6% 88.5% 88.1% 95.2% 92.1% 92.5% 101% 101% 99.0% 84LCS High-1 NA NA 87.0% 83.9% 92.3% 69.2% [a] 105% 93.8% 91.2% 84.2% 89.6% 84LCS High-2 NA NA 85.7% 79.3% 84.4% 68.5% [a] 102% 91.6% 87.4% 82.8% 82.9% 81LCS High-3 NA NA 86.0% 83.9% 87.0% 68.1% [a] 103% 91.6% 85.9% 85.0% 84.8% 82

j130125a(PFBA, PFPHxA) and Be130128a 

LCS Low-1 110% 100% 89.7% 96.0% 94.7% 97.4% 101% 99.4% 100% 98.1% 94.7% 93LCS Low-2 105% 97.1% 91.4% 92.1% 91.4% 92.0% 91.7% 90.8% 99.5% 98.1% 96.1% 87LCS Low-3 107% 99.6% 89.0% 96.7% 96.3% 96.5% 93.7% 92.6% 105% 103% 97.7% 87LCS Mid-1 98.6% 95.4% 85.5% 92.1% 90.5% 89.2% 87.5% 86.0% 94.4% 104% 93.7% 83LCS Mid-2 97.2% 94.3% 80.8% 91.1% 89.5% 87.4% 89.2% 88.2% 91.1% 99.3% 96.5% 82LCS Mid-3 88.8% 85.8% 75.1% 82.1% 80.9% 81.1% 78.9% 78.0% 84.9% 87.2% 80.4% 76LCS High-1 NA NA 86.9% 85.9% 85.4% 84.1% 105% 92.2% 80.2% 108% 87.7% 81LCS High-2 NA NA 85.2% 83.5% 81.8% 80.1% 102% 90.7% 79.0% 95.8% 82.4% 81LCS High-3 NA NA 82.8% 84.1% 83.2% 79.1% 101% 89.2% 76.8% 90.6% 86.3% 77
T

31 

Table 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 EtFO

j130128a (PFBA, PFPHxA) and Be130130a 

LCS Low-1 96.7% 87.9% 82.1% 90.3% 87.2% 85.2% 86.3% 85.4% 93.3% 114% 93.1% 84LCS Low-2 104% 95.1% 89.6% 97.6% 95.2% 91.3% 91.3% 91.3% 96.6% 117% 96.0% 93LCS Low-3 97.7% 90.0% 84.9% 92.4% 87.0% 88.3% 90.6% 89.4% 96.8% 97.3% 100% 87LCS Mid-1 98.7% 93.7% 82.0% 87.8% 85.9% 82.2% 85.8% 86.1% 87.8% 99.7% 89.6% 82LCS Mid-2 NA NA 84.8% 93.1% 88.6% 88.3% 93.5% 93.3% 93.2% 102% 96.9% 87LCS Mid-3 NA NA 74.0% 80.9% 78.9% 78.4% 80.7% 80.1% 80.4% 95.2% 83.5% 79LCS High-1 NA NA 78.7% 83.2% 79.6% 80.4% 94.9% 83.8% 76.8% 88.4% 84.8% 77LCS High-2 NA NA 84.5% 90.7% 87.3% 84.7% 101% 89.3% 81.2% 105% 85.2% 83LCS High-3 NA NA 83.9% 89.7% 85.1% 85.7% 101% 90.0% 79.5% 86.4% 88.4% 80

g130128a (PFBA, PFPHxA) and j130201a 

LCS Low-1 101% 80.2% 88.8% 92.0% 89.6% 89.3% 97.7% 89.6% 125% 88.8% 87.1% 81LCS Low-2 102% 92.6% 87.7% 91.3% 86.5% 85.4% 91.2% 82.9% 116% 84.1% 85.6% 78LCS Low-3 96.2% 83.2% 82.9% 91.9% 81.0% 85.1% 94.1% 85.7% 106% 84.8% 87.4% 78LCS Mid-1 96.6% 83.8% 79.5% 83.4% 76.6% 78.5% 98.1% 89.2% 86.8% 81.3% 81.2% 75LCS Mid-2 120% 98.4% 103% 102% 92.1% 94.4% 116% 106% 100% 96.7% 94.4% 92LCS Mid-3 100% 86.7% 84.3% 87.3% 80.0% 82.2% 96.8% 87.9% 93.8% 83.3% 81.6% 77LCS High-1 110% 87.9% NA NA NA NA 130% 75.6% NA 75.1% 77.9% 76LCS High-2 111% 93.0% NA NA NA NA 148% 79.0% NA 77.7% 76.9% 76LCS High-3 105% 85.6% NA NA NA NA 105% 67.4% NA 71.0% 74.3% 72
T

32 

Table 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 EtFO

m130204a (PFBA, PFPHxA) and j130201a 

LCS Low-1 87.5% 84.1% 85.0% 90.8% 83.6% 85.9% 102% 101% 80.5% 84.8% 90.8% 80LCS Low-2 88.1% 87.2% 84.7% 95.1% 91.2% 85.7% 107% 104% 82.2% 81.9% 95.8% 79LCS Low-3 96.4% 93.6% 95.9% 99.5% 96.5% 93.5% 112% 108% 99.7% 94.5% 100% 85LCS Mid-1 89.4% 87.1% 89.8% 98.6% 86.2% 86.1% 99.4% 95.4% 93.8% 90.6% 82.3% 81LCS Mid-2 95.2% 91.4% 89.8% 99.9% 99.0% 94.5% 105% 101% 103% 93.7% 92.3% 82LCS Mid-3 95.2% 92.4% 94.4% 100% 88.5% 94.2% 105% 100% 93.5% 94.4% 89.5% 86LCS High-1 80.9% 93.4% 75.9% 78.0% NA 73.5% 100% 75.9% 85.5% 92.5% 74.5% 65.3LCS High-2 81.4% NA 80.9% 76.8% NA 71.3% 88.7% 70.0% 85.6% 89.6% 75.9% 63.2LCS High-3 88.1% NA NA 93.4% NA 88.5% 104% 80.8% 103% 96.6% 78.1% 73

g130204a (PFBA, PFPHxA) and g130201a 

LCS Low-1 110% 95.7% 90.6% 93.0% 92.7% 96.3% 101% 96.8% 93.4% 90.4% 91.0% 90LCS Low-2 120% 94.8% 93.0% 104% 100% 100% 104% 99.5% 99.5% 94.8% 103% 92LCS Low-3 110% 93.6% 93.1% 102% 96.7% 95.1% 98.4% 92.5% 105% 94.1% 96.7% 90LCS Mid-1 113% 89.4% 94.6% 99.1% 90.9% 94.7% 97.8% 93.1% 95.5% 94.2% 92.5% 88LCS Mid-2 99.1% 78.8% 85.0% 83.8% 83.8% 81.6% 85.9% 81.9% 88.2% 81.4% 79.0% 74LCS Mid-3 110% 83.4% 91.5% 98.0% 93.0% 96.6% 99.9% 95.2% 104% 93.9% 93.5% 89LCS High-1 104% NA 87.2% 87.1% NA 82.8% 107% 77.8% 85% 109% 86.0% 71LCS High-2 124% NA NA 100% NA 93.4% 115% 86.0% 103% 126% 94.3% 80LCS High-3 112% NA NA 90.3% NA 84.4% 98.3% 75.6% 99.80% 118% 81.4% 79
T

33 

Table 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 EtFO

Low LCS Average 105% 95.0% 90.6% 95.0% 94.8% 93.5% 99.5% 96.2% 102% 97.0% 98.1% 89Low-LCS Std. Dev. 8.1% 6.0% 12% 11% 6.9% 7.1% 9.4% 7.9% 17% 9.8% 6.6% 7.Low LCS RSD 7.7% 6.3% 14% 12% 7.3% 7.6% 9.4% 8.3% 17% 10% 6.8% 8.Low LCS (n) 30 30 30 30 30 30 30 30 30 30 30 3Mid-LCS Average 104% 95.5% 90.9% 96.8% 92.5% 92.0% 98.1% 94.9% 95.7% 96.5% 94.5% 87Mid-LCS Std. Dev. 8.1% 8.5% 10% 8.8% 9.5% 7.9% 11% 9.5% 18% 7.6% 8.5% 8.Mid-LCS RSD 7.8% 8.9% 11% 9.1% 10% 8.6% 11% 10% 19% 7.8% 9.0% 9.Mid-LCS RSD (n) 28 28 30 30 30 30 30 30 30 30 30 3High-LCS Average 103% 96.8% 84.9% 89.0% 84.8% 81.8% 106% 83.7% 82.3% 93.3% 86.5% 81High-LCS Std. Dev. 13% 9.1% 3.5% 8.2% 7.1% 9.5% 12% 11.7% 20% 13% 8.5% 9.High LCS RSD 13% 9.4% 4.2% 9.2% 8.3% 12% 11% 14% 25% 14% 9.8% 12High LCS (n) 12 7 18 24 18 27 27 30 27 24 30 3Overall Study Average 104% 95.4% 89.4% 93.9% 91.6% 89.4% 101% 91.6% 93.6% 95.8% 93.0% 86Standard Deviation 9.0% 7.4% 10% 10% 8.9% 9.6% 11% 11% 20% 10% 9.2% 9.RSD 8.6% 7.8% 12% 11% 9.7% 11% 11% 12% 21% 11% 9.9% 11n (Total Number of LCS Results) 70 65 78 84 78 87 87 90 87 84 90 9Analytical Method Uncertainty (95%CI) + 18% + 15% + 21% + 20% + 18% + 19% + 22% + 23% + 40% + 20% + 18% + 1[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-PF

NA; No value determined for the LCS either due to instrument error or value outside of the calibration curve 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, laboratory matrix spike (LMS) samples were prepared 

for ~10% of the study samples. Approximately every 10th 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 was 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. All four replicates were then fortified with ISs, 

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

samples received in the study (12%) had a set of LMSs 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 Wellington Laboratories), and were quantified for 

total PFOS using L-PFOS calibration, as described earlier. 

For sample analytes PFBA, PFHpA, PFOA, PFBS and PFHS 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 samples (i.e. appropriate fortification is considered 

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

results for PFOS and FBSA the high- LMS level was the 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 following equation: 

*100%

SpikeConcentration

(Determine dConcentration of LMS DeterminedConcentration of FieldSample) LMSRecovery −

=

6.7 NIST SRM 1947 Results 

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

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

samples. The results of the analysis of the NIST SRM 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 value 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 rinseate blanks were collected and shipped 

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

Environmental Laboratory method ETS-8-044.1 and showed 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 results 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.
35 

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

SAMPLE ID LIMS ID 

Concentration (ng/L; parts per trillion) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA 

EBLK01 ISO12-01-01-04-019 <5.00 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK02 ISO12-01-01-04-036 <5.00 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK03 ISO12-01-01-04-054 5.69 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK04 ISO12-01-01-04-071 <5.00 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK005 ISO12-01-01-04-089 5.09 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK006 ISO12-01-01-04-107 <5.00 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK07 ISO12-01-01-04-125 <5.00 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK08 ISO12-01-01-04-143 <5.00 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK09 ISO12-01-01-04-161 <5.00 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK10 ISO12-01-01-04-179 <5.00 <2.00 <2.00 <4.80 <5.00 <5.00 <0.927 <2.00 <2.00 EBLK11 ISO12-01-01-04-186 <5.00 <2.00 <2.00 <4.80 <5.00 <5.00 1.40 <2.00 <2.00 

*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 + 17for 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 chemicals (PFCs) measured in fish collected 

from the Tennessee River in the Fall of 2012 were reported. 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 October 2012. 

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

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

Wilson and Wheeler Reservoirs of the Tennessee River, 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 removed 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 extraction and quantitative analysis of PFCs. 

An additional six process controls for evaluating the 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 (PFHpA), perfluorooctanoate (PFOA), 

perfluorobutane sulfonate (PFBS), perfluorohexanesulfonate (PFHS), perfluorooctanesulfonate 

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

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

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

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

known quantities of target analytes. The LMS recovery 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 sample 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 control bluegill fillet tissue at 3 levels, each 

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

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

LCS levels) were used to determine the analytical method 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 overall 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 standards (SRSs) 13C4-PFOA and 13C4-PFOS 

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

tissue samples analyzed (n = 175) were 85.3% for 13C4-PFOA and 88.1% for 13C4-PFOS. The 

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

precision with RSDs of 5.2% for 13C4-PFOA and 3.8% for 13C4-PFOS. 

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

highest frequency in Tennessee River fish samples and with the highest concentrations 

measured. PFOS concentrations ranged from <0.366 ng/g (ww) to 599 ng/g (ww). FBSA 

T

38 

Attachment A 

PFC Results from Analysis of Bass & Catfish Control Materials Used for Fillet-Pr

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

SAMPLE ID 

Average Concentration (ng/g; wet wt.) 

PFBA PFHxA PFHpA PFOA PFBS PFHS PFOS* FBSA FOSA 

TN12-0364-1/13-LM Bass-Right Side Fillet <0.250 <1.00 <0.0500 <0.100 <0.250 <0.100 4.27 <0.100 <0.250 TN12-0364-1/13-LM Bass-Right Side Fillet <0.250 <1.00 <0.0500 <0.100 <0.250 <0.100 4.12 <0.100 <0.250 TN12-0364-1/13-LM Bass-Right Side Fillet <0.250 <1.00 <0.0500 <0.100 <0.250 <0.100 3.85 <0.100 <0.250 TN12-0365-1/13-Catfish-Right Side Fillet <0.250 <1.00 <0.0500 <0.100 <0.250 <0.100 <0.100 <0.100 <0.250 TN12-0365-1/13-Catfish-Right Side Fillet <0.250 <1.00 <0.0500 <0.100 <0.250 <0.100 <0.100 <0.100 <0.250 TN12-0365-1/13-Catfish-Right Side Fillet <0.250 <1.00 <0.0500 <0.100 <0.250 <0.100 <0.100 <0.100 <0.250 [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 tissue sample extracted and analyzed Results 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 f
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Attachment B 

Preparation Forms, Raw Data and Chromatograms 

(Available Upon Request)