# 3

Testing Certificate #2052.01 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 tests results included in this report are covered by this accreditation following method ETS-8-045.1 

## Final Report Concentrations of PFOS in Fish Fillet Tissues from Largemouth Bass Collected in the Tennessee River in April 2012


# Study Title Analytical Study ISO12-01-01-01: Analysis of PFOS f rom Tennessee River Fish – Bakers Creek Area: April 2012 

# Data Requirement ISO17025 

# Project Director Charles Young Weston Solutions, Inc. 

# Principal Investigator Cleston C. Lange, Ph.D. 3M Environmental Laboratory 

# Report Completion Date

May 02, 2012 

# Analytical Laboratory 3M Environmental Health and Safety Operations

Environmental Laboratory 3M Center, Bldg 260-05-N-17 St. Paul, MN 55144 

# 3M LIMS Project Identification

ISO12-01-01-01
Total Number of Pages

25

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

2 

# T ABLE OF 

# C ONTENTS

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

................................................... ................................................... 2
List of Tables ....................................

................................................... ................................................... .... 3
1 Study Information .................................

................................................... ............................................ 4
2 Summary ...........................................

................................................... ............................................... 5
3 Introduction ......................................

................................................... ................................................. 7
4 Reference Substances ..............................

................................................... ...................................... 8

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

................................................... ........................................ 8
5.1 Methods ...........................................

................................................... .................................... 8
5.2 Sample Receipt ....................................

................................................... ............................... 9
5.3 Sample Preparation ................................

................................................... ............................. 9
5.4 LC/MS/MS Analysis .................................

................................................... .......................... 10
6 Analytical Results ................................

................................................... ........................................... 12
6.1 Calibration .......................................

................................................... ................................... 12
6.2 Limits of Quantitation (LOQs) .....................

................................................... ...................... 13
6.3 Continuing Calibration ............................

................................................... ........................... 14
6.4 Blanks ............................................

................................................... ..................................... 14
6.5 Laboratory Control Spikes (LCSs) ..................

................................................... .................. 14
6.6 Laboratory Matrix Spikes (LMSs) ...................

................................................... .................. 19
6.7 NIST SRM 1947 Results .............................

................................................... ...................... 20
6.8 Individual Sample Results .........................

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

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

................................................... .............................................. 23
8 Data/Sample Retention .............................

................................................... .................................... 24
9 List of Attachments ...............................

................................................... ......................................... 24
10 Signatures ........................................

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

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

3 

# L IST OF 

# T

ABLES Table 1. PFOS Results for Tennessee River Largemou th Bass Fillet Tissues, April 2012 ................ .. 6 Table 2. Analytical Reference Substances .........

................................................... .................................. 8
Table 3. Instrument Information ..................

................................................... ......................................... 11 Table 4. Gradient Liquid Chromatography Conditions (ETS-8-045) ...................................... .............. 11
Table 5. Mass Transitions ........................

................................................... ............................................ 11 Table 6. Analytical Batches for PFOS in Tennessee River Fish (April 2012) ........................... ........... 12
Table 7. L-PFOS Solvent Calibration Results ......

................................................... ............................... 13
Table 8. L-PFOS LCSs and Matrix Blank Results ....

................................................... .......................... 15
Table 9. brPFOS LCSs and Matrix Blank Results ....

................................................... .......................... 17 Table 10. Homogenization Process Control Results .

................................................... ......................... 20
Table 11. Individual PFOS Results for Fillet Tissu es from Fish Collected in the Tennessee River, April 2012 .................................

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

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

4 

## 1 Study Information
Sponsor 3M Company 

### Project Requestor Gary Hohenstein Environmental Manager Special Projects, EHS Operations Bldg 224-5W-03 St. Paul, MN 55144 Phone: (651) 737-3570 gahohenstein@mmm.com 

### Sampling Coordinator Charles Young Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 610-701-3787 charles.young@westonsolutions.com 

### Principal Analytical Investigator Cleston 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 Environmental Laboratory Building 260-5N-17 St. Paul, MN 55144 

### Study Personnel (3M Environmental Laboratory) Marlene Heying, Analyst (Pace Analytical, Professio nal Services) Kelly Ukes, Analyst (Pace Analytical, Professional Services) Jon Steege, Analyst (Pace Analytical, Professional Services) William Reagen, Ph.D., 3M Environmental Laboratory Management 

### Study Dates Study Initiation: April 10, 2012 Interim Analytical Initiation (PFOS): April 16, 20 12 Interim Analytical Completion (PFOS): April 24, 20 12 Interim Report Completion: April 30, 2012

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

5 

## 2 Summary
This analytical report provides the perfluorooctane sulfonate (PFOS) concentrations measured for twenty-nine (29) homogenized fillet tissues (skin r emoved) that were obtained from twenty-six (26) largemouth bass collected from the Tennessee River in April 2012 near Decatur, Alabama. This analysis was conducted in support of the Weston Sol utions, Inc. project plan, titled “Work Plan for Fi sh Sampling and Analysis for PFOS in Largemouth Bass f rom the Tennessee River in the Vicinity of the 3M Decatur Facility, Decatur, Alabama”. The analytica l aspects reported herein were conducted in the 3M Environmental Laboratory as 3M study ISO12-01-01-01

. The 26 largemouth bass were collected from the Tenn essee River in early April 2012 and were filleted i n the field (skin removed) by Weston Solutions, Inc. personnel. The 26 right side fillets from each fis h, and 6 left side fillets from six random fish as blind f ield duplicates, were sent to the Michigan State Un iversity Wildlife Toxicology Laboratory (MSU-WTL) for homoge nization. The processed fish homogenates from 26 right side fillets and 3 of the left side fillet s were then packaged in baggies and shipped frozen with 3 process controls and 1 standard reference material (NIST SRM 1947) to the 3M Environmental Laboratory for quantitative analyses of PFOS. The other 3 left side fillet homogenates (skin removed) , along with one aliquot of NIST SRM 1947, were sent to Axys Labs for analysis of PFOS as independent verification of the 3M Environmental Laboratory res ults. The remaining 20 whole fillets, skin on and not homogenized, were sent directly to the 3M Environme ntal Laboratory by Weston Solutions, Inc. field personnel to be retained frozen as “archive” sample s for potential future use. The twenty-nine (29) homogenized fish fillet tissue s were received on April 14, 2012 and April 17, 201 2 as two separate shipments. The analytical laborato ry was blind to the true identities of the Tennesse e River fish with regard to sex, age and specific sam pling location, which was only known to Weston Solutions, Inc. and were only labeled with unique, generic IDs. In addition to the largemouth bass tissues, three (3) process control fish tissues (pr e-study, mid-study and post-study controls) and one (1) standard reference material (NIST SRM 1947; Lake Mi chigan fish tissue) were received with the fish samples from MSU-WTL. Those control materials wer e previously provided by the 3M Environmental Laboratory to MSU-WTL prior to fish collection and processing to serve as controls for evaluating the homogenization process and evaluating the PFOS resu lts relative to a NIST SRM 1947 fish tissue PFOS reference value. In contrast to Tennessee Riv er fish samples, the four control tissues were identifiable by the laboratory in order to evaluate them for quality assurance purposes, as discussed herein. Once received at the 3M Environmental Laboratory, s amples were logged into the electronic laboratory information management system (LIMS) where each sam ple was assigned a unique 3M LIMS sample ID under project ISO12-01-01-01. Fish homogenates were stored frozen until preparation for analysis. Fish homogenates were re-homogenized, extracted by protein precipitation method and analyzed by liquid chromatography with triple quadrapole mass s pectrometric detection (LC/MS/MS) following the validated and published 3M Environmental Laboratory analytical method ETS-8-045.1. Quantitation was performed by internal standard (IS ) method using stable isotope labeled 13 

### C 8 

### -PFOS as the IS and utilizing linear isomer of PFOS (L-PFOS) solvent calibration. Each sample was extracted in duplicate along with corresponding laboratory matri x spikes (LMSs) to evaluate sample-specific PFOS recovery. As additional quality control during th e analysis, each sample received a fixed quantity o f a stable isotope labeled surrogate reference standard (SRS) 13 

### C 4 

### -PFOS prior to extraction; results of which were used as a secondary measure of PFOS reco very from each sample. Laboratory control spikes (LCSs) were employed to e valuate the method performance. Two sets of laboratory control spikes (LCSs) were prepared with each analytical batch in a control bass fillet homogenate; each set with three levels of PFOS fort ification at nominal 10.0, 100 and 1000 ng/g, each level prepared in triplicate. One set of LCSs was prepared with L-PFOS, and the second was prepared with a mixed linear plus branched isomer PFOS (brPF OS) to evaluate potential isomer effects on quantitation. Additionally, for analytical batches which contained diluted samples, high-level LCSs w ere similarly diluted to incorporate some measure of di lution accuracy and precision into the method performance evaluations. The LCS results were used for determining the method performance during

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

6 the study. The LCS (L- PFOS) results are reported in Table 8 and LCS (br PFOS) results are shown in Table 9 and demonstrated excellent analytical method perfo rmance with accuracies of within 100 + 25% for all LCS results and RSDs < 20%. Statistical ev aluation of the LCS results provided method uncertainties (95% CI) of + 19% for L-PFOS and + 11% for brPFOS. The concentrations of PFOS in pre-study, mid-study and post-study homogenization process controls were determined at 0.815 ng/g, 0.909 ng/g and 0.912 ng/g respectively. Those values compared well to the determined 0.804 ng/g average PFOS concentratio n for the same control tissue which did not travel to MSU-WTL and was not homogenized by MSU-WTL. The RPDs between results for the unprocessed versus MSU-processed control tissues were less than 15% for each and showed that PFOS was not significantly introduced to samples as a result of any sample contamination during processing or shipping of the fish tissue samples from MSU-WTL to the laboratory. The average determined PFOS concentration for the d uplicate prepared analytical samples are shown in Table 1, along with the RPD of the duplicate results for ea ch. The measured concentrations of PFOS ranged from 52.5 ng/g to 4770 ng/g. The reco veries of fortified PFOS from the LMS prepared for each at a level comparable to the endogenous PFOS l evel in the corresponding Tennessee River fish sample ranged from 76.7% to 125%, with an overall m ean recovery and standard deviation of 98.9% + 10%, as shown in Table 1 . The overall recovery of PFOS demonstrated excell ent sample specific PFOS measurement accuracy. Additionally, the prec ision of each result was excellent with RPDs less than 20%, and typically less than 10%. The recoveri es of the added stable isotope labeled SRS ( 13 

### C 4 

### - PFOS) were within 100 + 20% for all samples, and with excellent precision (RPDs < 20%) for all samples, as shown in Table 11

. The measured PFOS concentration for a NIST SRM 1947 was 6.71 ng/g (4.0% RPD, 95.3% LMS recovery), and was accurate to 13% RPD with respect to the published SRM 1947 mean reference value and standard deviation of 5.90 ng/g.

Table 1. PFOS Results for Tennessee River Largemou th Bass Fillet Tissues, April 2012
3M LIMS ID Sample ID Avg. PFOS Concentration (ng/g) [b] RPD (%) LMS PFOS Recovery
ISO12-01-01-01-021 Process Control #1 (Pre-Study) 0.815 2.0% 102% ISO12-01-01-01-022 Process Control #2 (Mid-Study) 0.909 [a] 34% 99.1% ISO12-01-01-01-023 MSF 01 120403 4770 1.4% 

### 96.5% ISO12-01-01-01-024 MSF 02 120403 4240 5.3% 

### 92.4% ISO12-01-01-01-025 MSF 03 120403 70.7 1.9% 

### 81.0% ISO12-01-01-01-026 MSF 04 120403 576 6.5% 

### 108% ISO12-01-01-01-027 MSF 05 120403 471 4.9% 

### 100% ISO12-01-01-01-028 MSF 06 120403 156 5.4% 

### 76.7% ISO12-01-01-01-029 MSF 07 120403 2920 1.6% 

### 98.7% ISO12-01-01-01-030 MSF 08 120403 1360 0.13% 

### 99.8% ISO12-01-01-01-031 MSF 09 120403 2020 5.2% 

### 104% ISO12-01-01-01-032 MSF 10 120403 1840 10% 

### 105% ISO12-01-01-01-033 MSF 21 120404 671 6.1% 

### 86.0% ISO12-01-01-01-034 MSF 22 120404 4660 3.5% 

### 99.6% ISO12-01-01-01-035 MSF 23 120404 52.5 0.0056% 

### 99.7% ISO12-01-01-01-036 MSF 24 120405 437 12% 

### 90.7% ISO12-01-01-01-037 MSF 25 120405 306 3.8% 

### 125% ISO12-01-01-01-038 MSF 26 120405 2090 1.0% 

### 94.2% ISO12-01-01-01-039 MSF 27 120403 2120 5.4% 

### 106% ISO12-01-01-01-040 MSF 28 120404 180 9.2% 

### 124% ISO12-01-01-01-041 MSF 29 120405 2050 0.063% 

### 102% ISO12-01-01-01-042 Process Control #3 (Post Study) 0.912 16% 

### 102%

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

7 Table 1. PFOS Results for Tennessee River Largemou th Bass Fillet Tissues, April 2012
3M LIMS ID Sample ID Avg. PFOS Concentration (ng/g) [b] RPD (%) LMS PFOS Recovery
ISO12-01-01-01-043 NIST SRM1947 6.71 4.0% 

### 95.3% ISO12-01-01-01-044 MSF 11 12-04-03 2940 3.9% 

### 93.5% ISO12-01-01-01-045 MSF 12 12-04-03 526 4.2% 

### 101% ISO12-01-01-01-046 MSF 13 12-04-03 950 1.7% 

### 99.0% ISO12-01-01-01-047 MSF 14 12-04-03 3370 0.50% 

### 102% ISO12-01-01-01-048 MSF 15 12-04-03 754 9.6% 

### 99.4% ISO12-01-01-01-049 MSF 16 12-04-04 1520 3.2% 86.9% ISO12-01-01-01-050 MSF 17 12-04-04 165 0.75% 84.2% ISO12-01-01-01-051 MSF 18 12-04-04 701 2.7% 103% ISO12-01-01-01-052 MSF 19 12-04-04 675 0.86% 104% ISO12-01-01-01-053 MSF 20 12-04-04 1180 0.82% 104% Notes: The average result and relative percent difference (RPD) was determined from duplicate analytical samp les for each fish homogenate. All reported concentration results are rounded to three significant figures wh ile statistical values are rounded to two significant figures. The analytical method data uncertainty was + 11% for brPFOS (30.6% branched isomer) and + 19% for L-PFOS (>99% linear isomer) based on LCS results. [a] RPD > 20% [b] The PFOS result is the cumulative concentration of branched and linear isomers of PFOS.



## 3 Introduction
A scientific investigation of PFOS levels in fish a nd surface water collected from the Tennessee River near the 3M Decatur facility was conducted in the S pring of 2012 per the study work plan titled: “Work Plan for Fish Sampling and Analysis for PFOS in Lar gemouth Bass from the Tennessee River in the Vicinity of the 3M Decatur Facility, Decatur, Alaba ma”. The analytical aspects reported herein are fo r the analyses of the fish tissues as conducted by the 3M Environmental Laboratory under the 3M study ISO12-01-01-01. The overall purpose of this study w as to provide additional site-specific information on PFOS level in fish and surface waters of the Tennes see River near the confluence of Baker’s Creek, located near the 3M Decatur facility. This report describes the sample receipt, extractio n and quantitative LC/MS/MS analysis procedures performed for the measurement of PFOS in the fish t issues harvested from largemouth bass collected in early April of 2012 from the Tennessee River, and p rovides the PFOS concentration results for those samples. Additionally included in this report are the assessment of the precision and accuracy of th e results based on several parameters: (1) The precis ion of sample results was determined from preparation and analysis of duplicate analytical sa mples for each homogenate; (2) the recovery of PFOS from laboratory matrix spikes (LMS) samples prepare d for each sample was a measure of samplespecific accuracy; (3) two LCS sets were prepared w ith each analytical batch, one fortified with L-PFO S and one with br-PFOS in a control largemouth bass f illet homogenate (Osage Catfisheries, Inc.) to evaluate analytical method precision and accuracy; and (4), the recovery of a stable isotope labeled SRS (13C4-PFOS) from each sample was determined and used as a secondary measure of samplespecific analyte recovery, and accuracy and precisi on. Personnel from Weston Solutions, Inc were responsib le for coordinating all field aspects of the study such as site selection, site parameters, fish and w ater collection procedures, handling and storage of collected specimens, documentation of individual fi sh information, filleting, and shipment of intact f ish fillets to the MSU-WTL for homogenization. The MSU -WTL personnel were responsible for proper homogenization of fillets, relabeling and distribut ion of homogenates to the appropriate analytical laboratories. The 3M Environmental laboratory was responsible for ensuring samples were received

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

8 appropriately, stored frozen and prepared and analy zed for PFOS. In addition, Axys Laboratories was to receive a sub-set of duplicate fish tissue homog enates for 3 of the Tennessee River fish (10% split ) from MSU-WTL and one standard reference material (N IST SRM 1947) for independent verification of the PFOS results attained by 3M, and will be report ed separately. This was a laboratory-blinded study and the fish ag es, sizes and specific river collection locations w ere not provided to the analytical laboratories. Tenne ssee River fish samples were only identifiable at t he laboratory by coded IDs provided to them by Weston Solutions, Inc. personnel. Weston Solutions, Inc. is responsible for all final analysis of the data f or associating PFOS concentrations with collection location, fish species, age, weight, etc. This rep ort focuses solely on the extraction and quantitati ve LC/MS/MS analysis performed at the 3M Environmental Laboratory and reports the determined concentration results for PFOS in the fish tissues, along with a measure of analytical accuracy and precision of the results based on several quality c ontrol aspects. 

## 4 Reference Substances
The reference substances used during the analysis o f the fish tissues and the consumable supplies are listed in Table 3

. Table 2. Analytical Reference Substances

Reference Substance PFOS (linear isomer) PFO S (linear + branched isomers) M4-PFOS M8-PFOS

**Chemical Name** Potassium Perfluoro

- n
- octane Sulfonate Potassium Perfluorooctane Sulfonate 13 C 4 -PFOS 13 C 8 -PFOS
**Chemical Formula** C 8 F 17 SO 3 K C 8 F 17 SO 3 K 13 C 4 12 C 4 F 17 SO 3 Na 13 C 8 F 17 SO 3 Na

**Use** Calibration, LMSs & LCSs Linear + Branched Isomer s LCSs Surrogate Internal Standard (IS)
**Source** Wellington Laboratories Wellington Laboratories Wel lington Laboratories Wellington Laboratories **Expiration Date** 10/18/2018 12/1/2014 12/21/2014 6/17/2012 **Storage Conditions** Frozen Frozen Frozen Frozen **Chemical Lot** LPFOSKBM06 br-PFOSK1111 MPFOS1211 M8PFOS0609 **TCR Number** TCR08-0001-1/1 TCR11-0041-1/19, -7/19 TCR12-0003-1 /14 TCR09-0051-9/15 **Physical Description** White Powder White Powder Methanol Solution [a] Met hanol Solution [a] **Purity** 99.8% >98% >98% >98% [a] Stable isotope labeled materials are provided a s methanol solutions by the vendor (source); the ve ndor’s reported concentration is used for calculating concentrations in samples.



## 5 Method Summary

5.1 Methods The re-homogenization of fish tissue homogenates fr om MSU-WTLs, and the sample extractions and quantitative LC/MS/MS analyses were performed follo wing validated and previously published 3M Environmental Laboratory method ETS-8-045.1 [Malins ky et. al. 2011, Analytica Chemica Acta 683 :pp248-257]. Sample analyses for fish samples occu rred from April 17th through April 24, 2012. The analytical runs are identified by the initial o f the instrument name used and the date and a lette r describing the order of the runs on that day (i.e. j120417a describes instrument Jonas on April 17, 20 12 and was the first analytical run of the day on that instrument; j120417b would describe the second run of the day on that same instrument). The identities o f the instruments used are shown in Table 3 . A summary of the analytical runs for reporting is sho wn in Table 6

.

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

9 

## 5.2 Sample Receipt
On April 12, 2012 the 3M Environmental Laboratory r eceived twenty (20) frozen whole fillets (skin on, not homogenized) directly from the field as “archiv e” samples which were stored frozen upon receipt. Those samples were received with 3M chains of custo dy (COCs) # 19436 and #19437. The archive samples were not analyzed as part of this study and were retained frozen for potential future use, but associated with this study. The whole fillet samp les were logged into the laboratory LIMS system and consecutively numbered based on their order on the COCs as ISO12-01-01-01-001 through -020. On April 14, 2012 nineteen (19) homogenized fillets and two process controls (pre-study and mid-study controls) were received at the 3M Environmental Lab oratory from MSU-WTL with COCs #14933 and #14940 and one MSU-WTL COC for the process controls . On April 17, 2012 an additional ten (10) Tennessee River fish tissue homogenates were receiv ed with 3M COC #14934, and the post-study process control and NIST SRM 1947 material were rec eived with a MSU-WTL COC. The three process control tissue homogenates (3 ~10 -25 gram aliquots of a control bluegill fillet homogenate TN11-0184-1/1) had been provided to MSU- WTL earlier by the 3M Environmental Laboratory for quality assurance purposes to monito r potential sample contamination during the homogenization process. Additionally, two ~5-10 gra m aliquots of a National Institutes of Standards an d Technology (NIST) standard reference material SRM 1 947 (Lake Michigan fish tissue homogenate) was also provided to MSU-WTL, to be re-packaged and one aliquot sent to each laboratory for analysis. The twenty-nine Tennessee River fish tissue homogenates , three process controls and one NIST SRM 1947 reference were logged into the Laboratory’s electro nic LIMS system in the consecutive order they were received, and in the order listed on the chains of custodies as ISO12-01-01-01-021 through ISO12-01- 01-01-053.


## 5.3 Sample Preparation
Each frozen fish tissue homogenate was removed from its polypropylene sample bag and rehomogenized with dry ice, then transferred back in to the same sample bag and left to stand overnight in a refrigerator to allow the dry ice to sublime (dis sipate). Then for each sample, duplicate nominal 0.5 gram aliquots of tissue homogenate were each accura tely weighed into a centrifuge tube for extraction. Additionally, a third and fourth aliquot of each wa s aliquoted and one fortified with L-PFOS at nomina l 50 ng/g and the other at 500 ng/g as laboratory matrix spikes (LMSs). Later, some samples were determined to be above the calibration range and we re re-prepared with a single higher LMS at 5000 ng/g and re-analyzed after being appropriately dilu ted. A stable isotope labeled PFC internal standar d ( 13 

### C 8 

### -PFOS) and stable isotope labeled surrogate ( 13 

### C 4 

### -PFOS) were added to each sample aliquot prior to solvent extraction, final concentration of each was nominal 1.0 ng/mL in final extract. Samples we re extracted by addition of 5.0 mL acetonitrile, brief ly mixed and then placed into a freezer for approximately 1 hr to facilitate precipitation of f at and protein solids. Then, samples were centri fuged to remove the solids. A 1.0 mL aliquot of each extrac t was transferred to an autosampler vial with 0.010 mL formic acid, capped and then analyzed by LC/MS/M S. In some instances, samples were diluted 10- fold or 100-fold appropriately with acetonitrile co ntaining IS and SRS at the same levels as previousl y added to samples during extraction. The first sample preparations began on April 16, 20 12 and the last preparations occurred on April 23, 2012. Samples were always prepared as analytical b atches, with one analytical batch typically comprised of a set of14 solvent calibration standar ds ranging from 0.025 ng/mL to 250 ng/mL; two sets of LCSs as described below, several method and solv ent blanks, and sample extracts representing approximately10 study samples (an analytical sample , analytical sample duplicate and LMSs were included per sample). All the analytical batches f or this study are listed in Table 6

. The typical calibration range for each analytical b atch data set was from 0.025 to 100 ng/mL (equivale nt

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

10 to nominal 0.25 to 1000 ng/g of fish tissue concent ration), with dropping of the highest prepared calibration standard of 250 ng/mL. In several inst ances the sample results exceeded the response of the 100 ng/mL calibrant and in those instances the 50 ng/g and 500 ng/g LMS fortification levels were determined to be insufficient relative to the endog enous analyte level because typically the ideal LMS fortifications are between 0.5-times and 10-times t he endogenous analyte level per 3M Environmental SOP requirements. In those instances, the samples were re-prepared with a higher LMS fortification at nominal 5000 ng/g concentration with dilutions, as appropriate, and the original sample results ignore d and only the re-prepared sample results reported. Exception(s): In one instance in run g120418b, the sample result for ISO12-01-01-01-049 sample and its LMSs were above the revised upper limit of quan titation (ULOQ) of 100 ng/mL (i.e. > 1000 ng/g), a nd a dilution with a 5000 ng/g LMS was accidentally no t prepared for that sample. In that instance, the original analysis result along with the high-LMS re sult (500 ng/g fortification) was reported, despite the fortification level being slightly less than 0.5-ti mes the endogenous level and determined from a high range calibration by inclusion of the 250 ng/mL sta ndard and exclusion of the calibration standards le ss than 7.5 ng/mL (eq. 75 to 2500 ng/g fish tissue); s ee analytical run/batch data for g120418b-high rang e results for that sample’s results. This fact was f ootnoted in the appropriate results tables.

Laboratory control spike (LCS) samples were prepare d in sets with three levels of nominal 10.0, 100 an d 1000 ng/g of PFOS and each level prepared in tripli cate. Each batch prepared had two sets of LCSs, normal LCSs fortified with L-PFOS the same as calib ration standards, and the second set prepared with brPFOS to verify the quantitative accuracy for PFOS typically found in environmental samples which are comprised of a mixture of linear and branched isome rs. The LCSs were prepared by fortifying nominal 0.5 gram aliquots of control largemouth bass fillet homogenate (TN08-0193-1/1), purchased from Osage Catfisheries, Inc. (Osage Beach, MO). Equivalent p repared non-fortified method blanks were also prepared with the control bass tissue to establish the endogenous control matrix PFOS level for subtraction from LCS results during LCS recovery ca lculations. Results of LCSs were used to evaluate the overall method performance during the analyses. Exception(s): In analytical batch m120423a, the typical set of LC Ss and method blanks was not prepared and analyzed with the diluted samples anal yzed. That run was conducted as a cleanup set for analysis of diluted samples which earlier results w ere greater than the ULOQ. This analytical batch/r un included only equivalently diluted high-level LCSs from the batches those samples were originally prepared and analyzed. Those dilution LCS results are included in the LCS tables and the statistics f or the method performance evaluations.


## 5.4 LC/MS/MS Analysis
The analysis of the fish extracts for PFOS was perf ormed by LC/MS/MS as described in method ETS-8-045.1. The relative percent difference (RPD) of the duplicate sample results was a measure of the sample-specific analytical precision . The determined recovery of fortified PFOS from LMS samples was used as a measure of sample-sp ecific analytical accuracy. Recovery of the surrogate ( 13 

### C 4 

### -PFOS) from duplicate analytical samples was additi onally used as an indirect measure of PFOS recovery and data precision from ea ch sample result. 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 3 , Table 4 and Table 5 . Additionally, a summary of all analytical runs c onducted for collection of the PFOS data reported herein are shown in Table 6

.

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

11 Table 3. Instrument Information
Instrument Name(s) ETS-MaryAnn (m), ETS-Ginger (g), ETS-Jonas (j) Analytical Method ID ETS-8-045.1 Liquid Chromatograph Agilent 1100 with Binary Pump Guard column Pre-Autosampler; Prism RP (2 x 50 mm, 5 μ m) Extraction column Waters Corp. Oasis HLB Online Column (3 x 20 mm, 5 μ m); 30°C Analytical column Betasil C18 (100 x 2.1 mm, 25 μ m); 30°C Column Switching Valve 0 to 5 min (waste); 5 minute s to 21 minutes to analytical column Injection Volume 40 μ L Mass Spectrometer API 5000 Triple Quadrapole Ion Source TurboIon Spray (Electrospray) Polarity Negative Ion Software Analyst 1.4.2


### Table 4. Gradient Liquid Chromatography Conditions (ETS-8-045)
Step Number Total Time

[min] Flow Rate

[mL/min] Percent A

[2 mm Aqueous Ammonium Acetate] Percent 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



### Table 5. Mass Transitions

Analyte Mass Transitions Monitored PFOS 499>130, 499>99, 499>80
13 C 8 -PFOS (IS) 507>80

13 C 4 -PFOS (Surrogate) 503>80 Note: Multiple mass transitions are summed to give the instrument response for PFOS

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

12 Table 6. Analytical Batches for PFOS in Tennessee River Fish (April 2012)
Analytical Batch # Analytical Run ID Report Decision Samples Analyzed/Reported (ISO12- 01-01-01-xxx)

[a] 1 j120417a Data Reported -033, -035, -036, -037, -0 40 2 m120417b Data Reported -022, -025 3 g120418b Data Reported -021, -042, -043, -045, -0 46, -048, -050 4 g120418b-high range Partial Data Set Reported -049 5 g120420a Data Reported -038, -039, -041, -044, -047, -051, -052, -053 6 m120420a Data Reported -023, -024, -026, -027, -028, -029, -030, -031, -032, -034 7 m120423a Data Reported Select dilutions [a] Samples listed only indicate which analytical r un the reported result for the analytical sample an d sample duplicate were determined and may not indica te where the corresponding LMS results were attained since several required dilution and re-ana lysis in different analytical batches. See the raw data for the identity of the exact analytical batch used for the reported LMS results.



## 6 Analytical Results
All reported concentration results are rounded to t hree significant figures. Statistical values are rounded to two significant figures. Because of roun ding to obtain the limited significant figure value s, reported results may vary slightly from those resul ts found in the raw data.


## 6.1 Calibration
Calibration standards were prepared by spiking vary ing known quantities of L-PFOS and SRS ( 13 

### C 4 

### - PFOS), and a fixed quantity of IS ( 13 

### C 8 

### -PFOS) into acetonitrile. A set of 14 calibration standards ranging from nominal 0.0250 ng/mL to 250 ng/mL were prepare d; equivalent to nominal 0.250 ng/g to 2500 ng/g for fish tissue quantitation. Following analysis o f the calibration standards, the nominal concentrat ion of each standard was plotted versus the measured analy te peak area/IS peak area response ratio. A quadratic equation with 1/x weighting was used to f it the calibration response data. Calibration curv es were not forced through zero. Calculating the dete rmined standard concentration using the resultant calibration curve and comparing to the actual 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 (r 2 

### ) was greater than 0.990 for all calibration curves . The summary for the calibration standards for each of the analytical da ta sets used for reporting is shown in Table 7

. Exceptions: Typically all calibration standards were analyzed, but the 250 ng/mL standard dropped during construction of the calibration curve. In analytical run g120418b-high range, only the 7.5 ng /mL to 250 ng/mL standards were used to construct the c alibration curve to quantitate sample ISO12-01-01- 01-49 within that range. In analytical run m120423 a, the 250 ng/mL standard was not analyzed.

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

13 Table 7. L-PFOS Solvent Calibration Results

**Analytical Run ID**  **PFOS (ng/mL)**

**r**

**STD 1 STD 2 STD 3 STD 4 STD 5 STD 6 STD 7 STD 8 STD 9**  **STD 10**  **STD 11**  **STD 12**  **STD 13**  **STD 14**

**STD CONC. 0.025 0.05 0.1 0.25 0.35 0.5 1 2.5 7.5 10 15 25 100**  **250**
j120417a 0.0222 0.0459 0.0989 0.261 0.367 0.509 1.04 2.58 7. 94 9.92 15 24.3 100 excluded 0.9998

m120417b 0.0234 0.049 0.0977 0.254 0.354 0.502 1.01 2.6 7.71 9.85 15.8 23.7 101 excluded 0.9995
jg120418b 0.0219 0.0475 0.0999 0.255 0.361 0.515 1.04 2.61 7. 6 9.99 15.4 24.1 100 excluded 0.9998g120418bhigh range excluded excluded excluded excluded excluded exclud ed excluded exclude d 6.43 9.63 16.7 27.6 94.8 253 0.9978
g120420a 0.0228 0.0488 0.101 0.254 0.354 0.505 1.01 2.61 7.6 2 9.93 15.4 24.2 100 excluded 0.9998

m120420a 0.0243 0.05 0.0977 0.247 0.355 0.506 0.999 2.57 7.5 4 10.1 15.3 24.3 101 excluded 0.9999

m120423a 0.0237 0.0489 0.101 0.253 0.35 0.496 0.997 2.68 7.4 2 10 15.7 24 101 excluded 0.9996 **Average Value**  **0.0231 0.0484 0.0994 0.254 0.357 0.506 1.02 2.61 7. 47 9.92 15.6 24.6 99.7 253 0.9995** 

**Std Dev. 0.00092 0.0014 0.0015 0.0045 0.0061 0.0064 0.019 0.**  **039 0.48 0.15 0.55 1.34 2.21 NA 0.00074**

**RSD 4.0% 3.0% 1.5% 1.8% 1.7% 1.3% 1.9% 1.5% 6.5% 1.5% 3**  **.5% 5.4% 2.2% NA 0.074% Average**  **Recovery 92.2% 96.7% 99.4% 102% 102% 101% 102% 104% 99.5% 99**  **.2% 104% 98.4% 99.7% 101% NA**

**n 6 6 6 6 6 6 6 6 7 7 7 7 7 1 7** 



## 6.2 Limits of Quantitation (LOQs)
The lower limit of quantitation (LLOQ) was the low est non-zero calibration standard in the curve that met linearity and accuracy requirements (100 + 30%) and for which the area counts were at least 2 X that of the average response determined from solven t blanks. The LLOQ for PFOS in this study was typically the nominal concentration 0.0250 ng/mL ac etonitrile standards, and correlating to a 0.25 ng/ g LLOQ in fish tissues when accounting for the ~10-fo ld sample dilution which occurs during extraction (nominal 0.5 g tissue diluted with 5 mL acetonitril e). Typically, the 250 ng/mL standard was dropped and t he upper limit of quantitation (ULOQ) was revised to the 100 ng/mL calibration standard (i.e. upper l imit of nominal 1000 ng/g for PFOS in fish tissues)

. However, for one run (g120418b) a 6-point high rang e calibration from 7.5 ng/mL to 250 ng/mL was employed to quantify the results for one sample (IS O-01-01-01-049) which was accidentally not diluted to be captured with a 100 ng/mL ULOQ. That curve m et the same calibration curve requirements as defined in section 6.1 and showed good LMS recovery for the high-LMS for that result and was therefore reported.

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

14 

## 6.3 Continuing Calibration
During the course of each analysis, continuing cali bration verifications (CCVs) were performed by regular injection of low- to mid-level calibration standards during the analytical run. The back calc ulated concentration results were used to verify that the instrument response and the initial calibration cur ve were still in control during the analysis of the en tire batch. All CCVs bracketing reported data for PFOS met the method criteria of 100% ± 25% accuracy and all CCVs for 13 

### C 4 

### -PFOS surrogate were within 100 + 25%. The results of CCVs are summarized for each analytical batch in the raw data. Exception(s): In analytical batch/run g120418b, one CCV result f or PFOS was 163% recovery due to an irregularly low IS response which was apparently not reflected in the PFOS response for that injection. That CCV immediately followed the analy sis of the calibration standards and LCSs in the beginning of the run and only flanked one side of a sample set. The remaining CCVs in that run were a ll acceptable and therefore the data was reported with a documented method deviation. The reason for the unusual low IS response was not determined.



## 6.4 Blanks
Three types of blanks were analyzed during this ana lysis: method blanks were prepared with control largemouth bass fillet homogenate (TN08-0193-1/1) a nd were spiked with IS and surrogate to determine the endogenous PFOS concentration in the control ma trix for the LCSs in each analytical batch; (2) a bluegill “sunfish” control matrix (TN11-0194-1/1) w as analyzed in analytical runs g120420a and m120420a to ascertain endogenous PFOS levels in tha t control matrix, to compare to process control from MSU-WTL which were the same matrix that passed through the homogenization process; and (3) acetonitrile solvent blanks (with and without IS & surrogate) were also included for evaluations. All blank results were carefully evaluated to deter mine any effects from instrument contamination and analytical carry over and served to aid in defining the LLOQs for quantitation. All of the blank samp le results were considered acceptable upon evaluation of the raw data based on the intended use of the different types of blanks. The PFOS results for the bluegill (TN11-0194-1/1) c ontrol matrix blank tissue analyses was 0.804 + 0.089 ng/g (n = 6) and that result was used for com paring the homogenization process control sample results.


## 6.5 Laboratory Control Spikes (LCSs)
Laboratory control spikes (LCSs) were prepared by f ortifying known quantities of PFOS into 0.5 gram aliquots of a control bluegill fillet tissue. The LCS sets were prepared at three levels (nominal 10, 100 and 1000 ng/g fish tissue), and each level was prep ared in triplicate. Two sets of LCSs were prepared for these analyses, one with L-PFOS (same as calibr ation standards) and the second set fortified with brPFOS (30.6% branched isomer content). The LCS results are used to evaluate the method performance during the study and overall used for c alculation of the analytical method uncertainty for the study, as discussed in the conclusion section. The LCS results and the analytical method uncertaintie s determined from the statistical evaluation of LCS w ith L-PFOS and with br-PFOS are shown in Table 8 and Table 9 , respectively. The method acceptance criteria of 100 ± 30% was met for all LCSs, with a few exceptions as noted in the LCS results tables. The LCS recoveries were calculated using the follow ing equation:



### 100% * ion Concentrat Spike LCS)

of ion Concentrat d (Determine Recovery LCS =

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

15 Table 8. L-PFOS LCSs and Matrix Blank Results **Analytical Run ID**  **LCS Sample I.D. Measured PFOS**  **Concentration (ng/g) Endogenous PFOS**  **Conc. (ng/g) Fortified** 

**Conc. (ng/g) PFOS** 

**Recovery Average** 

**Recovery Recovery** 

**RSD**j120417a 120417-2-002MB 0.545

0.567 0.00 NA NA NA 120417-2-003MB 0.576 120417-2-004MB 0.580 120417-2-LowLCS 12.8 10.5 116% 114% 1.5% 120417-2-LowLCSD 13.1 10.9 115% 120417-2-LowLCST 11.9 10.1 113% 120417-2-MidLCS 108 102 106% 111% 6.0% 120417-2-MidLCSD 123 103 119% 120417-2-MidLCST 112 101 110% 120417-2-HighLCS >ULOQ 994 NA NA NA 120417-2-HighLCSD >ULOQ 1060 NA 120417-2-HighLCST >ULOQ 941 NAm120417b 120417-1-001MB 0.590

0.595 0.00 NA NA NA 120417-1-002MB 0.596 120417-1-003MB 0.598 120417-1-LowLCS 28.9 25.7 110% 107% 4.7% 120417-1-LowLCSD 27.0 26.1 101% 120417-1-LowLCST 25.6 23.0 109% 120417-1-MidLCS [a] 102 NA NA NA 120417-1-MidLCSD [a] 99.5 NA 120417-1-MidLCST [a] 101 NA 120417-1-HighLCS 953 1040 91.4% 87.5% 3.9% 120417-1-HighLCSD 794 933 85.1% 120417-1-HighLCST 901 1050 86.0%g120418b 120418-001MB 0.605

0.594 0.00 NA NA NA 120418-002MB 0.621 120418-003MB 0.555 120418-LowLCS 12.0 10.4 110% 110% 0.53% 120418-LowLCSD 11.8 10.3 110% 120418-LowLCST 12.8 11.0 111% 120418-MidLCS 115 106 109% 106% 2.4% 120418-MidLCSD 102 96.0 106% 120418-MidLCST 107 103 104% 120418-HighLCS 933 1040 90.1%
86.1% 4.4% 120418-HighLCSD 900 1050 85.8% 120418-HighLCST 867 1050 82.5%g120418b-HighRange 120418-MidLCS 113

0.594 106 106% 103% 2.9% 120418-MidLCSD 100 96.0 103% 120418-MidLCST 104 103 100% 120418-HighLCS 929 1040 89.7% 87.2% 2.8% 120418-HighLCSD 913 1050 87.0% 120418-HighLCST 891 1050 84.8%G120420a 120420-1-001MB 0.643

0.572 0.00 NA NA NA 120420-1-002MB 0.537 120420-1-003MB 0.535 120420-1-LowLCS 12.2 10.6 109% 109% 4.2% 120420-1-LowLCSD 12.1 10.1 114% 120420-1-LowLCST 10.9 9.88 105% 120420-1-MidLCS 100 95 105% 108% 2.8%

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

16 Table 8. L-PFOS LCSs and Matrix Blank Results **Analytical Run ID**  **LCS Sample I.D. Measured PFOS**  **Concentration (ng/g) Endogenous PFOS**  **Conc. (ng/g) Fortified** 

**Conc. (ng/g) PFOS** 

**Recovery Average** 

**Recovery Recovery** 

**RSD** 120420-1-MidLCSD 120 108 111% 120420-1-MidLCST 114 106 108% 120420-1-HighLCS 1090 929 117% 117% NA 120420-1-HighLCSD >ULOQ 916 NA 120420-1-HighLCST >ULOQ 950 NA 120420-1-HighLCS DF:100 10.1 929 108% 108% 2.4% 120420-1-HighLCSD DF:100 10.1 916 110% 120420-1-HighLCST DF:100 10.0 950 105%m120420a 120420-1-001MB 0.643

0.565 0.00 NA NA NA 120420-1-002MB 0.545 120420-1-003MB 0.508 120420-1-LowLCS 12.3 10.6 110% 108% 4.4% 120420-1-LowLCSD 11.9 10.1 112% 120420-1-LowLCST 10.7 9.88 103% 120420-1-MidLCS 96.6 95 101% 104% 3.4% 120420-1-MidLCSD 117 107.6 108% 120420-1-MidLCST 111 106 105% 120420-1-HighLCS 721 929 77.6% 89.2% 13% 120420-1-HighLCSD 816 916 89.1% 120420-1-HighLCST 960 950 101% 120420-1-HighLCS DF:100 8.88 929 95.5% 101% 4.9% 120420-1-HighLCSD DF:100 9.25 916 101% 120420-1-HighLCST DF:100 10.0 950 105%m120423a 120420-1-HighLCS df:10 948 0.565 [b] 929 102% 105% 2.9% 120420-1-HighLCSD df:10 953 916 104% 120420-1-HighLCST df:10 1030 950 108% 120417-2-HighLCS df:10 1050 0.571 [b] 1040 101% 99.9% 0.82% 120417-2-HighLCSD df:10 934 933 100% 120417-2-HighLCST df:10 1040 1050 99.0%



### Average Recovery (Low LCS) 110% Standard Deviation (Low LCS) 4.2% RSD (Low LCS) 3.8%

n 15 Average Recovery (Mid LCS) 107% Standard Deviation (Mid LCS) 4.5% RSD (Mid LCS) 4.2%

n 15 Average Recovery (High LCS) 89.8% Standard Deviation (High LCS) 10% RSD (High LCS) 11%

n 13 Average Recovery (High LCS-Diluted) 103% Standard Deviation (High LCS-Diluted) 4.4% RSD (High LCS-Diluted) 4.2%

n 12

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

17 Table 8. L-PFOS LCSs and Matrix Blank Results **Analytical Run ID**  **LCS Sample I.D. Measured PFOS**  **Concentration (ng/g) Endogenous PFOS**  **Conc. (ng/g) Fortified** 

**Conc. (ng/g) PFOS** 

**Recovery Average** 

**Recovery Recovery** 

**RSD** 

### Average Recovery (All Levels LCSs) 103% Standard Deviation (All Levels LCSs) 9.7% RSD (All Levels LCSs) 9.4%

n 55 2 x Std. Dev. (Analytical Method Uncertainty for L -PFOS) 19% Note: The endogenous level is the average result fr om three analyzed matrix blanks for the bass contro l tissue TN08-0193 used for LCS preparations. NA; not applicable [a] IS accidentally was not added to the preparatio n and LCS value not determined [b] Endogenous level was not determined in the anal ytical run, so previous result was used (when more than one result was available, preference was given to the endogenous PFOS result obtained on the same instrument)



### Table 9. brPFOS LCSs and Matrix Blank Results
**Analytical Run ID**  **LCS Sample I.D. Measured PFOS**  **Concentration (ng/g)**  **Endogenous PFOS**

**Conc. (ng/g) Fortified** 

**Conc. (ng/g) PFOS** 

**Recovery Average** 

**Recovery Recovery** 

**RSD**j120417a 120417-2-002MB 0.545

0.567 0.00 NA NA NA 120417-2-003MB 0.576 120417-2-004MB 0.580 120417-2-BLLowLCS 12.6 10.3 117% 113% 4.1% 120417-2-BLLowLCSD 10.6 9.29 108% 120417-2-BLLowLCST 11.4 9.64 113% 120417-2-BLMidLCS 112 95.6 117% 121% 3.4% 120417-2-BLMidLCSD 113 92.0 122% 120417-2-BLMidLCST 122 97.2 125% 120417-2-BLHighLCS >ULOQ 1000 NA NA NA 120417-2-BLHighLCSD >ULOQ 934 NA 120417-2-BLHighLCST >ULOQ 1020 NAm120417b 120417-1-001MB 0.548

0.595 0.00 NA NA NA 120417-1-002MB 0.549 120417-1-003MB 0.615 120417-1-BLLowLCS 12.7 10.5 115% 113% 2.4% 120417-1-BLLowLCSD 12.5 10.8 110% 120417-1-BLLowLCST 11.9 9.97 114% 120417-1-BLMidLCS 119 104 114% 112% 1.9% 120417-1-BLMidLCSD 103 92.7 111% 120417-1-BLMidLCST 116 105 110% 120417-1-BLHighLCS >ULOQ 1020 NA NA NA 120417-1-BLHighLCSD >ULOQ 999 NA 120417-1-BLHighLCST >ULOQ 975 NAg120418b 120418-001MB 0.605

0.594 0.00 NA NA NA 120418-002MB 0.621 120418-003MB 0.555 120418-BLLowLCS 12.8 10.7 114% 110% 3.2% 120418-BLLowLCSD 12.2 10.6 110% 120418-BLLowLCST 11.8 10.5 107%

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

18 Table 9. brPFOS LCSs and Matrix Blank Results
**Analytical Run ID**  **LCS Sample I.D. Measured PFOS**  **Concentration (ng/g)**  **Endogenous PFOS**

**Conc. (ng/g) Fortified** 

**Conc. (ng/g) PFOS** 

**Recovery Average** 

**Recovery Recovery** 

**RSD** 120418-BLMidLCS 125 109 114% 113% 1.4% 120418-BLMidLCSD 107 95.8 111% 120418-BLMidLCST 108 94.9 113% 120418-BLHighLCS >ULOQ 981 NA NA NA 120418-BLHighLCSD >ULOQ 1030 NA 120418-BLHighLCST >ULOQ 1010 NAg120418b-HighRange 120418-BLMidLCS 126

0.594 109 98.5% 96.5% 2.1% 120418-BLMidLCSD 107 95.8 94.4% 120418-BLMidLCST 108 94.9 96.5% 120418-BLHighLCS 1140 981 115% 121% 4.4% 120418-BLHighLCSD 1300 1030 125% 120418-BLHighLCST 1250 1010 123%G120420a 120420-1-001MB 0.643

0.572 0.00 NA NA NA 120420-1-002MB 0.537 120420-1-003MB 0.535 120420-1-BLLowLCS 11.5 9.76 112% 109% 2.3% 120420-1-BLLowLCSD 10.3 9.19 107% 120420-1-BLLowLCST 11.4 9.95 109% 120420-1-BLMidLCS 113 104 108% 111% 6.9% 120420-1-BLMidLCSD 113 93.5 120% 120420-1-BLMidLCST 117 110 106% 120420-1-BLHighLCS >ULOQ 948 NA NA NA 120420-1-BLHighLCSD >ULOQ 958 NA 120420-1-BLHighLCST >ULOQ 976 NAm120420a 120420-1-001MB 0.643

0.565 0.00 NA NA NA 120420-1-002MB 0.545 120420-1-003MB 0.508 120420-1-BLLowLCS 11.1 9.76 108% 105% 2.4% 120420-1-BLLowLCSD 9.98 9.19 103% 120420-1-BLLowLCST 11.0 9.95 105% 120420-1-BLMidLCS 111 104 106% 109% 6.9% 120420-1-BLMidLCSD 110 93.5 117% 120420-1-BLMidLCST 114 110 103% 120420-1-BLHighLCS >ULOQ 948 NA NA NA 120420-1-BLHighLCSD >ULOQ 958 NA 120420-1-BLHighLCST >ULOQ 976 NAm120423a 120420-1-BLHighLCS df:10 1005 0.565 [b] 948 106% 105% 1.0% 120420-1-BLHighLCSD df:10 996 958 104% 120420-1-BLHighLCST df:10 1030 976 105% 120417-2-BLHighLCS df:10 1110 0.571 [b] 1020 109% 105% 4.1% 120417-2-BLHighLCSD df:10 1050 999 105% 120417-2-BLHighLCST df:10 979 975 100% Average Recovery (Low LCS) 

### 110% Standard Deviation (Low LCS) 

### 4.0% RSD (Low LCS) 

### 3.6%
n 

### 15 Average Recovery (Mid LCS) 

### 113% Standard Deviation (Mid LCS) 

### 6.3%

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

19 Table 9. brPFOS LCSs and Matrix Blank Results
**Analytical Run ID**  **LCS Sample I.D. Measured PFOS**  **Concentration (ng/g)**  **Endogenous PFOS**

**Conc. (ng/g) Fortified** 

**Conc. (ng/g) PFOS** 

**Recovery Average** 

**Recovery Recovery** 

**RSD** RSD (Mid LCS) 

### 5.6%
n 

### 15 Average Recovery (High LCS) 

### NA Standard Deviation (High LCS) 

### NA RSD (High LCS) 

### NA
n 

### 0 Average Recovery (High LCS-Diluted) 

### 105% Standard Deviation (High LCS) 

### 2.8% RSD (High LCS-Diluted) 

### 2.7%

n 

### 6 **Average Recovery (All Levels LCSs)** 

### 110% **Standard Deviation (All Levels LCSs)** 

### 5.7% **RSD (All Levels LCSs)** 

### 5.1%

**n** 

### 36 **2 x Std. Dev. (Analytical Method Uncertainty for L -PFOS)**  

### 11% Note: The endogenous level is the average result fr om three analyzed matrix blanks for the bass contro l tissue TN08-0193 used for LCS preparations. NA; not applicable [a] IS accidentally not added to the preparation an d LCS value not determined [b] Endogenous level was not determined in the anal ytical run, so previous result was used (when more than one result was available, preference was given to the endogenous PFOS result obtained on the same instrument) 

## 6.6 Laboratory Matrix Spikes (LMSs)
Laboratory matrix spikes (LMSs) were generated by f ortifying known quantity of L-PFOS to an aliquot of fish fillet tissue and then preparing the sample th e same as regular non-fortified samples. For this study, each sample had at least two LMSs prepared, and sev eral had a third prepared at a higher level following initial analyses. The original two LMSs w ere fortified at nominal 50 and 500 ng/g, and for approximately 1⁄2 of the samples either the 50 ng/g o r 500 ng/g LMS was sufficient relative to the endogenous level to evaluate the sample-specific PF OS recovery. However, for the other 1⁄2 of samples the levels were significantly higher and required a LMS fortified at 5000 ng/g to sufficiently evaluat e recovery. The most appropriate LMS fortification f or evaluating recovery is typically between 0.5-tim es and 10-times the endogenous level determined for th e non-fortified sample. Exception(s): For the three homogenization process control fish tissues, the nominal 50 ng/g LMS was used to evaluate PFOS recovery, but was ~ 50-times the endogenous PFOS level in the corresponding non-fortified samples. Additionally, for sample IS O-12-01-01-049, the 500 ng/g LMS result was used to evaluate PFOS recovery, but was slightly less than 0.5-times the endogenous level of the corresponding non-fortified sample result. A SOP deviation was i ncluded in the raw data to accommodate these exceptions. Overall, the LMS recoveries for L-PFOS fortified in to the fish samples ranged from 76.7% to 125%, with an overall mean recovery + standard deviation of 98.9% + 10% (n=29 Tennessee river fish fillet homogenates). The LMS recoveries for the pre-, mi d- and post-study process controls were 102%, 99.1% and 102%, respectively. The LMS recovery fro m the analysis of the NIST SRM-1947 Lake

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

20 Michigan fish tissue homogenate was 95.3%. The met hod acceptance criteria of 100 ± 30% was met for all LMS results. The LMS recovery results are s ummarized in Table 1 and the individual sample results used for calculating LMS recoveries are sho wn in Table10

. The calculation of PFOS recoveries used the following 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
The MSU-WTL was provided two aliquots of a Lake Mic higan trout standard reference material (SRM 1947) purchased from the National Institutes of Sta ndards and Technology (NIST). The reference value of 5.9 + 0.39 ng/g PFOS was recently published for NIST SRM 1947 [Reiner et. al. 2012. Anal Bioanal Chem ; DOI 10.1007/s00216-012-5943-5]. Two aliquots of NIST SRM 1947 were provided to the MSU-WTL prior to the commencement of the study. One non-processed aliquot was subsequently returned to the 3M Environmental Laboratory with the processed Tennessee River fish samples and proc ess controls for analysis. The second aliquot of NIST SRM 1947 (not-processed) was sent to Axys Labo ratories by MSU-WTL to be analyzed with the three field duplicates. The 3M Environmental Laboratory’s determined averag e + RPD value for the NIST SRM 1947 sample was 6.71 ng/g + 0.27 ng/g PFOS for duplicate analytical sample res ults. The nominal 50 ng/g LMS recovery for the SRM 1947 was 95.3%. The results s how that the average PFOS concentration determined for SRM 1947 was accurate to 114% with r espect to the NIST SRM 1947 reference value.



## 6.8 Process Control Results
The MSU-WTL was provided three ~25 g aliquots of 3M -homogenized control bluegill fillet tissue TN11- 0194-1/1 that was purchased from Osage Catfisheries , Inc. (Osage Beach, Mo) prior to the study. One aliquot was planned to be homogenized at MSU prior to the homogenization of Tenn. River Fish (prestudy process control), one near the middle of the process of homogenizing the Tenn. River fish (midstudy process control), and one after the homogeniz ation of the Tenn. R. fish was completed (post-stud y process control). Those homogenization process con trols were returned with the sample shipments for analysis of PFOS and compared to the PFOS results o f for the same control bluegill tissue, but which did not travel to MSU-WTL for processing. The resu lts of those comparisons are shown in Table 10

. The results showed that the RPD was less than 15% f or each and that no significant contamination occurred during the homogenization process and ship ment process that would have affected the PFOS results of this study.

Table 10. Homogenization Process Control Results
**Sample ID Sample Description PFOS (ng/g) RPD**  TN11-0194-1/1 Bluegill fillet control tissue TN11-0 194-1/1 0.804 NA

ISO12-01-01-01-021 Pre-Study Process Control (TN11-0194-1/1) 0.815 1.3%

ISO12-01-01-01-022 Mid-Study Process Control (TN11-0194-1/1) 0.909 12%

ISO12-01-01-01-042 Post-Study Process Control (TN11-0194-1/1) 0.912 13% Relative percent difference (RPD) was calculated fo r the control tissue result relative to the process control result.

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

21 

## 6.9 Individual Sample Results
The individual analytical sample results are shown in Table 11 . A summary of the average concentrations with their RPDs and LMS recoveries d etermined from these results are provided in Table 1

. Table 11. Individual PFOS Results for Fillet Tissues from Fis h Collected in the Tennessee River, April 2012

**LIMS Sample ID Sample Description PFOS**  **Conc. (ng/g)**  **Avg. PFOS**

**Concentration (ng/g)**  **RPD (%) Surrogate**  **( 13**  **C 4**  **-PFOS) Recovery**  **[a] LMS Spike** 

**Concentration (ng/g)**  **LMS Spike**

**Recovery (%)**

ISO12-01-01-01-021 Pre-Study, Process Control #1 (TN11-0194-1/1) 0.806 0.815 2.0% 100% NA NA ISO12-01-01-01-021 Dup Pre

- Study, Process Control #1 Dup (TN11

- 0194

- 1/1) 0.823 100%

ISO12-01-01-01-021 LMS Pre-Study, Process Control #1 LMS (TN11-0194-1/1) 52.2 52.2 NA 102% 50.5 [d] 102%

ISO12-01-01-01-022 Mid-Study, Process Control #2 (TN11-0194-1/1) 1.07 0.909 [b] 34% 105% NA NA ISO12-01-01-01-022 Dup Mid-Study, Process Control #2 Dup (TN11-0194-1/1) 0.752 105%

ISO12-01-01-01-022 LMS Mid-Study, Process Control #2 LMS (TN11-0194-1/1) 53.2 53.2 NA 107% 52.8 [d] 99.1%
ISO12-01-01-01-023 MSF 01 120403 4810 4770 1.4% 98.6% NA NA ISO12-01-01-01-023 Dup MSF 01 120403 Dup 4740 107% ISO12

- 01

- 01

- 01

- 023 LMS High MSF 01 120403 LMS High 9340 9340 NA 101% 4730 96.5% ISO12-01-01-01-024 MSF 02 120403 4120 4240 5.3% 107% NA NA ISO12-01-01-01-024 Dup MSF 02 120403 Dup 4350 103% ISO12

- 01

- 01

- 01

- 024 LMS High MSF 02 120403 LMS 8580 8580 NA 98.4% 4700 92.4% ISO12-01-01-01-025 MSF 03 120403 71.4 70.7 1.9% 101% NA NA ISO12-01-01-01-025 Dup MSF 03 120403 Dup 70.0 102% ISO12-01-01-01-025 LMS MSF 03 120403 LMS 108 108 NA 101% 46.0 81.0% ISO12-01-01-01-026 MSF 04 120403 595 576 6.5% 100% NA NA ISO12-01-01-01-026 Dup MSF 04 120403 Dup 558 102% ISO12-01-01-01-026 LMS MSF 04 120403 LMS 1150 1150 NA 98.9% 524 108% ISO12-01-01-01-027 MSF 05 120403 460 471 4.9% 102% NA NA ISO12-01-01-01-027 Dup MSF 05 120403 Dup 483 100% ISO12-01-01-01-027 LMS MSF 05 120403 LMS 955 955 NA 100% 482 100% ISO12-01-01-01-028 MSF 06 120403 152 156 5.4% 103% NA NA ISO12-01-01-01-028 Dup MSF 06 120403 Dup 160 105% ISO12-01-01-01-028 LMS MSF 06 120403 LMS 581 581 NA 107% 554 76.7% ISO12-01-01-01-029 MSF 07 120403 2900 2920 1.6% 103% NA NA ISO12-01-01-01-029 Dup MSF 07 120403 Dup 2950 101% ISO12-01-01-01-029 LMS MSF 07 120403 LMS 8140 8140 NA 101% 5280 98.7% ISO12-01-01-01-030 MSF 08 120403 1350 1360 0.13% 101% NA NA ISO12-01-01-01-030 Dup MSF 08 120403 Dup 1360 104% ISO12-01-01-01-030 LMS MSF 08 120403 LMS 6270 6270 NA 100% 4920 99.8% ISO12-01-01-01-031 MSF 09 120403 2070 2020 5.2% 104% NA NA ISO12-01-01-01-031 Dup MSF 09 120403 Dup 1970 106% ISO12-01-01-01-031 LMS MSF 09 120403 LMS 7480 7480 NA 100% 5280 104% ISO12-01-01-01-032 MSF 10 120403 1740 1840 10% 94.8% NA NA ISO12-01-01-01-032 Dup MSF 10 120403 Dup 1930 91.0% ISO12-01-01-01-032 LMS MSF 10 120403 LMS 7170 7170 NA 100% 5060 105% ISO12-01-01-01-033 MSF 21 120404 651 671 6.1% 89.7% NA NA ISO12-01-01-01-033 Dup MSF 21 120404 Dup 692 96.3%

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

22 Table 11. Individual PFOS Results for Fillet Tissues from Fis h Collected in the Tennessee River, April 2012

**LIMS Sample ID Sample Description PFOS**  **Conc. (ng/g)**  **Avg. PFOS**

**Concentration (ng/g)**  **RPD (%) Surrogate**  **( 13**  **C 4**  **-PFOS) Recovery**  **[a] LMS Spike** 

**Concentration (ng/g)**  **LMS Spike**

**Recovery (%)**

ISO12

- 01

- 01

- 01

- 033 LMS MSF 21 12040 4 LMS 108 0 1080 NA 90.1% 478 86.0% ISO12

- 01

- 01

- 01

- 034 MSF 22 120404 457 0 4660 3.5% 90.7% NA NA ISO12-01-01-01-034 Dup MSF 22 120404 Dup 4740 94.1% ISO12-01-01-01-034 LMS MSF 22 120404 LMS 9650 9650 NA 98.3% 5010 99.6% ISO12

- 01

- 01

- 01

- 035 MSF 23 120404 52.4 52.5 0.0056% 97.8% NA NA ISO12

- 01

- 01

- 01

- 035 Dup MSF 23 120404 Dup 52.5 102% ISO12

- 01

- 01

- 01

- 035 LMS Low MSF 23 120404 LMS 552 552 NA 91.9% 502 99.7% ISO12

- 01

- 01

- 01

- 036 MSF 24 120405 464 437 12% 102% NA NA ISO12

- 01

- 01

- 01

- 036 Dup MSF 24 120405 Dup 409 87.1% ISO12

- 01

- 01

- 01

- 036 LMS MSF 24 120405 LMS 884 884 NA 98.8% 494 90.7% ISO12

- 01

- 01

- 01

- 037 MSF 25 120405 312 306 3.8% 95.8% NA NA ISO12

- 01

- 01

- 01

- 037 Dup MSF 25 120405 Dup 300 101% ISO12

- 01

- 01

- 01

- 037 LMS MSF 25 120405 LMS 999 999 NA 91.0% 554 125% ISO12

- 01

- 01

- 01

- 038 MSF 26 120405 2 100 2090 1.0% 94.0% NA NA ISO12

- 01

- 01

- 01

- 038 Dup MSF 26 120405 Dup 20 80 100% ISO12

- 01

- 01

- 01

- 038 LMS MSF 26 120405 LMS 68 30 6830 NA 100% 5030 94.2% ISO12

- 01

- 01

- 01

- 039 MSF 27 12040 3 206 0 2120 5.4% 95.1% NA NA ISO12

- 01

- 01

- 01

- 039 Dup MSF 27 12040 3 Dup 21 80 96.1% ISO12

- 01

- 01

- 01

- 039 LMS MSF 27 12040 3 LMS 738 0 7380 NA 102% 4950 106% ISO12

- 01

- 01

- 01

- 040 Dup MSF 28 120404 Dup 172 180 9.2% 98.2% NA NA ISO12

- 01

- 01

- 01

- 040 MSF 28 120404 188 107.9% ISO12

- 01

- 01

- 01

- 040 LMS MSF 28 120404 LMS 783 783 NA 97.1% 487 124% ISO12

- 01

- 01

- 01

- 041 MSF 29 12040 5 20 50 2050 0.063% 92.0% NA NA ISO12

- 01

- 01

- 01

- 041 Dup MSF 29 12040 5 Dup 20 50 94.4% ISO12

- 01

- 01

- 01

- 041 LMS MSF 29 12040 5 LMS 710 0 7100 NA 102% 4953 102%
ISO12-01-01-01-042 Post Study , Process Control #3 0.984 0.912 16% 101% NA NA ISO12-01-01-01-042 Dup Post Study, Process Control #3 Dup (TN11-0194-1/1) 0.839 99.0%

ISO12-01-01-01-042 LMS Post Study, Process Control #3 LMS (TN11-0194-1/1) 56.3 56.3 NA 103% 54.1 [d] 102%
ISO12

- 01

- 01

- 01

- 043 Reference Tissue (NIST SRM 1947) 6.85 6.71 4.0% 98.8% NA NA ISO12

- 01

- 01

- 01

- 043 Dup Reference Tissue Dup (NIST SRM 1947) 6.58 102% ISO12

- 01

- 01

- 01

- 043 LMS Reference Tissue 1/2 LMS (NIST SRM 1947) 54.9 54.9 NA 100% 50.6 95 .3 % ISO12

- 01

- 01

- 01

- 044 MSF 11 12

- 04

- 03 299 0 2940 3.9% 100% NA NA ISO12

- 01

- 01

- 01

- 044 Dup MSF 11 12

- 04

- 03 Dup 2880 101% ISO12

- 01

- 01

- 01

- 044 LMS MSF 11 12

- 04

- 03 LMS 75 80 7580 NA 101% 4970 93.5% ISO12

- 01

- 01

- 01

- 045 MSF 12 12

- 04

- 03 537 526 4.2% 103% NA NA ISO12

- 01

- 01

- 01

- 045 Dup MSF 12 12

- 04

- 03 Dup 515 99.0% ISO12

- 01

- 01

- 01

- 045 LMS MSF 12 12

- 04

- 03 LMS 545 0 5450 NA 104% 4880 101% ISO12

- 01

- 01

- 01

- 046 MSF 13 12

- 04

- 03 9 42 950 1.7% 96.5% NA NA ISO12

- 01

- 01

- 01

- 046 Dup MSF 13 12

- 04

- 03 Dup 9 58 102% ISO12

- 01

- 01

- 01

- 046 LMS MSF 13 12

- 04

- 03 LMS 58 10 5810 NA 100% 4910 99.0 % ISO12

- 01

- 01

- 01

- 047 MSF 14 12

- 04

- 03 33 60 3370 0.505 100% NA NA ISO12

- 01

- 01

- 01

- 047 Dup MSF 14 12

- 04

- 03 Dup 337 0 97.6% ISO12

- 01

- 01

- 01

- 047 LMS MSF 14 12

- 04

- 03 LMS 82 70 8270 NA 100% 4810 102% ISO12

- 01

- 01

- 01

- 048 MSF 15 12

- 04

- 03 718 754 9.6% 98.7% NA NA ISO12

- 01

- 01

- 01

- 048 Dup MSF 15 12

- 04

- 03 Dup 790 102% ISO12

- 01

- 01

- 01

- 048 LMS MSF 15 12

- 04

- 03 LMS 56 70 5670 NA 102% 4940 99.4% ISO12

- 01

- 01

- 01

- 049 MSF 16 12

- 04

- 04 1550 [e] 1520 3.2% 100% NA NA ISO12-01-01-01-049 Dup MSF 16 12-04-04 Dup 1500 [e] 99.0% ISO12-01-01-01-049 LMS MSF 16 12-04-04 LMS 1950 [e] 1950 NA 100% 493 [c] 86.9% ISO12-01-01-01-050 MSF 17 12-04-04 165 165 0.75% 101% NA NA

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

23 Table 11. Individual PFOS Results for Fillet Tissues from Fis h Collected in the Tennessee River, April 2012

**LIMS Sample ID Sample Description PFOS**  **Conc. (ng/g)**  **Avg. PFOS**

**Concentration (ng/g)**  **RPD (%) Surrogate**  **( 13**  **C 4**  **-PFOS) Recovery**  **[a] LMS Spike** 

**Concentration (ng/g)**  **LMS Spike**

**Recovery (%)**

ISO12

- 01

- 01

- 01

- 050 Dup MSF 17 12

- 04

- 04 Dup 166 100% ISO12

- 01

- 01

- 01

- 050 LMS MSF 17 12

- 04

- 04 LMS 581 581 NA 100% 493 84.2% ISO12

- 01

- 01

- 01

- 051 MSF 18 12

- 04

- 04 710 701 2.7% 100% NA NA ISO12

- 01

- 01

- 01

- 051 Dup MSF 18 12

- 04

- 04 Dup 691 101% ISO12

- 01

- 01

- 01

- 051 LMS MSF 18 12

- 04

- 04 LMS 6080 6080 NA 99.0% 5220 103% ISO12

- 01

- 01

- 01

- 052 MSF 19 12

- 04

- 04 672 675 0.86% 101% NA NA ISO12

- 01

- 01

- 01

- 052 Dup MSF 19 12

- 04

- 04 Dup 678 102% ISO12

- 01

- 01

- 01

- 052 LMS MSF 19 12

- 04

- 04 LMS 5620 5620 NA 100% 4780 104% ISO12

- 01

- 01

- 01

- 053 MSF 20 12

- 04

- 04 1170 1180 0.82% 101% NA NA ISO12

- 01

- 01

- 01

- 053 Dup MSF 20 12

- 04

- 04 Dup 1180 100% ISO12

- 01

- 01

- 01

- 053 LMS MSF 20 12

- 04

- 04 LMS 6150 6150 NA 101% 4780 104% All concentration values are rounded to 3 significa nt figures for reporting. Statistical results are rounded to 2 significant figures. As a result, ra w data values may vary slightly from those reported NA; not applicable [a]Surrogate was spiked into sample aliquots at nom inal 1.00 ng/g, result is shown for the least dilut ed sample result [b] RPD > 20% [c] The LMS spike concentration was less than 1⁄2 of the endogenous PFOS concentration for the non-forti fied sample results [d] The LMS spike concentration was greater than 10 -times of the endogenous PFOS concentration for the non-fortified sample results [e] Value was determined from a six point high-rang e calibration curve from 7.5 ng/mL to 250 ng/mL for quantifying PFOS in tissues from nominal 75.0 to 2 500 ng/g. All other reported results were obtained wit h calibration from 0.025 ng/mL to 100 ng/mL and the 250 ng/mL standard excluded, and samples were typically diluted to within that range when they ex ceeded 1000 ng/g.



## 7 Conclusion
The determination of perfluorooctane sulfonate (PFO S) concentrations for 29 largemouth bass fillet tissues (skin removed) collected from the Tennessee River in April 2012 near Decatur, Alabama was successfully completed. In addition, 3 homogenizat ion process controls and one NIST SRM 1947 standard reference material were analyzed. This an alysis was conducted in support of the Weston Solutions, Inc. project plan, titled “Work Plan for Fish Sampling and Analysis for PFOS in Largemouth Bass from the Tennessee River in the Vicinity of th e 3M Decatur Facility, Decatur, Alabama”. The analytical aspects reported herein were conducted i n the 3M Environmental Laboratory as 3M study ISO12-01-01-01. Fish fillets (skin removed) were prepared in the fi eld by Weston Solutions, Inc. personnel after being collected from the Tennessee River and the shipped to the Michigan State University Wildlife Toxicolog y Laboratory where fish fillet homogenates were prepa red. The frozen homogenates were then shipped to the 3M Environmental Laboratory in two shipments that were received on April 14, 2012 and April 17, 2012. Quantitative analysis for PFOS was performe d from April 16, 2012 through April 24, 2012 following analytical method ETS-8-045.1. The determined average concentrations of PFOS in th e Tennessee River fish tissue samples ranged from 52.5 ng/g to 4770 ng/g. The recoveries of fo rtified PFOS from the LMS samples prepared for each Tennessee River fish tissue samples were within 100 + 25% recovery, ranging from 76.7% to 125%, with an overall average + standard deviation of 98.9% + 10%. Additionally, the average recovery and standard deviation for a surrogate recovery standar d ( 13 

### C 4 

### -PFOS) for all of the Tennessee River fish samples was 99.5% + 4.3%. The determined average PFOS value and relative diff erence for the NIST SRM 1947 fish tissue that was also received was 6.71 + 0.27 ng/g (4.0% RPD); a 114% recovery relative to the NIST reference value of 5.90 + 0.39 ng/g.

### Study: ISO12-01-01-01 Analysis for PFOS in Fish Collected from the Tennes see River in April 2012

25 

# Attachment A Preparation Forms, Raw Data and Chromatograms

(Available Upon Request)