Document 3m65nOZdQZqw4292L1011pL3

A R V Lk'W l PFOS: AN EARLY LIFE-STAGE TOXICITY TEST WITH THE FATHEAD MINNOW (.Pimephales promelas) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-108 3M LAB REQUEST NO. U2723 U.S Environmental Protection Agency "Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1400 OECD Guideline 210 AUTHORS: Kurt R. Drottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: July 2, 1999 STUDY COMPLETION DATE: April 26,2000 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 72 000337 W il d l if e In t e r n a t io n a l ltd. - 2- PROJECT NO.: 454A-108 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: An Early Life-Stage Toxicity Test with the Fathead Minnow (Pimephalespromelas) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-108 STUDY COMPLETION: April 26,2000 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792,17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: The test substance was not characterized in accordance with full GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being m aintained in the 3M archives. The test substance is being recharacterized in accordance with GLP. The stability o f the test substance under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Senior Biologist SPONS DATE C039S W il d l if e In t e r n a t io n a l ltd. -3 - PROJECT NO.: 454A-108 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: "t)ATE CONDUCTED: DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: Test Substance Preparation Analytical Sampling, DO Measurements and Egg Separation for Test Initiation Observations Analytical Data and Draft Report Biological Data and Draft Report Final Report August 2,1999 August 2,1999 August 5,1999 September 9,1999 August 5,1999 September 9,1999 December 3,6 and 7,1999 December 7,1999 January 3-5,2000 April 24 and 25,2000 January 5,2000 April 26,2000 August 4,1999 August 9,1999 September 10,1999 December 15,1999 January 7,2000 April 26,2000 Marshall (L Hynson Quality Assurance Program S 000999 W il d l if e In t e r n a t io n a l ltd. -4 - REPORT APPROVAL PROJECT NO.: 454A-108 SPONSOR: 3M Corporation TITLE: PFOS: An Early Life-Stage Toxicity Test with the Fathead Minnow (Pimephalespromelas) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-108 STUDY DIRECTOR: MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants DATE O O IO O O W il d l if e International ltd. -5 - PROJECT NO.: 454A-108 TABLE OF CONTENTS Title/Cover Page.................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement...............................................................................................2 Quality Assurance Statement................................................................................................................................3 Report Approval....................................................................................................................................................4 Table o f Contents..................j '........................................................................................................................... 5 Summary................................................................................................................................................................ 7 Introduction........................................................................................................................................................... 8 O b jectiv e........................................................................................................................... 8 Experimental Design.............................................................................................................................................8 Materials and Methods............................................................................................................ 9 Results and Discussion..................................................................................................................... C o nclusions...................................................................................................................................... R eferen ces........................................................................................................... 14 15 TABLES Table 1 - Summary o f Analytical Chemistry Data........................................................................................17 Table 2 - Specific Conductance, Hardness and Alkalinity o f the Test solutions........................................ 18 Table 3 - pH o f W ater in the Test Chambers.............................................................................................. 19 Table 4 - Dissolved Oxygen Content (mg/L) o f Water in the Test Chambers.......................................... 20 O O lC O l W il d l if e In t e r n a t io n a l ltd. 6- - PROJECT NO.: 454A-108 TABLE OF CONTENTS - Continued - TABLES (Cont'd.) Table 5 - Temperature (C ) o f W ater in the Test Chambers.......................................................................... 21 Table 6 - Hatching Success o f Fathead Minnow Embryos Exposed to the Test Substance........................................................................................................................ 22 Table 7 - Survival o f Fathead Minnow Larvae Exposed to the Test Substance for 42 Days Post-H atch.................................................................................................................... 23 Table 8 - Total Length, W et W eight and Dry Weight o f Fathead Minnow Larvae at the End o f the 42-Day Post-Hatch Observation Period............................................................. 24 APPENDICES Appendix I - Dilution W ater Chemistry Parameters Measured During the 4-Week Period Immediately Preceding the Test.................................... .......................25 Appendix II- Analyses o f Pesticides, Organics, Metals and Other Inorganics in W ildlife International Ltd. Well W ater........................................................................ 26 Appendix H I- The Analysis o f PFOS in Freshwater in Support of Wildlife International Ltd. Project N o.: 454A-108....................................................................... 27 Appendix IV - Cumulative Percent M ortality and Treatment-Related E ffects.........................................47 Appendix V - Fish Total Length (mm )..................................................... 56 Appendix VI - Fish W et W eight (mg)..........................................................................................................61 Appendix VII - Fish Dry Weight (m g)..........................................................................................................66 Appendix VIII - Changes to Protocol.............................................................................................................71 Appendix IX - Personnel Involved in the Study........................................................................................72 001002 W il d l if e In t e r n a t io n a l ltd. PROJECT NO.: 454A-108 1 1 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: F t EST ORGANISM: SOURCE OF TEST ORGANISMS: . AGE OF TEST ORGANISMS: NO-OBSERVED-EFFECTCONCENTRATION: LOWEST-OBSERVED-EFFECTCONCENTRATION: MAXIMUM ACCEPTABLE TOXICANT CONCENTRATION: SUMMARY 3M Corporation Susan A. Beach Wildlife International Ltd. Easton, Maryland 21601 454A-108 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: An Early Life-Stage Toxicity Test with the Fathead Minnow (.Pimephales promelas) Negative Control, 0.14,0.29,0.57,1.1,2.3 and 4.6 mg a.i./L Negative Control, 0.15,0.30,0.60,1.2,2.4 and 4.6 mg a.i./L Experimental Start - August 5,1999 Biological Termination - September 21,1999 Experimental Termination - September 22,1999 47 Days Fathead Minnow (Pimephales promelas) Wildlife International Ltd. Cultures Easton, Maryland 21601 Eggs <24 hours old at test initiation 0.30 mg a.i./L 0.60 mg a.i./L 0.42 mg a.i./L 001003 W il d l if e In te r n a tio n a l ltd. - 8- PROJECT NO.: 454A-108 INTRODUCTION This study was conducted by W ildlife International Ltd. for 3M Corporation atthe Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase o f the test was conducted from August 5, 1999 to September 21,1999. Raw data generated by Wildlife International Ltd. and a copy ofthe finalreport are filed under Project Number 454A-108 in archives located on the Wildlife International Ltd. site. v OBJECTIVE The objective o f this study was to evaluate the effects of PFOS on early life-stage development o f the fathead minnow, Pimephales promelas. Time to hatch, hatching success, survival and growth were evaluated during the 47-day study. EXPERIMENTAL DESIGN Fathead minnow embryos were exposed to a geometric series o f six test concentrations and a negative (dilution water) control under flow-through conditions. Four replicate test chambers were maintained in each treatment and the control group. Each test chamber contained one incubation cup with 20 embryos,resulting in a total o f 80 embryos per treatment, th e exposure period included a 5-day embryo hatching period, and a 42-day post-hatch juvenile growth period. The Sponsor requested that the study be extended past the normal 28-day post-hatch growth period to determine if survivorship would be affected. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon acute toxicity data. Nominal test concentrations were 0.14,0.29,0.57,1.1,2.3 and 4.6 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples of test water collected from each treatment and the control group at the beginning ofthe test, on Day 4, at weekly intervals during the test, and at test termination. To start the test, embryos less than 24 hours old were collected from cultures and groups of 1 and 2 individuals were impartially distributed among incubation cups until each cup contained 20 embryos. One cup was then placed in each treatment and control test chamber. Twice during the next twenty-four hours and daily thereafter, all dead embryos were counted and removed from the cups to avoid contaminating viable embryos. All 001004 W ild life International ltd. -9 - PROJECT NO.: 454A-108 eggs that remained were considered viable. Dead embryos continued to be removed daily. After hatching, the larvae were counted and released into the test chambers, where exposure continued until test termination. Observations o f mortality and other clinical signs were made daily during the test. Time to hatch, hatching success, growth, and survival were monitored in each treatment and control group. Data were evaluated to determine the no-observed-effect-concentration (NOEC), the lowest-observed-effect-concentration (LOEC) and the maximum acceptable toxicant concentration (MATC). MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: An Early LifeStage Toxicity Test with the Fathead Minnow (Pimephalesprometas)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.1400 (1); OECD Guidelinefo r Testing o f Chemicals, 210: Fish Early-Life Stage Toxicity Test (2); ASTM Standard E1241-88, Standard Guidefor Conducting Early Life-Stage Toxicity Tests with Fish (3) and Standard Evaluation Procedure, Fish Early Life-Stage Test (4). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity of 90.49%, and an expiration date o f 2008. The test substance was stored at ambient room temperature. Preparation of Test Concentrations One stock solution was prepared for each o f the six concentrations tested. A primary stock solution was prepared by dissolving PFOS in dilution water at a concentration o f 88.4 mg a.i./L. The primary stock solution was stirred with an electric stainless steel top-down mixer until all the test substance was dissolved. The primary stock solution was proportionally diluted with dilution water to prepare five secondary stock solutions at concentrations o f 44.2, 22.1,11.0,5.52 and 2.76 mg a.i./L. Stock solutions were prepared every three to four days during the test. The five stocks were injected into the diluter mixing chambers (at a rate o f 6.0 mL/minute) 001005 W il d l if e Intern atio na l ltd. -10- PROJECT NO.: 454A-108 where they were mixed with dilution water (at a rate o f 116 mL/minute) to achieve the desired test concentrations. The resultant test concentrations were adjusted for purity of the active ingredient in the test substance (90.49%). Test Organism The fathead minnow, Pimephales promelas, was selected as the test species for this study. Fathead minnows are representative of an important group o f aquatic vertebrates and were selected for use in the study based upon past history o f use and ease o f culturing in the laboratory. Fathead minnow embryos used in this test originated from cultures maintained by Wildlife International Ltd., Easton, Maryland. The embryos were removed from spawning substrates and examined under the dissecting microscope to select healthy specimens at approximately the same stage o f development. Embryos collected for use in the test were from six individual spawns, and were less than 24 hours old when the test was initiated. Newly-hatched larvae were fed live brine shrimp nauplii {Artemia sp.) 3 times per day during the first 7 days post-hatch. On Days 8 through 40 post-hatch, all fish were fed live brine shrimp nauplii 3 times daily on weekdays and 2 times daily on weekends. Brine shrimp nauplii were obtained by hatching cysts purchased from Bonneville Artemia International Inc., Salt Lake City, Utah. Fish were not fed for at least 48 hours prior to the termination o f the test to allow for clearance o f the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted each week to account for losses due to mortality. The average wet weight o f 16 fish in the negative control (Replicate A) measured at the end of the test was 0.16 g. Biomass loading (the total wet weight of the fish per liter o f test water) at the end of the test was 0.062 g o f fish per liter o ftest water that passed throughthe test chamber during a 24-hour period. Instantaneous loading (the total wet weight o f fish per liter o f water in the tank) at the end of the test was 0.37 g o f fish per liter o f test water. Test Apparatus A continuous-flow diluter was used to deliver each concentration o f the test substance and a negative (dilution water) control. A peristaltic pump (Cole-Parmer Instrument Company) was used to deliver the six test substance stock solutions into mixing chambers assigned to each treatment group. The stock solutions were diluted with well water in the mixing chambers in order to obtain the desired test concentrations. The flow o f 001006 W il d l if e In ter n a tio n a l ltd. - 11- PROJECT NO.: 454A-108 dilution water to the mixing chambers was controlled by rotameters. Rotameters were calibrated prior to test initiation and at weekly intervals thereafter. The flow of test water from each mixing chamber was split and allowed to flow into four replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test, and at weekly intervals thereafter to ensure that flow rates varied by no more than 10% o f the mean o f the four replicates. The diluter was adjusted so that each test chamber received approximately 6.0 volume additions o f test water every 24 hours. The test substance delivery pump was calibrated before the test and at weekly intervals thereafter. The general operation o f the diluter was checked visually at least two times per day during the test and at least once at the beginning and end o f the test. The test chambers were 9-L glass aquaria filled with approximately 7 L o ftest solution. The depth ofthe test water in a representative test chamber was approximately 17 cm. The test chambers were impartially positioned in a temperature-controlled environmental chamber designed to maintain a temperature o f 252C. Test chambers were labeled with the project number, test concentration and replicate. The embryo incubation cups were suspended in the water column of each test chamber and attached to a rocker arm. The reciprocating motion o f the rocker arm (approximately 2 rpm) facilitated circulation oftest water around the embryos during incubatioii. The incubation cups were constructed from glass cylinders approximately 50 mm in diameter with 425 /m nylon screen mesh attached to the bottom with silicone sealant. Dilution W ater The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the W ildlife International Ltd. site. The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity, and pH o f the well water during the four-week period immediately preceding the test are presented in Appendix I. The well water was passed through a sand filter to remove particles greater than approximately 25 /urn, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to delivery to the diluter system, the water again was filtered (0.45 pm), then passed through a UV sterilizer to remove 001007 Wil d l if e Internatio nal ltd. -12- PROJECT NO.: 454A-108 microorganisms and particles. The results o f periodic analyses performed to measure the concentrations of selected contaminants in well water used by Wildlife International Ltd. are presented in Appendix n. Environmental Conditions Lighting used to illuminate the cultures and test chambers during culturing and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod of 16 hours o f light and 8 hours o f dark was controlled with an automatic timer. A 30-minute transition period of low light intensity was provided\vhen lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the surface o f the negative control (replicate A) at test initiation, at test termination and at weekly intervals during the test. Light intensity ranged from 581 to 619 lux. Temperature was measured in each test chamber at the beginning and end o f the test and at weekly intervals during the test using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control replicate using a Fulscope ER/C Recorder. The target test temperature during the study was 25 2C. The pH was measured at the beginning and end o f the test and at weekly intervals during the test in alternating replicates o f each treatment and the control group. Dissolved oxygen concentrations were measured daily in alternating replicates o f each treatment and the control group during the first 7 days o f the test, at weekly intervals during the test, and at test termination. Hardness, alkalinity and specific conductance were measured in the negative control and the highest treatment group with surviving organisms at the beginning o f the test, once a week during the test, and at test termination. Measurements o f pH were made using a Fisher Accumet Model 915 pH meter and dissolved oxygenwas measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Hardness and alkalinity were measured based on procedures in Standard Methods fo r the Examination o f Water and Wastewater (5). Biological Observations and Measurements Observations o f mortality were made twice during the first 24 hours o f the embryo exposure period and daily until hatch. After hatching, larvae were observed daily to evaluate the number ofmortalities and the number o f individuals exhibiting clinical signs o f toxicity or abnormal behavior during the 42-day post-hatch exposure 001008 W il d l if e In ter n a tio n a l ltd. -13- PROJECT NO.: 454A-108 period. From these observations, hatching success, time to hatch, and post-hatch growth and survival were evaluated. Hatching success was estimated as the percentage o fviable embryos that hatched successfully. Post hatch survival was estimated as the number of larvae surviving to test termination divided by the number o f embryos which hatched successfully. Post-hatch growth o fthe fathead minnows was evaluated atthe conclusion o f the 42-day post-hatch exposure period. Total lengths for each surviving fish were determined using a digitizing tablet with the SIGMA SCANTM scientific measurement system and wet and dry weights were measured using an analytical balance. Data Analyses Hatching success, post-hatch survival, wet weight, dry weight and total length o f thejuvenile fish were test endpoints that were analyzed statistically. Data collected during this test were either continuous-variable data (e.g., weight and length) or discrete-variable data (e.g., mortality proportions). Discrete-variable data were analyzed using 2 X 2 contingency tables to identify treatment groups that showed a statistically significant difference (psO.05) from the negative control group. All continuous-variable data were evaluated for normality using Shapiro-Wilk's test and for homogeneity ofvariance using Bartlett's test Analysis o f variance and Dunnett's test were used to evaluate differences between treatment and control means. The results o f the statistical analyses were used to aid in the determination o f the NOEC, LOEC and MATC. The MATC was estimated as the geometric mean of the NOEC and LOEC. All statistical tests were performed on a personal computer using TOXSTAT 3.5 (6) and SPSS/PC Version 2.0 (7) statistical software. Analytical Chemistry Prior to test initiation, one set o fpretest water samples was collected from two replicate test chambers of each treatment group and the negative control to evaluate diluter performance. On Days 0, 4,7,14,21,28,35, 42 and at test termination, samples were collected from two alternating replicate test chambers o f each treatment and the control group to measure concentrations o f the test substance. Samples were collected at mid-depth from each test chamber. Samples were collected in 30-mL Nalgene bottles and analyzed immediately. Analytical procedures used in the analysis o f the samples are provided in Appendix HI. 001009 W il d l if e Intern atio na l ltd. -14- PROJECT NO.: 454A-108 RESULTS AND DISCUSSION Verification o f Test Concentrations Results o f analyses to measure concentrations o f PFOS in water samples collected during the test are presented in Table 1 and in the analytical chemistry report (Appendix HI). Nominal concentrations selected for use in this study were 0.14, 0.29, 0.57, 1.1,2.3 and 4.6 mg a.i./L. When measured concentrations of samples analyzed during the test were averaged, the mean measured concentrations were 0.15,0.30,0.60,1.2,2.4 and 4.6 mg a.i./L, which represented 107,103, 105,109, 104 and 100% o f the nominal concentrations, respectively. Mean measured concentrations were used to express the NOEC, LOEC and MATC. Pre-testsamples collectedto verify diluter operation (Appendix HI) were not used to calculate mean measured values. Physical and Chemical Measurements o f Water Measurements o f the specific conductance, hardness and alkalinity made on the negative control and treatment water are presented in Table 2. No apparent differences in these parameters existed between the treatments and the control group. Measurements o f pH, dissolved oxygen, and temperature are presented in Tables 3, 4, and 5, respectively. W ater pH measurements were consistent over time and showed no obvious differences between control water and the treatment groups. Measurements o f pH ranged from 8.0 to 8.4. Dissolved oxygen concentrations remained *7.6 mg/L (92% of saturation). All temperature measurements were within the desired range o f 252 C.' Hatching Success and Time to Hatch All viable fathead minnow embryos hatched on Day 4 or 5 o f the test (Table 6). There wereno apparent differences between the time to hatch in the negative control and the PFOS treatmentgroups. Hatching success in the negative control was 93%. Hatching success in the PFOS treatment groups was 2:90%. Hatching success was not significantly different in any PFOS treatment group in comparison to the negative control (p > 0.05). Post-Hatch Survival and Clinical Observations After 42 days o f post-hatch exposure, survival in the negative control was 88% (Table 7). Post-hatch survival in the 0.15, 0.30, 0.60, 1.2, 2.4 and 4.6 mg a.i./L treatment groups was 79, 81, 66, 5.4, 0 and 0%, respectively. Analysis o f post-hatch survival data by 2 X 2 contingency tables showed that survival was significantly different in the 0.60, 1.2, 2.4 and 4.6 mg a.i./L treatment groups in comparison to the negative 001010 W il d l if e In ter n a tio n a l ltd. -15- PROJECT NO.: 454A-108 control (p i 0.05). All fathead minnows surviving to test termination appeared normal with no overt signs of sublethal toxicity. Fathead minnows which did not survive generally appeared to be swimming erratically prior to death (Appendix IV). Growth Growth data for each treatment and the control group are summarized in Table 8. The individual measurements are provided in Appendices V, VI and VII. Growth was evaluated at testtermination by measuring the total length, wet weight and dry weight o f each surviving fish. The statistical analyses o f growth data did not include PFOS treatment groups which had a statistically significant effect on survival. Fatheadminnows exposed to PFOS at concentrations o f 0.15 or 0.30 mg a.i./L for 42 days post-hatch showed no statistically significant reductions in total length, wet weight or dry weight in comparison to the negative control (p > 0.05). CONCLUSIONS Fathead minnows (jPimephales promelas) exposed to PFOS at concentrations 0.30 mg a.i./L for 42 days post-hatch showed no statistically significant reductions in time to hatch, hatching success, survival or growth. The most sensitive endpoint in this study was post-hatch survival. Fathead minnows exposed to PFOS at concentrations s 0.60 mg a.i./L had statistically significant differences in post-hatch survival in comparison to the negative control. Consequently; the LOEC for this study, based on post-hatch survival, was 0.60 mg a.i./L. The NOEC was 0.30 mg a.i./L and the MATC was calculated to be 0.42 mg a.i./L. O O lO ll W il d l if e In t e r n a t io n a l ltd. -16- PROJECT NO.: 454A-108 REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines {draft), OPPTS Number 850.1400: Fish Early-Life Stage Toxicity Test. 2 OECD G uideline for T esting of Chem icals. 1992. Guideline 210: Fish Early-Life Stage Toxicity Test. 3 ASTM S tandard E1241-88. 1988. Standard Guidefor Conducting Early Life-Stage Toxicity Tests with Fish. American-Society for Testing and Materials. 4 U.S. Environm ental Protection Agency. 1986. Standard Evaluation Procedure, Fish Early LifeStage Test. Office of Pesticide Programs. Hazard Evaluation Division. EPA 540/9-86-138. 5 . APHA, AWWA, W PCF. 1989. StandardMethodsfo r the Examination o f Water and Wastewater. 17th Edition, American Public Health Association. American W ater Works Association. Water Pollution Control Federation, New York. 6 W est, Inc. and D.D. Gulley. 1996. TOXSTAT 3.5. Western EcoSystems Technology, Inc. Cheyenne, Wyoming. 7 SPSS Inc. 1988. SPSS/PC+Version 2.0. Chicago, Illinois. 001012 PROJECT NO.: 454A-108 - 17- Table 1 Summary o f Analytical Chemistry Data Sponsor: Test Substance: Test Organism: 3M Corporation PFOS Fathead Minnow, Pimephales promelas ^^^^^^21=====^^^^^====**TMTM====^============================-TM*TM===============================-TMTM======== Mean Percent Nominal Test Measured of Concentration Concentration Nominal (mg a.i./L) Day 0 Day 4 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 47 (mg a.i./L) Negative Control <LOQ` <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ -- 0.14 0.147 0.141 0.144 0.134 0.153 0.160 0.179 0.157 0.147 0.15 0.160 0.140 0.148 0.135 0.143 0.158 0.173 0.160 0.155 107 0.29 0.287 0.270 0.292 0.269 0.307 0.343 0.311 0.319 0.296 0.30 0.277 0.289 0.296 0.266 0.315 0.341 0.325 0.313 0.276 103 0.57 0.571 0.619 0.597 0.539 0.608 0.639 0.646 0.575 0.545 0.60 0.576 0.659 0.642 0.535 0.580 0.617 0.644 0.576 0.543 105 1.1 1.14 1.21 1.13 1.03 1.19 1.30 1.30 1.14 1.13 1.13 1.25 1.23 1.10 1.24 1.31 1.31 1.19 1.09 2.3 2.21 2.52 2.43 2 __ __ _ 2.27 2.46 2.38 - - - --- 4.6 4.56 4.79 4.46 __2 _ .M. __ _ 4.40 ,4.79 4.76 ----- *The limitofquantitation (LOQ) was 0.0458 mg a.i./L. .M...e..a"su"rements disco^ntinued due to 100% morta--liit*.y...... 1.2 2.4 4.6 109 104 100 001C13 - 18- PROJECT NO.: 454A-108 Table 2 Specific Conductance, Hardness and Alkalinity o f the Test Solutions Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Parameter Specific Conductance (mhos/cm) Hardness (mg/L as CaC03) Alkalinity (mg/L as CaC03) Test Chamber Replicate `4.6 mg a.i./L 22.4 mg a.i./L 31.2mg a.i./L Test Group Negative Control PFOS Treatment Negative Control PFOS Treatment Negative Control PFOS Treatment 0 315 3301 136 1401 176 1781 A 7 330 3102 140 1402 178 1782 D 14 300 3153 140 1443 178 1763 A 21 300 3003 136 1403 178 1783 B 28 290 3003 144 1363 178 1763 C 35 300 3003 136 1403 176 1763 A 42 290 3003 140 1403 178 1803 B 47 290 3153 136 1363 174 1763 C 001014 W il d l if e International ltd. -19- Table 3 pH o f Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Mean Measured Test Concentration (mg a.i./L) 0 7 14 Test Day 21 28 Negative Control 8.1 V 8.2 8.2 8.3 8.4 0.15 8.2 8.2 8.2 8.3 8.4 0.30 8.2 8.2 8.2 8.3 8.4 0.60 8.2 8.2 8.2 8.3 8.4 1.2 8.2 8.2 8.2 8.3 8.4 2.4 8.2 8.2 1 - - 4.6 8.2 8.2 1 __ _ Replicate A D ABC 'Measurements discontinued due toT00% mortality. PROJECT NO.: 454A-108 35 42 47 8.0 8.1 8.1 8.0 8.1 8.1 8.1 8.1 8.2 8.1 8.1 8.2 8.0 8.2 8.2 --- ---- ABC 001015 - 20- PROJECTNO.: 454A-108 Table 4 Dissolved Oxygen Content (mg/L) o f W ater in the Test Chambers1 Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Fathead Minnow, Pimephales promelas Dilution Water: Well Water_________________________________________________ Mean Measured ______ Test Day Test Concentration (mg a.i./L) 0 12 3 4 5 6 7 / 14 ' 21 28 35 42 47 Negative Control 8.1 8.0 8.1 8.1 8.0 8.0 8.0 8.2 8.1 8.1 8.2 7.8 7.6 8.0 0.15 8.1 8.0 8.1 8.1 8.0 8.0 8.0 8.2 8.2 8.1 8.2 7.6 7.8 8.0 0.30 8.1 8.1 8.2 8.1 8.0 8.0 7.9 8.1 8.2 8.1 8.2 7.6 7.8 8.0 0.60 8.1 8.0 8.2 8.1 8.1 8.0 8.0 8.1 8.1 8.1 8.2 7.8 7.6 8.0 1.2 8.2 8.0 8.1 8.0 8.0 7.9 8.0 8.2 8.2 8.0 8.2 7.6 7.9 8.2 2.4 8.0 8.0 8.1 8.0 8.0 8.1 8.0 8.2 __2 -- - -- - - 4.6 8.1 8.0 8.1 8.1 8.1 8.0 8.0 8.1 2- -- - -- -- Replicate A BC DA B CDAB CAB C 'A dissolved oxygen concentration o f 4.9 mg/L represents 60% saturation at 25 C in freshwater. Measurements discontinued due to 100% mortality. 9T0T00 W il d l if e In te r n a tio n a l ltd. - 21- PROJECT NO.: 454A-108 Table 5 Temperature (C) of W ater in the Test Chambers Sponsor: 3M Corporation Test Substance: Test Organism: PFOS Fathead Minnow, Pimephales promelas Dilution Water: Well Water Mean Measured Test Day Test Concentration (mg a.i./L) Replicate, 0 7 14 21 28 35 42 47 Negative Control A ' 24.4 24.7 24.7 24.5 24.5 24.7 24.5 24.6 B 24.4 24.7 24.7 24.5 24.5 24.7 24.4 24.5 C 24.4 24.7 24.6 24.6 24.5 24.7 24.5 24.5 D 24.5 24.7 24.7 24.5 24.5 24.7 24.5 24.5 0.15 A 24.4 24.7 24.7 24.6 24.4 24.7 24.5 24.4 B 24.5 24.6 24.7 24.6 24.5 24.8 24.6 24.4 C 24.4 24.7 24.7 24.6 24.5 24.7 24.5 24.5 D 24.5 24.7 24.6 24.5 24.5 24.7 24.4 24.4 0.30 A 24.4 24.7 24.7 24.6 24.5 24.8 24.4 24.4 B 24.4 24.7 24.7 24.5 24.5 24.7 24.4 24.4 C 24.4 24.7 24.6 24.5 24.4 24.7 24.5 24.4 D 24.5 24.7 24.6 24.5 24.5 24.7 24.4 24.4 0.60 A 24.4 24.7 24.6 24.5 24.4 24.6 24.4 24.4 B 24.4 24.6 24.5 24.4 24.4 24.7 24.3 24.3 C 24.3 24.6 24.6 24.5 24.4 24.6 24.3 24.4 D 24.3 24.6 24.5 24.4 24.4 24.6 24.4 24.4 1.2 A 24.5 24.5 24.5 24.4 24.4 24.5 24.3 24.3 B 24.4 24.6 24.5 24.5 24.5 24.6 24.4 24.4 C 24.4 24.6 24.6 24.5 24.5 24.6 24.4 24.4 D 24.5 24.7 24.6 24.5 24.5 24.6 24.5 24.4 2.4 A 24.5 24.7 _2 .. ... _ _ B 24.6 24.7 __2 -- -------- C 24.6 24.7 ---- ------ D 24.5 24.7 __2 -- -- -- ---- 4.6 A 24.5 24.6 _2 .. __ B 24.4 24.6 --2 -- -- - - ~ C 24.5 24.5 _2 -- -- -- -- -- D 24.5 24.5 __2 -- -- -- -- -- 1A continuous recording of temperature in the negative control replicate A showed that the temperature rangedfrom 24.0 to 25.0C throughout the test. Measurements discontinued due to 100% mortality. 001017 Wild life Internatio nal ltd. - 22- PROJECT NO.: 454A-108 Table 6 Hatching Success o f Fathead Minnows Exposed to the Test Substance _______________ Sponsor 3M Corporation Test Substance: PFOS Test Organism: Fathead Minnow, Ptmephalespromelas Dilution Water Well Water B B B aaS ^B S B ^^B 8 B aB B S S S S aS S S S 8 S aB B B ^^aS B B aaX S aB 9 B B B B S B S 3 B E aB B aB rB 8 S B S 3 S aB B a8 B B X B B aS B aaB aS B S aB B B aS S ^B :^^B aB B B S aaB aB ^B B ^B B S B B B S B S :^^^B B S aS S B B B aB Mean Measured Number Hatched Total Treatment Test Concentration Number of =*=*l==l*= = = *= **^ = = = = = = = = = = = = = = ^ Number Percent (mg a.i./L) Replicate Eggs Exposed Dayl Day 2 Day 3 Day 4 Day 5 Hatched Hatching Success1 Negative Control A B C D 20 0 0 0 7 18 74 20 0 0 0 5 18 20 0 0 0 6 20 20 0 0 0 2 18 93 0.15 A 20 0 0 0 S 18 76 B 20 0 0 0 5 19 C 20 0 0 0 2 19 D 20 0 0 0 3 20 95 0.30 A 20 0 0 0 4 17 72 B 20 0 0 0 1 18 C 20 0 0 0 2 18 D 20 0 0 0 7 19 90 0.60 A 20 0 0 0 5 18 76 B 20 0 0 0 6 19 C 20 0 0 0 10 20 D 20 0 0 0 7 19 95 1.2 A 20 000 9 18 74 B 20 0 0 0 7 19 C 20 0 0 0 4 18 D 20 0 0 0 5 19 93 2.4 A 20 0 0 0 7 18 75 B 20 0 0 0 3 19 C 20 0 0 0 4 . 20 D 20 0 0 0 2 18 94 4.6 A 20 0 0 0 3 19 74 B 20 0 0 0 4 17 C 20 0 0 0 1 - 19 D 20 0 0 0 6 19 1 Percent Hatching Success Number of Eggs Hatched X 100 Number of Eggs Exposed 93 C01018 W il d l if e In t e r n a t io n a l ltd. -23- PROJECT NO.: 454A-108 Table 7 Survival o f Fathead Minnow Larvae Exposed to the Test Substance for 42 Days Post-Hatch Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Fathead Minnow, Pimephales promelas Dilution Water: Well Water Mean Measured Initial Approximate Live Counts1 Test Number of (Days Post-Hatch) Concentration (mg a.i./L) Replicate Larvae 7 14 21 28 35 Negative Control A' 18 17 17 17 17 16 B 18 15 15 15 15 15 C 20 19 18 18 17 17 D 18 18 18 18 18 17 42 16 15 17 17 Replicate Percent Survival 89 83 85 94 Treatment Percent Survival 88 0.15 A 18 17 17 16 15 15 15 83 79 B 19 18 18 17 15 15 15 79 C 19 17 16 16 15 15 15 79 D 20 17 17 16 15 15 15 75 0.30 A 17 15 15 15 15 15 15 88 81 B 18 18 17 16 16 16 16 89 C 18 15 15 15 13 11 11 61 D 19 19 18 18 16 16 16 84 0.60 A 18 17 16 16 15 13 12 -- 67 66* B 19 17 17 16 14 14 13 68 C 20 19 19 18 13 13 13 65 D 19 18 18 16 13 13 12 63 1.2 A 18 12 12 7 3 3 2 11 5.4* B 19 11 10 4 2 2 1 5.3 C 18 10 7 2 1 1 1 5.6 D 19 7 6 0 0 o - 0 0.0 2.4 A 18 0 00000 0.0 0.0* B 19 0 00000 0.0 C 20 000000 0.0 D 18 000000 0.0 4.6 A 19 000000 0.0 0.0* B 17 0 00000 0.0 C 19 0 00000 0.0 D 19 000000 0.0 `Live counts made on Day 2 through 21 post-hatch were variable due to the difficulty in counting large numbers of living fish. The counts made on Day 42 were exact counts of all fish surviving at the end of the test. Indicates a significant difference from the negative control using a 2 X 2 contingency table (p <. 0.05)._________ 001019 W il d l if e Inter n a tio n a l ltd. -24- PROJECT NO.: 454A-108 Table 8 Total Length, W et Weight and Dry Weight of Fathead Minnow Larvae at the End o f the 42-Day Post-Hatch Observation Period Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Fathead Minnow, Pimephales promelas Dilution Water: Well Water Mean Measured Number of Test Concentration (mg a.i./L) Surviving Larvae Total Length (mm) Mean (SD) Negative Control 65 26.5 (0.721) 0.15 60 26.6 (0.208) 0.30 58 26.6 (0.813) Wet Weight (mg) 158 (9.10) 160 (3.10) 167 (11.9) Dry Weight (mg) Mean (SD) 32.5 (1.20) 33.3 (0.900) 34.2 (2.70) 0.601 1.2* 50 26.5 (0.399) 166 (11.3) 33.5 (2.70) 4 26.7 (2.02) 185 (33.8) 35.4 (6.66) 2.41 0 4.6' 0 - -- -,, 1 Not analyzed statistically due to a significant (p <. 0.05) effect on survival. C01020 W il d l if e Internatio nal ltd. -25- PROJECT NO.: 454A-108 APPENDEXI Dilution W ater Chemistry Parameters Measured During the 4-W eek Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Specific Conductance (mhos/cm) v Mean 321 (N = 4) Range 315-330 Hardness (mg/L as C aC 03) 126 (N = 4) 124 -128 Alkalinity (mg/L as C aC 03) 172 (N = 4) 170-172 pH 8.2 (N = 4) 8.2 - 8.3 001021 W il d l if e In ter n a tio n a l ltd. -26- PROJECT NO.: 4S4A-108 APPENDIXII Analyses o f Pesticides, Organics, Metals and Other Inorganics in Wildlife International Ltd. Well W ater1 ANALYSIS MEASURED CONCENTRATION Miscellaneous Measurements Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon3 Total Cyanide 286 < < < 0.050 1.0 10.0 mg/L mg/L mg/L Mg/L Organochlorines and PCBs Aldnn Alpha BHC Beta BHC Delta BHC Gamma BHC (Lindane) Chlordane DDD, pp' DDE, pp` DDT, pp' Dieldnn EndosuIfan,A Endosulfan, B Endosulfan Sulfate Endrin Endrin Aldehyde Heptachlor Methoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < < 0.005 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260 < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L M g/L m&l Mg/L Mg/L Mg/L Mg/L Mg/L Mg Mg/L Mg/L Mg/L Mg/L Mg/L Mg Mg/L Mg/L Mg/L Mg/L Mg/L Mg/L Metals and Other Inorganics Aluminum Arsenic3 Beryllium3 Cadmium3 Calcium3 Chromium3 Cobalt3 Lead3 Magnesiunr Manganese3 Mercury Molybdenum3 Nickel3 Potassium3 Selemniuumm' Silvie r Sodium Zinc3 < 100 Mg/L < 25.0 Mg/L < 0.50 ' Mg/L < 1.0 Mg/L 35.0 mg/L < 2.0 Mg/L < 1.0 Mg/L < 20.0 Mg/L < 100 Mg/L < 10.0 Mg/L 13.5 mg/L < 1.0 Mg/L < 0.20 Mg/L < 2.0 Mg/L < 2.0 Mg/L 6.62 mg/L < 25.0 Mg/L < 1.0 Mg/L 21.3 mg/L < 20.0 Mg/L `Analyses performed by QST Environmental, Gainesville, Florida for samples collected on November 3 through November 7,1997. Analyses performed by Wildlife International Ltd. for the sample collected on November 5,1997. Analyses performed by Wildlife International Ltd. for samples collected on November 5 through 7,1997. C01022 W il d l if e Internatio nal ltd. -27- APPENDIX IE PROJECT NO.: 454A-108 THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-108 001023 W il d l if e In ter n a tio n a l ltd. -28- PROJECT NO.: 454A-108 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: An Early Life-Stage Toxicity Test with the Fathead Minnow (Pimephales promelas) WILDLIFE INTERNATION\ A' L LTD. PROJECT NO.: 454A-108 PRINCIPAL INVESTIGATOR: ion A. MacGregor, B S. Scientist MANAGEMENT: W illard B. Nixon, Ph.Dr Manager, Analytical Chemistry DATE 001024 W il d l if e Internatio nal ltd. -29- PROJECT NO.: 454A-108 Introduction Freshwater samples were collected from an early life-stage toxicity study designed to determine the effects o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the fathead minnow (Pimephales promelas). This study was conducted by W ildlife International Ltd. and identified as Project No.: 454A-108. The analyses of these water samples were performed at W ildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis between August 4,1999 and September 21,1999 and were typically analyzed on each sample receipt day. Test Substance and Internal Standard The test substance used for this study was W ildlife International Ltd. identification number 4675. The test substance was used to prepare calibration and m atrix fortification samples. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned'Wildlife International Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical A bstract Number: 27619-97-2. The standard was stored under ambient conditions. Analytical Method The method used for the analysis o f the freshwater samples was developed at W ildlife International Ltd. and entitled "Analytical Method for the Determination o f PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II o f W ildlife International Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure w ater solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range o f the PFOS methodology. Concentrations o f the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC M ass Spectrometer equipped with a Perkin- 001025 W il d l if e In ter n a tio n a l ltd. -30- PROJECT NO.: 454A-108 Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C i8 analytical column (50 mm x 2 mm I.D ., 3 pm particle size). The instrument param eters are summarized in Table 1. A method flowchart is provided in Figure 1. Calibration Curve and Limit o f Quantitation Calibration standards o f PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.000915 to 0.00915 mg a.i./L, were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis o f individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms o f low and high calibration standards are presented in Figures 3 and 4, respectively. The method limit of quantitation (LOQ) for these analyses was set at 0.0458 mg a.i./L calculated as the product o f the lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution factor o f the matrix blank samples (50). M atrix Blank and Fortification Samples Nine matrix blank samples were analyzed to determine possible interference. N o interferences were observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram o f a matrix blank is presented in Figure 5. Freshwater was fortified at 0.0915, 0.915 and 7.32 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean procedural recovery o f 102%. A representative ion chromatogram o f a m atrix fortification is presented in Figure 6. 001026 W il d l if e International ltd. -31 - PROJECT NO.: 454A-108 Example Calculations Sample number 454A-108-3, nominal concentration o f 0.14 mg a.i./L in freshwater. Peak Area Ratio = Analyte Peak Area/Intemal Standard Peak Area Concentration Ratio = Concentration o f Analyte/Concentration o f Internal Standard Internal Standard Concentration: 0.100 mg/L Initial Volume: 0.500 mL Final Volume: 25.0 mL Dilution Factor: 50.0 PFOS Peak Area: 244330 Internal Standard Peak Area: 2316753 Peak Area Ratio: 0.10546 Calibration curve equation. Slope: 3.44236 Intercept: 0.00427 Curve is weighted (1/x) Peak area ratio - (Y-intercept) PFOS (mg a.i./L) at instrument = Slope x Internal Standard Concentration 0.10546 - 0,00427 3.44236 * 0.100 = 0.00294 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at instrument x Dilution Factor = 0.00294 x 50.0 = 0.147 C01027 W il d l if e Inter n a tio n a l ltd. -32- PROJECT NO.: 454A-108 PFOS (mg a.i./L) in sample Percent o f Nominal Concentration = PFOS (mg a.i./L) nominal x 100 0.147 0.14 x 100 = 105% RESULTS Sample Analysis Freshwater samples were collected from the early life-stage toxicity study with the fathead minnow (Pimephales promelas) at pretest, August 4, 1999, at test initiation, August 5, 1999 (Day 0), on August 9,1999 (Day 4), on August 12, 1999 (Day 7) and weekly throughout the test until test termination on September 21, 1999 (Day 47). The measured concentrations o f PFOS in the samples collected at pre test ranged from 91.4 to 105% o f the nominal concentrations (Table 4). The measured concentrations of PFOS in the samples collected at initiation of exposure o f the test organisms (Hour 0) ranged from 95.5 to 114% o f the nominal concentrations (Table 5). Samples collected at Day 4, DayJ , Day 14, Day 21, Day 28, Day 35 and Day 42 had measured concentration ranges o f 93.1 to 116%, 97.0 to 113%, 91.7 to 100%, 102 to 113%, 108 to 119%, 107 to 128% and 101 to 114% o f nominal values, respectively. Samples collected at test termination (Day 47) had a measured concentration range o f 95.2 to 111% of nominal values. A representative ion chromatogram o f a test sample is shown in Figure 7. 001028 W i l d l i f e In t e r n a t i o n a l ltd. -33- p r o j e c t n o .: 4 5 4 A-1 0 8 Table 1 Typical HPLC/MS Operational Parameters INSTRUMENT: v ANALYTICAL COLUMN:' Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). Keystone Betasil Cig column (50 mm x 2 mm I.D., 3 pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 5.00 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 72.0% Methanol : 28.0% NANOpure Water containing 0.1% Formic Acid INJECTION VOLUME: 25.0 pL PFOS RETENTION TIME: Approximately 4.3 minutes INTERNAL STANDARD RETENTION TIME: Approximately 3.0 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu 001029 W i l d l i f e In t e r n a t i o n a l ltd. -34- PROJECT NO.: 454A-108 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-108-) MAB-1 Sample Type Matrix Blank Measured Concentration of PFOS1 (mg a.i./L) <LOQ MAB-2 v Matrix Blank <LOQ MAB-3 Matrix Blank <LOQ MAB-4 Matrix Blank <LOQ MAB-5 Matrix Blank <LOQ MAB-6 Matrix Blank <LOQ MAB-7 Matrix Blank <LOQ MAB-8 Matrix Blank <LOQ MAB-9 Matrix Blank <L0Q 1 The limit of quantitation (LOQ) was 0.0458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0,000915 mg a.i./L) and the dilution factor of the matrix blank samples (50). 001020 W i l d l i f e In t e r n a t i o n a l ltd. -35- PROJECT NO.: 454A-108 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-108-) MAS-1 MAS-4 MAS-7 MAS-10 MAS-13 MAS-16 M AS-19 MAS-22 MAS-25 MAS-2 MAS-5 MAS-8 MAS-11 MAS-14 MAS-17 MAS-20 MAS-23 MAS-26 MAS-3 MAS-6 MAS-9 MAS-12 MAS-15 M AS-18 MAS-21 MAS-24 MAS-27 Concentrations o f PFOS (mg a.i./L) Fortified Measured 0.0915 0.0915 0.0915 0.0915 0.0915 0.0915 0.0915 0.0915 0.0915 0.0908 0.0924 0.0893 0.0942 0.0884 0.0886 0.103 0.0933 0.0970 Percent Recovered 99.2 101 97.6 103 96.6 96.8 113 102 106 0.915 0.915 0.915 0.915 0.915 0.915 0.915 0.915 . 0.915 0.894 0.908 0.899 0.961 0.988 0.912 0.988 0.909 0.933 97.7 99.2 . 98.3 105 108 99.7 108 99.3 102 7.32 7.59 7.32 7.53 7.32 7.75 7.32 7.33 7.32 7.66 7.32 7.36 7.32 7.51 7.32 7.47 7.32 7.13 104 103 106 100 105 101 103 102 97.4 Mean = 102 Standard Deviation= 3.99 CV = 3.91% N = 27 001031 W i l d l i f e In t e r n a t i o n a l ltd. -36- PROJECT NO.: 454A-108 Table 4 Measured Concentrations o f PFOS in Pre-test Diluter Verification Samples from an Early Life-Stage Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 (Negative Control) 0.14 .0.29 Sample Number (454A-108-) PT-1 PT-2 PT-3 PT-4 PT-5 PT-6 Sampling Time (Hours) -24 -24 -24 -24 -24 -24 PFOS Measured Concentration (mg a.i./L)u < LOQ < LOQ 0.141 0.147 0.276 0.265 Percent of Nominal -- -- 101 105 95.2 91.4 0.57 PT-7 -24 PT-8 -24 0.565 0.561 99.1 98.4 1.1 PT-9 -24 PT-10 -24 1.10 100 1.09 99.1 2.3 PT-11 -24 PT-12 -24 2.13 92.6 2.17 94.3 4.6 PT-13 -24 4.48. 97.4 PT-14 -24 4.47 97.2 1 The limit o f quantitation (LOQ) was 0.0458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution factor of the matrix blank samples (50). 2 Measured values of matrix blanks and matrix fortification samples analyzed concurrently with pre-test samples were <LOQ, 101, 91.1 and 112 percent of nominal concentrations.__________________ _ 001032 W i l d l i f e In t e r n a t i o n a l ltd. -37- PROJECT NO.: 454A-108 Table 5 Measured Concentrations o f PFOS in Freshwater Samples from a Fathead Minnow Early Life-Stage Toxicity Test Nominal Test Concentration (mg a.i./L) 0 .0 (Negative Control) Sample Number (454A-108-) 1 2 M5 16 29 30 43 44 53 54 63 64 73 74 83 84 93 94 Sampling Time (Day) 0 0 4 4 7 7 14 14 21 21 28 28 35 35 42 42 47 47 PFOS Measured Concentration1 (mg a.i./L) < LOQ < LOQ < LOQ < LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Percent of Nominal -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 0.14 3 4 0 0 0.147 0.160 105 114 17 4 18 4 31 7 32 7 0.141 0.140 0.144 0.148 101 100 103 106 45 14 46 14 55 21 56 21 65 28 66 28 75 35 76 35 85 42 86 42 95 47 96 47 0.134 0.135 0.153 0.143 0.160 0.158 0.179 0.173 0.157 0.160 0.147 0.155 95.7 96.4 109 102 114 113 128 124 112 114 105 111 1 The limit of quantitation (LOQ) was 0.0458 mg a.i./L based upon the product of the lowest calibration standard analyzed (0,000915 mg a.i./L) and the dilution factor o f the matrix blank samples (5 0 ).___________ CG1033 W i l d l i f e In t e r n a t i o n a l ltd. -38- PROJECT NO.: 454A-108 Table 5 (continued) Measured Concentrations of PFOS in Freshwater Samples from a Fathead Minnow Early Life-Stage Toxicity Test Nominal Test Concentration (mg a.i./L) 0.29 Sample Number (454A-108-) 5 6 M9 20 33 34 47 48 57 58 67 68 77 78 87 88 97 98 Sampling Time (Day) 0 0 4 4 7 7 14 14 21 21 28 28 35 35 42 42 47 47 PFOS Measured Concentration1 (mg a.i./L) 0.287 0.277 0.270 0.289 0.292 0.296 0.269 0.266 0.307 0.315 0.343 0.341 0.311 0.325 0.319 0.313 0.296 0.276 -- Percent of Nominal 99.0 95.5 93.1 99.7 101 102 92.8 91.7 106 109 118 118 . 107 112 110 108 102 95.2 0.57 7 0 80 21 4 22 4 35 7 36 7 49 14 50 14 59 21 60 21 69 28 70 28 79 35 80 35 89 42 90 42 99 47 100 47 0.571 0.576 .619 0.659 0.597 0.642 0.539 0.535 0.608 0.580 0.639 0.617 0.646 0.644 0.575 0.576 0.545 0.543 100 101 109 116 105 113 94.6 93.9 107 102 112 108 113 113 101 101 95.6 95.3 1 The limit o f quantitation (LOQ) was 0.0458 mg a.i./L based upon the product of the lowest calibration standard analyzed (0,000915 mg a.i./L) and the dilution factor of the matrix blank samples (50).____________ 001034 W i l d l i f e In t e r n a t i o n a l ltd. -39- PROJECT NO.: 454A-108 Table 5 (continued) Measured Concentrations o f PFOS in Freshwater Samples from a Fathead Minnow Early Life-Stage Toxicity Test Nominal Test Concentration (mg a.i./L) Sample Number (454A-108-) 9 10 N23 24 37 38 51 52 61 62 71 72 81 82 91 92 101 102 Sampling Time (Day) 0 0 4 4 7 7 14 14 21 21 28 28 35 35 42 42 47 47 PFOS Measured Concentration1 (mg a.i./L) 1.14 1.13 1.21 1.25 1.13 1.23 1.03 1.10 1.19 1.24 1.30 1.31 1.30 1.31 1.14 1.19 1.13 1.09 Percent of Nominal 104 103 110 114 103 112 93.6 100 108 113 118 119 118 119 104 108 103 99.1 11 12 ` 25 . 26 39 40 0 0 4 4 7 7 2.21 96.1 2.27 98.7 2.52 110 2.46 107 2.43 106 2.38 103 13 0 4.56 99.1 14 0 27 4 4.40 95.7 4.79 104 28 4 4.79 104 41 7 4.46 97.0 42 7 4.76 103 1 The limit of quantitation (LOQ) was 0.0458 mg a.i./L based upon the product of the lowest calibration standard analyzed (0,000915 mg a.i./L) and the dilution factor o f the matrix blank samples (50), ________ 001035 W i l d l i f e In t e r n a t i o n a l ltd. -40- PROJECT NO.: 454A-108 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER Prepare matrix fortification samples by spiking the requisite volume o f PFOS stock solutions directly into filtered freshwater using gas-tight syringes and Class A volumetric flasks. N/ 4* Dilute matrix fortification and test samples into the range of the calibration standards by partially filling Class A volumetric flasks with 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). Add the Appropriate volume o f sample and bring the flask to volume with the dilution solvent. Process the matrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis o f PFOS in freshwater. 001036 W i l d l i f e In t e r n a t i o n a l ltd. -41 - PROJECT NO.: 454A-108 Concentration (Ratio) Figure 2. A typical calibration curve for PFOS. Slope = 3.44236; Intercept = 0.00427; r = 0.9994. Curve is weighted (1/x). 001037 W il d l if e In t e r n a t io n a l ltd -42- PROJECT NO.: 454A-108 MaoOuan. n u s to a t .1 Print** Tua, Oat I I . I tM 04:4 cate n a o n N Cat m o o ocm jo m M a t a m ioolc iC.iiiifTiiiiii iA444Atoa.odAixJ4aCom ma.K v 4B4A*tOt 7HM BUS - DAY I M U OM 4.98 in ^ parted 4 l> f0 6 u m m Ia m u I S t a n d a rd l : 4 .M O l M l, air aoa 4 3 1 .7 Notea Thiaa. a.o OuAnt Thraa. 0 .3 M itt W Uth 3 M Ut. W kfth B u a . W Uth R T vm n. (mob) Sm ooth Eapaeiad RT 40 SO a Z.M Ai m 2 4 3 7 7 1 0 H aight 111141 Start Tim a End T ina I n ia g ra tto n WVdtfi 2.84 LU air RatanUon TYna Ituagm Uon Typo 3 .0 0 A*OO T h u . Aufl S . 1* M 14:3 100 0 so TO SO o 40 SO so 10 )%flt 71 . W Papa | | MMWMyi 10 1 0 4 1 fl|M MeOu*n. v -- lau i . PraHW. Tim . O al 1 . l i t t 0 M c*(M iw F i * 6i p f o s w u o a paaK A pM oetB in H a a fln ra w i n n ia in a j te P ir w r Oo h w u * 444A-104 PMM E L I OAY M A I M i P P O S _101 S T D 0.00100 T h u . A u g ft, 1M 0 1 4:41 4678A -004D O 3 j .00 ln 1 p a rto d PTCS intam ai S u n M flt 4 H 0 F 0 S U aaA raa I A baoiui* RacanSnn T in a : 4.* o o i m i. n r a a a n o 4 9 8 .6 N rtaa Thraa. Q uant Ttiraa. 12.0 2.0 M n. W W i 3 Mutt. WWU> S Baaa- W idth R T Win. (aoea) Sm ooth C a iim il RT 40 20 2 1J A na IT IIO H aigrit 5130 S tart Tima 4^6 EndTW ia 4 .7 3 Integration W idth M7 R atantlon Tim a 443 totagtatlon Typ* to o l 10 O TO SO so 40 SO SO to l 1. Figure 3. A representative ion chromatogram of a low-level (0.000915 mg a.i./L) PFOS standard. 001033 W il d l if e In ter n a tio n a l ltd -43- PROJECT NO.: 454A-108 MacOuan. vamlaa .# mmP n n tM Tim , Q al t* . 1 M I 04v*4 C U tf lM n CM PROP N M M N PMMc API 100LC H O I I I M a i W t.ft4M A 1W >.0A U aM ; CM tM X Il! 4A*A-10e PMM BL8 OAT 0 HUM IHM PR O S .108 STD *,A 1N 4873A -0040-40 F094.98 m 1 parted T h u , Aw q . 1 9 9 0 1 k W 1: 488 .7 Naiaa Ttva*. O uant Tiwa*. Win. W idth I M . WWl b*m . widen m w n (M ca) SnoM i Cxpaetad RT M.0 o88.a 40 SO a a.M A m 843*430 H aight 708881 S tan Tima End Tbna touagratiM W idth a.94 3 .8 * 0 .8 7 O tM ntlM H i m M tagraM A Typa 3 .0 8 A-aa Pa^a 1 M anaNyt 108801 op* U acCuuv anian 1.3 P nnlaa; T im . OM 18. M M M M CaH M iten P U i G al P R 0 8 O M U ttM PMM A H I00L C W M w aC hrefl tA aSaA W LO SA U O W : P P 0 8 .1 Q T O OJ3100 467S A -004O -40 pros4.90 in 1 paitod Inrwmal S ta n d a rd : 4 H P P O S T h u . AmQ &. 1 0 8 8 1 3 * R p i 8al t Figure 4. A representative ion chromatogram of a high-level (0.00915 mg a.i./L) PFOS standard. 001033 W il d l if e In te r n a tio n a l ltd -44- PROJECT NO.: 454A-108 Mm Ou m . a n io n 1. PrWad- Tu*. O d IB. IBM 0 * 4 CBlBraUen AM: CM PPQO UtAUOBB PMMAPI tM L C I H CornnoM- 4S4A-10 4 C tC - OAY 0 OkAUOBB I bbCWBW I.Ot44A1Qa .OIAUOM. T hu. A ug %, 1 9 0 0 11:31 W M B IrttamM StBOdBRl 1: 4 . M O ) Ml, 2 ? aaa 4CC .7 NoiM Tnrs. a .o Q uant Thru*. Min. w a r n M u tt WMIh 0 .2 a 0 M M . W wth 4 m w in . (m o b ) s o Sm ooth a Expectod AT 2 .M A/BB 2373144 MBigtU 191TTS S ta r t Tirrvo U4 Rod Tfcno Intkgration Widen M0427 Rotowien tim S4B <nigrai>on T y p A -e a PM* 1 M I WManaftpi 1 0 1 7 7 2 p MooOumv m raion * *M M d: Two. M I I , IB M 0 * 4 CMbraii* P4 * CM PPOO 0 4 M M N PMfc API 104LC HO:M aMCnmm IMMMAIBO.OtAUOW: CMncnum*; 4M A *iaS PHM CL* . DAY a OBAUOBB p ro * _ l0 7 M AS-1 Thu. Aug S. ISO# I* 2 1 kXOfnol S to n d e rtt 4 H P F O S U M Ark A b so lu t RMBMkM TMl t : 4.B a t Ml. 2 0 7 s c a n 40C . N ot* Tfxrm. Q uant Tbra. Min. W idth MuH. W k tin b m . w idm a t w in. (*} Sm ooth E x p ac tn rJR T 1 3 .0 t.O 3 40 SO S A 3* Amt o H iht 0 S ta n Tim E n d Tim fnlsgration W idth ftotsrxio* Tim -0.00 0.00 0.00 0.00 lnigrattcn Typa T ip M 2 iMmrnkr R40Cbob Figure 5. A representative ion chromatogram o f a matrix blank sample (454A-108-MAB-1). The arrow indicates the retention time o f PFOS. 001040 W il d l if e In t e r n a tio n a l ltd -45- PROJECT NO.: 454A-108 pr o s a i o T N i. Aug ft. 1 M i f J T M l M inum ai Standort t : 4 .M Q i m i. t% 1 4 S 4.7 Note* T htoa. Q u a r t Tha*. MM. W idth ao..oa a M a tt VYkJM S o so . W idth mrwwv :<Ma) Sm ooth 40 ao a At m 2340074 H ftlght >77853 Qlftrt Thai* End Tim o Irtagratwwi W idth ROtOftho T im o Irttogrolirvt Typ X. 5. ft. ft. A-1 M M Q uM vonlJl M M * r u t . c m 1. i m w+a * CMbTMMn M a: C S PPQ S OftAUOOO Pad* API -W0US HCfcMaaoOtvo l.ft4ftaA1<ftJ>ftA0Qftfti OnawooMo: 4S4Alft I W B J I - OAV 4 OSALO-- P o sato la Figure 6. A representative ion chromatogram o f a matrix fortification sample (454A-108-MAS-1). 001041 W il d l if e In te r n a tio n a l ltd -46- M w O w i.w w dM i.e N M C Tb. DO 1*. i m ( M c itfcm to p t*. c o p m otMjq w Patm m to etc HO'Maixinowi i*4W A iei j* * u e* s ( CMrmwMrn: aA>lQ F M I & I - O A T O M M M M PAO*_114 9 4.8ft in 1 p a tte d d-MAOS u M u internal Standard T h u . A ug ft. I S M 1 t ; 4.8ft Q i Ml. 2 8 7 sc * im 428 .7 NoiM ThrM . Qw4Mt TTu m . Min. W idin 2 .0 0 .2 3 Mull. W idth ft Ba m . W hitn 40 AT W in. IMC) 20 Sflteoih B f t t cWJ AT ft 2.8ft A nt 2318733 HMgM >8201 t l v l Tim* 834 C n d Tim I n te r a c to n W Wth n t w w lw i T*m* 3.S2 0.ft7 3 .0 8 M agfalion Typ A BS PROJECT NO.: 454A-108 M ftla fl IM 3 < N n . lu n U P lW M Tu*. Oct ift. tftfft 04:4 CftSftrMmn PIM; Cal PAOft MAUOI8 AaftR API 10QLC HOMm CM* l.4*4Al0ft_0ftAVCW : Cow w ot' PMM Lft - DAT OftAUOa* . A* I d l A F 0 3 _ 1 H 3 T tm , A u g ft, 1 8 8 8 1 * M ms4 3 0 in t p a tte d M m r u l S u u id aid : 4MPA D S Us* A ft AOaolw* n* tan tlo n Tim 1: 4.9ft Q l MI. 2ST a e o n s 48.a Note# Thraa. Q usni Thra*. 12.0 a.o Min. widwi M ult VAcm 8o m . W iom B T Win. (aaca) 3 ft 40 20 ftm o o tn 2 E Jtp a a a d RV 4JZft A nn 2 4 4 3 3 ft M sight iftO ftft 8l r t Tim a 4 .0 4 E nd Tim 4.T1 InMftfMtOn W idth Ba*nBn Tim 034 87 Initgraiten Typ A - 8M 10 Ift ft ft ft aotM tenafty: i f to r r - c p iai toi 1*1 a n 3 3 4 U 4 3 .0 8 3.8ft Figure 7. A representative ion chromatogram of a test sample (454A-108-3). 001042 - 47- PR0JECTN0.: 454A-108 APPENDIX IV Cumulative Mortality and Treatment-Related Effects1 Sponsor: Test Substance: Test Organism: Dilution W ater 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Mean Measured Test Concentration (mg a.i7L) Negative Control Replicate A B C D # Hatched 18 18 20 18 Day 5 # Dead 0 1 0 0 Effects 18AN 17AN 18AN 18AN Day 6 #Dead 0 0 0 0 Effects AN AN AN AN Day 7 # Dead 1 2 0 0 Effects AN AN AN AN 0.15 A 18 0 18AN 0 AN 1 AN B 19 0 19AN 0 AN 1 AN C 19 0 19AN 0 AN 1 AN D 20 0 20AN 0 AN 1 AN 0.30 A 17 0 17AN 0 AN 0 AN B 18 0 18AN 0 AN 0 AN C 18 0 18AN 0 AN 1 AN D 19 0 19AN 0 AN 0 AN 0.60 A 18 0 18AN 0 AN 0 AN B 19 0 19AN 0 AN 0 AN C 20 0 20AN 0 AN 0 AN D 19 0 19AN 0 AN 1 AN 1.2 A 18 0 18AN 1 AN 5 AN B 19 0 19AN 3 AN 7 AN C 18 0 18AN 2 AN 6 AN D 19 0 19AN 2 AN 9 AN 2.4 A 18 0 18AN 14 4E B 19 0 19AN >18 IE C 20 0 18AN, 2C ' 12 6 A N .2 E D 18 0 17AN, 1C 17 IE 4.6 A 19 0 19E.C 19 __ B 17 0 17E.C C 19 0 19E,C 17 -- 19 - D 19 2 17E.C 19 - ' Observed Effects: AN **Appears Normal; C = Lethargy, E = Erratic swimming; R = Lying on bottom. 18 19 20 18 19 17 19 19 -- -- -- - _ -- - Day 8 #Dead 1 2 1 0 1 1 1 1 1 0 1 0 0 1 0 1 5 7 8 9 18 19 20 18 19 17 19 ' 19 Effects AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN __ .. - -- -- Day 9 #Dead 1 2 1 0 1 1 1 2 1 0 2 0 1 2 1 1 5 7 8 11 18 19 20 18 19 17 19 19 Effects AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN _ - _ _ _ 0010-13 - 48- APPENDIX IV (Continued) Cumulative Mortality and Treatment-Related Effects1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promela] Well Water Mean Measured Test Concentration (mga.L/L) Replicate # Hatched Day 10 # Dead Effects Day 11 # Dead Effects Day 12 # Dead Effects Negative Control A 18 1 AN B 18 2 AN C 20 1 AN D 18 0 AN 1 AN 3 AN 1 AN 0 AN 1 AN 3 AN 1 AN 0 AN / 0.15 A 18 1 AN 1 AN 1 AN B 19 1 AN . 1 AN 1 AN C 19 2 AN 2 AN 2 AN D 20 3 AN 3 AN 3 AN 0.30 A 17 1 AN 2 AN 2 AN B 18 0 AN 0 AN 0 AN C 18 3 AN ' 3 AN 3 AN D 19 0 AN 0 AN 0 AN 0.60 A 18 1 AN 1 AN 1 AN B 19 2 AN 2 AN 2 AN C 20 1 AN 1 AN 1 AN D 19 1 AN 1 AN 1 AN T.2 A 18 5 AN 6 AN 6 AN . B 19 7 AN 8 AN 8 AN C 18 8 AN 8 AN 8 AN D 19 11 AN 12 AN 12 AN 2.4 A 18 18 18 __ 18 B 19 19 - 19 - 19 - C 20 20 - 20 - 20 - D 18 18 - 18 18 - 4.6 A 19 19 T9 19 _ B 17 17 - 17 17 C 19 19 - 19 - 19 - D 19 19 - 19 - 19 - 1 Observed Effects: AN = Appears Normal; C = Lethargy; E - Erratic swimming; R = Lying on bottom. o po PROJECTNO.: 454A-I08 Day 13 Mortality 1 3 1 0 1 1 2 3 2 0 3 0 1 2 1 1 6 8 8 12 18 19 20 18 19 17 19 19 Effects AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN - - _ - - Day 14 Mortality 1 3 1 0 1 1 2 3 2 0 3 0 1 2 1 1 6 8 8 12 18 19 20 18 19 17 19 19 Effects AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN -- -- - __ -- -- - - 49- APPENDIX IV (Continued) Cumulative Mortality and Treatment-Related Effects1 Sponsor: Test Substance: Test Organism: Dilution Water 3M Corporation PFOS Fathead Minnow, Pimephalespromelas Well Water Mean Measured Test Concentration (mga.i./L) Replicate # Hatched Day 15 # Dead Effects Day 16 # Dead Effects Day 17 # Dead Effects Negative Control A B C D 18 18 20 18 1 AN 3 AN 1 AN 0 AN 1 AN 3 AN 1 AN 0 AN 1 AN 3 AN 1 AN 0 AN j 0.15 A 18 1 AN 1 AN 1 AN B 19 1 AN ' 1 AN 1 AN C 19 2 AN 2 AN 2 AN D 20 3 AN 3 AN 3 AN 0.30 A 17 2 AN . 2 AN 2 AN B 18 0 AN . 0 AN 0 AN C 18 3 AN 3 AN 3 AN D 19 0 AN 0 AN 0 AN 0.60 A 18 1 AN 1 AN 1 AN B 19 2 AN 2 AN 2 AN C 20 1 AN 1 AN 1 AN D 19 1 AN 1 AN 1 AN 1.2 A 18 6 AN 6 AN B 19 8 AN 8 AN C 18 8 AN 8 AN D 19 12 AN 12 AN 2.4 A 18 18 _ 18 _ B 19 19 -- 19 - C 20 20 20 -- D 18 18 - 18 - 4.6 A 19 19 _ ' ` 19 B 17 17 -- 17 - C 19 19 - 19 - D 19 19 - 19 - , 1 Observed Effects: AN " AppearsNormal; C = Lethargy, E = Erratic swimming; R = Lying on bottom. 6 AN ' 8 AN 8 AN 12 AN 18 19 20 18 - 19 17 19 19 - PROJECTNO.: 454A-I08 Day 18 # Dead 1 3 1 0 1 1 2 3 2 0 3 0 1 2 1 1 6 9 9 12 18 19 20 18 19 17 19 19 Effects AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN - - -- - - - Day 19 Mortality 1 3 2 0 1 1 3 3 2 1 3 1 2 2 1 1 6 9 11 13 18 19 20 18 19 17 19 19 Effects AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN - - " _ - - - - 50- PROJECTNO.: 454A-108 Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mga.i./L) Negative Control 0.15 APPENDIX IV (Continued) Cumulative Mortality and Treatment-Related Effects1 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Replicate A B C D # Hatched 18 18 20 18 Day 20 # Dead 1 3 2 0 Effects AN AN AN AN _ Day 21 # Dead 1 3 2 0 Effects AN AN AN AN __________ Day 22__________ # Dead 1 3 2 0 Effects AN AN AN AN / A 18 1 AN 1 B 19 1 AN . 1 C 19 3 AN 3 D 20 3 AN 3 AN AN AN AN 2 AN 1 AN 3 AN 4 AN ____ _ _____ ____ Day 23__________ ___________Day 24 UDead 1 3 2 0 2 1 3 4 Effects AN AN AN AN AN AN AN AN # Dead 1 3 2 0 2 1 3 4 Effects AN AN AN AN AN AN AN AN 0.30 A 17 2 AN 2 AN 2 AN B 18 1 AN 1 AN 1 AN C 18 3 AN ' 3 AN 3 AN D 19 1 AN 1 AN 1 AN 0.60 A 18 2 AN 2 AN 2 AN B 19 2 AN 2 AN 3 AN C 20 1 AN 1 AN 1 AN D 19 1 AN 1 AN 1 AN 2 AN 1 AN 3 AN 1 AN 2 AN 3 AN 1 AN 1 AN 2 AN 2 AN 3 AN 1 AN 2 AN 3 AN 1 AN 1 AN 1.2 A 18 6 AN 6 AN B 19 9 AN 9 AN C 18 11 AN 12 AN D 19 13 AN 13 AN 2.4 A 18 18 __ 18 B 19 19 C 20 20 - -. 19 20 - D 18 18 - 18 " 4.6 A 19 19 ' 19 B 17 17 - 17 . ' - C 19 19 - 19 - D 19 19 . - 19 - 1 Observed Effects: A =Appears Normal; C =Lethargy, E = Erratic swimming; R = Lying on bottom. 6 AN 9 AN 12 AN 13 AN 18 19 20 18 - 19 _ 17 19 19 - 6 AN 10 AN 12 AN 13 AN 18 19 20 - 18 - 19 .. 17 19 19 - 7 AN 10 AN 12 AN 13 AN 18 19 20 18 - 19 _ 17 19 19 - 001046 -51 - Sponsor: Test Substance: Test Organism: Dilution Water. Mean Measured Test Concentration (mga.i./L) Negative Control 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Day 25 APPENDIX IV (Continued) Cumulative Mortality and Treatment-Related Effects1 Day 26 Day 27 Replicate A B C D # Hatched 18 18 20 18 # Dead 1 3 2 0 Effects AN AN AN AN # Dead 1 3 2 0 Effects AN AN AN AN # Dead 1 3 2 0 Effects AN AN AN AN j 0.15 A 18 2 AN 2 AN 2 AN B 19 1 AN 2 AN 3 AN C 19 3 AN 3 AN 3 AN D 20 4 AN 4 AN 5 AN 0.30 A 17 2 AN 2 AN 2 AN B 18 2 AN . 2 AN 2 AN C 18 3 AN 3 AN 3 AN D 19 1 AN 1 AN 1 AN 0.60 A 18 2 AN 2 AN 2 AN B 19 3 AN 3 AN 3 AN C 20 1 AN 2 AN 2 AN D 19 2 AN 3 AN 4 AN 1.2 A 18 9 AN 11 AN.2E 13 AN B 19 13 AN 15 AN 15 AN C 18 14 AN 16 AN 16 AN D 19 18 IE 19 " 19 2.4 A 18 18 18 B 19 19 - 19 - C 20 20 - ' 20 - D 18 18 - 18 - 4.6 A B 19 19 17 17 _ 1 19 L 17 - C D 19 19 19 19 - 19 -, 19 - 1 Observed Effects: AN = Appears Normal; C =Lethargy, E = Erratic swimming, R = Lying on bottom. 18 19 20 - 18 - 19 17 -19 19 - o PROJECT NO.: 454A-108 Day 28 # Dead 1 3 2 0 Effects AN AN AN AN 3 AN 3 AN 3 AN 5 AN 2 AN 2 AN 4 AN 3 AN 2 AN 3 AN 2 AN 4 AN 13 AN 15 AN 16 AN 19 - 18 .. 19 20 - 18 - 19 -- 17 19 19 - Day 29 # Dead 1 3 2 0 Effects AN AN AN AN 3 AN 3 AN 3 AN 5 AN 2 AN 2 AN 4 AN 3 AN 2 AN 3 AN 3 AN 4 AN 15 AN 16 AN 16 AN 19 - 18 _ 19 20 -- 18 - 19 17 19 19 _ - 52- Sponsor: Test Substance: Test Organism: Dilution Water Mean Measured Test Concentration (mg a.i7L) Negative Control 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Day 30 APPENDIXIV (Continued) Cumulative Mortality and Treatment-Related Effects1 Day 31 Day 32 Replicate A B C D # Hatched 18 18 20 18 # Dead 1 3 2 0 Effects AN AN AN AN # Dead 1 3 2 0 Effects AN AN AN AN ft Dead 1 3 2 0 Effects AN AN AN AN 0.15 A 18 3 AN 3 AN 3 AN B 19 3 AN 3 AN 3 AN C 19 3 AN 3 AN 3 AN D 20 5 AN 5 AN 5 AN 0.30 A 17 2 AN 2 AN 2 AN B 18 2 AN 2 AN 2 AN C 18 4 AN 4 AN 4 AN D 19 3 AN 3 AN 3 AN 0.60 A 18 2 AN 2 AN 3 AN B 19 4 AN 4 AN 4 AN C 20 3 AN 3 AN 4 AN D 19 4 AN 4 AN 4 AN 1.2 A 18 15 AN 15 AN 15 AN B 19 17 AN 17 AN 17 AN C 18 16 AN 17 AN 17 AN D 19 19 19 - 19 2.4 A 18 18 -- 18 18 __ B 19 19 - 19 - 19 - C 20 20 - 20 - 20 - D 18 18 - 18 - 18 - 4.6 A 19 19 .19 __ 19 B 17 17 - 17 - 17 - C 19 19 - 19 - 19 - D 19 19 - 19 - 19 - PROJECT NO.: 454A-108 555 5555 5555 5555 5555 Day 33 # Dead 1 3 3 0 3 4 4 5 2 2 5 3 3 5 7 6 15 17 17 19 18 19 20 18 19 17 19 19 Effects AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN - - __ - - Day 34 UDead 1 3 3 0 3 4 4 5 2 2 5 3 3 5 7 6 15 17 17 19 18 19 20 18 19 17 19 19 " -- - - _ - - 0010 - 53- Sponsor: Test Substance: Test Organism: Dilution Wat: Mean Measured Test Concentration (mga.L/L) Negative Control 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Day 35 APPENDIX IV (Continued) Cumulative Mortality and Treatment-Related Effects1 Day 36 Day 37 Replicate A B C D # Hatched 18 18 20 18 # Dead 1 3 3 0 Effects AN AN AN AN # Dead 1 3 3 0 Effects AN AN AN AN # Dead 2 3 3 1 Effects AN AN AN AN 0.15 A 18 3 AN 3 AN 3 AN B 19 4 AN 4 AN 4 AN C 19 4 AN 4 AN 4 AN D 20 5 AN 5 AN 5 AN 0.30 A 17 2 AN 2 AN 2 AN B 18 2 AN 2 AN 2 AN C 18 5 AN 5 AN.1R 7 AN D 19 3 AN 3 AN 3 AN 0.60 A 18 3 AN 4 AN 4 AN B 19 5 AN 5 AN 5 AN C 20 7 AN 7 AN 7 AN D 19 6 AN 6 AN 6 AN 1.2 A 18 15 AN 15 AN 15 AN B 19 17 AN 17 AN 17 AN C 18 17 AN 17 AN 17 AN D 19 19 - 19 19 - 2.4 A 18 18 _, 18 18 B 19 19 - 19 - 19 - C 20 20 - 20 - 20 - D 18 18 - 18 - 18 - 4.6 A 19 19 __ 19 B 17 17 - 17 _ C 19 19 - 19 D 19 19 - 19 -- ` Observed Effects: AN = Appears Nonnal; C = Lethargy, E = Erratic swimming; R Lying on bottom. 19 17 19 19 - - PR0JECTN0.: 454A-108 Day 38 # Dead 2 3 3 1 3 4 4 5 2 2 7 3 4 5 7 6 15 17 17 19 18 19 20 18 19 17 19 19 Effects AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN - - " _ - - Day 39 # Dead 2 3 3 1 3 4 4 5 2 2 7 3 4 5 7 6 15 17 17 19 18 19 20 18 19 17 19 19 Effects AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN - - - _ - -54- Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) Negative Control 3M Corporation PFOS Fathead Minnow, Ptmephales prometas Well Water Day 40 APPENDIX IV (Continued) Cumulative Mortality and Treatment-Related Effects1 Day 41 Day 42 Replicate A B C D # Hatched 18 18 20 18 # Dead 2 3 3 1 Effects AN AN AN AN # Dead 2 3 3 1 Effects AN AN AN AN # Dead 2 3 3 1 Effects AN AN AN AN 0.15 A 18 3 AN 3 AN 3 AN B 19 4 AN 4 AN 4 AN C 19 4 AN 4 AN 4 AN D 20 5 AN 5 AN 5 AN 0.30 A 17 2 AN 2 AN 2 AN B 18 2 AN 2 AN 2 AN C 18 7 AN 7 AN 7 AN D 19 3 AN 3 AN 3 AN 0.60 A 18 5 AN 5 AN 5 AN B 19 5 AN 5 AN 5 AN C 20 7 AN 7 AN 7 AN D 19 6 AN 6 AN 6 AN 1.2 A 18 15 AN 15 AN 15 AN B 19 17 AN 17 AN 17 AN C 20 17 AN 17 AN 17 AN D 19 19 - 19 " 19 - 2.4 A 18 18 --i 18 18 .. B 19 19 - 19 - 19 - C 20 20 - 20 - 20 - D 18 18 " 18 " 18 " 4.6 A 19 19 . .19 _ 19 B 17 17 - 17 _ 17 - C 19 19 - 19 - 19 - D 19 19 - 19 - 19 - 1 Observed Effects: AN **Appears Normal; C = Lethargy; E *Erratic swimming; R = Lying on bottom. O O cn PROJECTNO.: 454A-108 Day 43 # Dead 2 3 3 1 3 4 4 5 2 2 7 3 Effects AN AN AN AN AN AN AN AN AN AN AN AN 5 AN 5 AN 7 AN 6 AN 15 AN 17 AN 17 AN 19 18 __ 19 20 - 18 - 19 _ 17 19 19 - Day 44 # Dead 2 3 3 1 3 4 4 5 2 2 7 3 Effects AN AN AN AN AN AN AN AN AN AN AN AN 5 AN 5 AN 7 AN 6 AN 15 AN 17 AN 17 AN 19 - 18 19 -- 20 - 18 - 19 17 -- 19 -- 19 - Wildlife International ltd. -55- PROJECT NO.: 454A-108 555 APPENDIXIV (Continued) ___________________ Cumulative Percent Mortality and Treatment-Related Effects1 Sponsor. 3M Corporation Test Substance: PFOS Test Organism: Fathead Minnow, Pimephales promelas Dilution Water:______ Well Water______ __ _____________________________________ Mean Measured Test Concentration (mg a/L) Replicate # Hatched Day 45 # Dead Effects Day 46 #Dead Effects Negative Control A 18 2 B 18 3 C 20 3 D 18 1 AN AN AN AN 2 AN 3 AN 3 AN 1 AN 0.15 A 18 3 AN 3 AN B 19 4 AN 4 AN C 19 4 AN 4 AN D 20 5 AN 5 AN 0.30 A 17 2 AN 2 AN B 18 2 AN 2 AN C 18 7 AN 7 AN D 19 3 AN 3 AN 0.60 A 18 5 AN 6 AN B 19 5 AN 5 AN C 20 7 AN 7 AN D 19 6 AN 6 AN 1.2 A 18 15 AN 15 AN B 19 17 AN 17 AN C 18 17 AN 17 AN D 19 19 - 19 - 2.4 A 18 18 18 B 19 19 - 19 - C 20 20 -- 20 . -- D 18 18 - 18 - 4.6 A 19 19 19 B 17 17 -- 17 -- C 19 19 - 19 -- D 19 19 - 19 - 'Observed Effects: AN = Appears Normal; C " Lethargy, E** Erratic swimming; R ==Lying on bottom. Day 47 = ^= # Dead 2 3 3 1 3 4 4 5 2 2 7 3 6 6 7 7 16 18 17 19 18 19 20 18 19 17 19 19 001051 Wildlife International ltd. -56- PROJECT NO.: 454A-108 APPENDEX V Fish Total Length (mm) Negative=Contro|i>ra_ i^ ^ Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Fathead Minnow, Pimephales promelas Dilution Water: Well Water Fish Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 A S/ 26.0 28.6 26.5 31.0 31.1 25.0 25.5 27.1 27.2 27.1 . 25.3 32.4 24.6 26.8 27.2 21.4 -- -- -- -- B CD 29.6 31.7 27.8 26.1 25.7 27.6 28.3 28.3 28.2 30.9 27.7 30.4 25.2 27.8 25.1 26.2 23.0 28.3 22.3 28.6 27.0 26.5 26.3 28.2 29.7 26.6 20.6 30.2 25.7 22.9. 18.4 28.4 24.6 29.0 15.2 24.5 28.9 20.1 25.6 29.1 23.7 31.2 26.8 28.1 - 24.5 -- 28.2 25.5 -- 22.1 20.7 -- -- -- ---- -- ---- Replicate Mean Overall Mean (s) 27.1 27.1 25.7 26.0 26,5 ( 0.721) Note: Total length values were rounded for presentation. Manual calculations of means and standard deviation may differ slightly from those generated using TOXSTAT* 3.5. 001052 Wildlife International ltd. -57- PROJECT NO.: 454A-108 APPENDIX V (Continued) Fish Total Length (mm) 0.15 mga.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 v A 29.0 26.2 27.5 19.8 29.4 28.3 32.1 27.9 18.1 23.9 28.5 29.5 23.6 27.1 27.8 -- -- ~ -- -- Replicate BC 23.9 27.9 25.0 29.8 26.3 26.6 28.0 27.0 23.1 32.1 23.3 27.9 26.9 23.0 27.4 25.7 30.7 23.8 26.1 27.7 24.9 22.3 28.1 27.7 26.1 26.8 24.0 24.2 30.4 29.0 ---- -- -- -- ---- ---- D 26.8 25.7 29.6 26.8 25.2 25.8 20.1 . 26.1 27.3 29.0 27.6 25.8 29.4 24.1 30.0 -- .. -- -- -- . Replicate Mean Overall Mean (s) 26.6 26.3 26.8 26.6 ( 0.208) 26.6 Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT 3.5. S B S s s s s B B X s s a B B S S S S ^ ^ ^ B ^ a s B B S s B & B a B m e ^ ^ K a a s B a ^ B a S B a K n B a fia s s a a B a n a B B m s s s ^ s a s s s B s a B B 001053 Wildlife International ltd. -58- PROJECT NO.: 454A-108 APPENDIX V (Continued) Fish Total Length (mm) 0.30 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number A BC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 . 20 v 25.9 27.0 19.4 26.2 28.0 25.7 28.0 19.7 29.5 25.5 28.1 30.5 21.0 20.6 27.3 -- -- -- 30.8 27.8 25.2 27.2 24.0 29.1 27.6 25.0 26.1 27.9 27.8 28.5 22.9 28.3 26.2 28.8 24.7 24.5 26.3 25.6 28.2 25.4 27.8 -- 30.3 -- 25.0 -- 25.1 -- 24.2 -- ---- ---- ---- ---- 27.8 25.9 26.6 25.4 33.1 27.0 28.0 29.8 28.1 25.9 .27.3 27.9 30.4 23.1 - 22.4 28.0 -- -- -- -- Replicate Mean Overall Mean (s) 25.5 26.4 27.1 26.6 ( 0.813) 27.3 Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT* 3,5,__________ 001054 Wildlife International ltd. -59- PROJECT NO.: 454A-108 APPENDEX V (Continued) Fish Total Length (mm) 0.60 mg a.i./L & B 8 3 a s s s S B s s ssu u g B B ^ u sa m s3 a a i^ B a B e B ^ a a n ^ a B S S 3 8 S B B n sss ssssssB sss9 a a B a a a n & a B a s Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Fathead Minnow, Pimephales promelas Dilution Water: Well Water Fish Number __________________________ R eplicate_________________ A BC D 1 v 27.1 30.2 28.0 25.4 2 22.7 22.8 29.0 24.6 3 24.5 30.2 28.7 21.1 4 24.4 25.3 28.1 27.9 5 22.8 22.7 27.3 30.8 6 27.0 24.7 25.8 25.9 7 27.1 28.9 24.5 28.5 8 29.1 25.0 25.0 27.4 9 28.4 22.0 28.7 25.6 10 27.5 26.9 30.0 23.1 11 23.7 26.9 25.1 . 23.4 12 31.3 29.1 26.4 32.2 13 -- 26.3 25.4 -- 14 -- -- -- . -- 15 . . -- -- -- 16 -- -- -- 17 -- -- -- -- 18 -- -- -- -- 19 mmi -- -- -- 20 ---- -- -- Replicate Mean Overall Mean (s) 26.3 26.2 27.1 26.5 ( 0.399) 26.3 Note: Total length values were rounded for presentation. Manual calculations of means and standard deviation may differ slightly from those generated using TOXSTAT*3,5. C C 0 5 5 Wildlife International ltd. -60- PROJECT NO.: 454A-108 APPENDIX V (Continued) Fish Total Length (mm) Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephalespromelas Well Water Fish N=umber 2 3 4 5 6 7 8 A____________ B _ v 26.2 23.1 26.7 Replicate __________ C_____________ D 28.7 . 9 10 11 12 13 14 15 16 17 18 19 20 Replicate Mean 24.7 26.7 28.7 Overall Mean (is)_____________________________ 26.7 ( 2,02)__________________ ___ Note: Total length values were rounded for presentation. Manual calculations of means and standard deviation may differ slightly from those generated using TOXSTAT* 3.5. 001056 Wildlife International ltd. -61- PROJECTNO.: 454A-108 Negative Control Sponsor: Test Substance: Test Organism: Dilution Water: APPENDIX VI Fish Wet Weight (mg) 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number AB C D 1 148.1 247.9 184.1 172.9 2 200.5 158.2 182.9 201.4 3 148.9 187.6 206.2 164.5 4 205.6 227.9 162.8 225.0 5 231.0 143.0 173.9 142.4 6 129.1 137.5 124.9 209.2 7 130.1 82.4 210.4 165.0 8 151.1 127.8 139.9 173.9 9 153.1 169.0 181.5 78.7 10 154.6 238.7 142.0 104.6 11 132.6 64.5 215.6 98.3 12 275.1 180.1 28.5 124.2 13 135.5 202.2 60.4 124.7 14 147.9 196.3 145.9 286.8 15 147.6 163.8 165.0 49.9 16 89.1 -- 182.0 ' 144.0 17 -- 77.4 56.9 18 -- -- -- -- 19 -- - - -- -- 20 -- -- -- -- Replicate Mean Overall Mean (s) 161.2 168.5 152.0 158 (9.10) 148.4 Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT*13.5. 001057 Wildlife International ltd. -62- PROJECT NO.: 454A-108 APPENDIX VI (Continued) Fish Wet Weight (mg) 0.15mga.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephalespromelas Well Water Fish Number Replicate A B CD 1 182.5 118.0 200.1 195.5 2 139.7 133.9 242.4 122.9 3 166.6 124.4 160.4 204.7 4 67.6 199.8 148.0 142.4 5 222.8 89.0 290.1 125.3 6 205.9 119.0 167.2 173.0 7 240.0 165.0 86.6 58.4 8 176.8 195.9 128.2 146.6 9 55.8 200.8 76.6 177.3 10 104.0 180.1 181.7 198.7 11 198.2 139.1 70.0 177.5 ' 12 231.8 210.3 184.6 123.4 13 103.0 155.0 186.7 202.1 14 156.8 105.0 135.6 112.5 15 152.3 214.3 199.9 - 219.3 16 -- -- -- -- 17 .-- -- -- -- 18 -- -- -- -- 19 -- -- -- -- 20 -- -- -- - Replicate Mean Overall Mean (s) 160.3 156.6 163.9 160 (3.10) 158.6 Note: Total length values were rounded for presentation. Manual calculations of means and standard deviation may differ slightly from those generated using TOXSTAT 3.5. C0105S Wildlife International ltd. -63- PROJECT NO.: 454A-108 APPENDIX VI (Continued) Fish Wet Weight (mg) fr30jngju7L ==s=s===^ ^ Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number A Replicate B C D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 . 20 v 144.5 161.6 62.4 184.3 203.7 126.7 163.1 75.6 256.3 125.6 198.9 268.4 83.4 53.7 189.8 -- -- -- --` -- 208.8 181.1 135.5 192.0 165.6 211.9 123.8 180.7 142.9 175.1 199.7 202.2 224.6 122.1 106.3 149.2 -- -- -- -- 206.7 208.4 193.2 147.1 185.6 209.1 201.7 220.1 126.5 145.4 150.9 -- -- -- .. -- -- -- -- -- 162.4 155.3 203.6 135.8 261.8 164.2 157.0 193.2 178.3 124.0 142.7 151.6 234.7 65.6 93.0 179.2 -- -- -- -- Replicate Mean Overall Mean (s) 153.2 170.1 181.3 167 (11.9) 162.7 Note: Total length values were rounded for presentation. Manual calculations of means and standard deviation may differ slightly from those generated using TOXSTAT 3.5. 001059 Wildlife International ltd. -64- PROJECT NO.: 454A-108 APPENDIX VI (Continued) Fish Wet Weight (mg) Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number A Replicate B C D 1 N206.7 223.7 180.5 161.2 2 96.2 122.1 225.4 125.6 3 113.2 221.0 200.6 85.1 4 131.0 160.7 189.0 201.3 5 93.3 103.3 174.9 272.7 6 169.8 141.1 156.7 132.8 7 163.2 205.3 148.2 169.3 8 231.5 130.6 172.5 175.3 9 223.1 84.0 231.4 137.2 10 201.2 152.9 236.0 88.0 11 136.2 159.4 151.8 85.9 12 176.3 212.0 177.3 251.8 13 -- 177.1 125.1 .. 14 -- -- -- -- 15 -- -- -- ' -- 16 -- -- -- 17 -- -- -- 18 -- -- -- - - 19 --` -- -- -- 20 -- -- -- -- Replicate Mean Overall Mean (s) 161.8 161.0 182.3 166 (11.3) 157.2 Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT 3.5. COIOGO Wildlife International ltd. -65- PROJECT NO.: 454A-108 APPENDEX VI (Continued) Fish Wet Weight (mg) L2m Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number A B CD 1 v 173.3 179.3 220.6 2 133.7 3 4 5 6 7 8 .. .. ~ 9 10 11 12 13 14 .. .. - .~ 15 -- -- -- 16 - - - '- 17 18 1- - -- 19 .. .. -' _________ 20________________ ^ _____________ =______________ =_____________ -- Replicate Mean 153.5 179.3 220.6 Overall Mean (s)_____________________ ._________185 (33.8)______________________ Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT* 3,5. CC1061 W il d l if e In te r n a tio n a l ltd. - 66- PROJECT NO.: 454A-108 APPENDIX VII Fish Dry Weight (mg) ^ g tiv e _ ^ n to l Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number A Replicate BC D 1 ^ 30.3 51.2 40.8 38.8 2 42.5 32.7 40.5 42.4 3 32.0 39.9 40.3 32.9 4 43.0 45.2 33.6 50.4 5 45.6 28.6 37.5 30.3 6 24.6 26.2 24.9 46.2 7 25.4 15.7 47.6 34.0 8 31.5 25.1 30.2 . 37.0 9 30.2 36.8 37.7 15.4 10 30.0 45.7 27.6 21.0 11 27.6 11.8 43.6 20.1 12 55.0 . 39.7 06.5 26.1 13 26.6 37.8 10.3 25.2 14 29.4 43.1 30.8 62.9 15 29.7 30.8 32.7 - -.09.3 16 19.4 -- 39.5 '29.9 17 -- -- 16.8 11.5 18 -- ---- -- 19 -- - -- -- 20 -- -- .. -- Replicate Mean Overall Mean (s) 32.7 34.0 31.8 31.4 32.5 ( 1.20) Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT*3,5. C010S2 Wildlife International ltd. -67- PROJECT NO.: 454A-108 APPENDIX VII (Continued) Fish Dry Weight (mg) Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas W ell W ater Fish Number A BC D 1 v 39.0 22.3 43.6 39.4 2 27.5 25.8 55.6 26.2 3 35.1 25.1 33.6 40.5 4 13.9 39.8 27.6 32.3 5 46.7 16.9 61.9 27.2 6 41.5 22.0 34.0 38.7 7 49.3 34.8 16.4 13.5 8 36.1 41.1 26.3 32.1 9 10.9 41.2 14.6 38.9 10 19.4 37.5 35.0 46.7 11 44.3 30.3 13.9 38.3 12 50.3 45.3 38.9 23.0 13 21.9 32.7 38.1 41.2 14 28.4 20.7 26.9 23.3 15 32.2 46.6 43.2 48.4 16 -- ---- -- 17 -- -- --. -- 18 -- ---- 19 - - ---- -- 20 -- ---- -- Replicate Mean Overall Mean (s) 33.1 32.1 34.0 33.3 ( 0.900) 34.0 Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT* 3.5. 00X0G 3 Wildlife International ltd. -68- PROJECT NO.: 454A-108 APPENDIX VII (Continued) Fish Dry Weight (mg) 0.30 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well W ater Fish Number A Replicate BC D 1 27.1 41.2 39.9 33.1 2 33.9 37.3 47.8 32.1 3 10.9 26.2 40.1 43.7 4 40.0 39.3 30.5 27.1 5 42.3 31.9 39.0 51.4 6 25.8 42.5 . 45.2 34.1 7 33.3 24.8 44.1 33.1 8 15.6 35.3 46.7 39.7 9 52.2 28.8 24.4 34.7 10 25.8 34.3 28.2 24.2 11 42.7 40.4 32.5 28:7 12 56.0 39.5 -- 32.1 13 18.2 45.8 -- 50.1 14 11.7 23.2 -- 14.5 15 41.0 20.1 -- 19.0 16 -- 27.1 -- 36.4 17 -- - - -- 18 -- -- -- -- 19 -- - - -- -- 20 ---- -- -- Replicate Mean Overall Mean (s) 31.8 33.6 38.0 34.2 ( 2.70) 33.4 Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT 3.5. 001064 W il d l if e Internatio nal ltd. -69- PROJECT NO.: 454A-108 APPENDIX VD (Continued) Fish Dry Weight (mg) 0.60 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number A B C D 1 v 43.9 44.7 38.5 28.8 2 18.8 23.1 46.3 23.9 3 23.1 44.4 39.9 15.8 4 25.1 32.7 36.9 40.0 5 18.2 20.6 35.1 54.2 6 33.0 25.3 30.1 24.6 7 31.3 42.1 27.4 34.5 8 48.6 26.9 32.5 35.8 9 49.0 14.3 46.8 27.9 10 42.9 30.4 49.1 17.1 11 27.0 31.7 29.6 16.3 12 59.8 42.4 37.9 51.1 13 -- 32.4 24.0 -- 14 -- -- 15 -- -- - - ' -- 16 -- -- -- -- 17 -- -- -- -- 18 -- ---- 19 . ,, ---- 20 -- -- -- -- Replicate Mean Overall Mean (s) 35.1 31.6 36.5 30.8 33.5 ( 2.70) Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT 3.5. B9SS^SSflS5B8B3S^BM SM HflBBKSI^n89SE8BHBB8S8flS5BBBBE8S9l0SSp35BBEfiE^^^9SSSSSsHBBS5K3SHHSiS&S&BaEflEBB)B9SEBESasS C01065 W il d l if e Intern atio na l ltd. -70- PROJECT NO,: 454A-108 APPENDEX VII (Continued) Fish Dry Weight (mg) 1.2m ga.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number ______ A _ Replicate JB_________ C_ D 1 - 33.3 34.2 42.6 2 25.6 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Replicate Mean Overall Mean (s) 29.5 34.2 42.6 35.4 ( 6.66) Note: Total length values were rounded for presentation. Manual calculations o f means and standard deviation may differ slightly from those generated using TOXSTAT* 3.5.__________ 0 0 1 .0 6 6 W il d l if e In t e r n a t io n a l l t d . -71- PROJECT NO.: 454A-108 APPENDEXVni Changes to Protocol V This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to add the proposed experimental start and termination dates. 2. The post-hatch growth period was extended to 42 days. 3. Nominal test concentratibns were 0.14,0.29,0.57, 1.1,2.3 and 4.6 mg a.i./L. qoxog^ Wil d l if e In ter n a tio n a l ltd. -72- PROJECT NO.: 454A-108 APPENDIX EX Personnel Involved in the Study The following key Wildlife International Ltd. personnel were involved in the conduct or management o fthis study: 1. Henry O. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. W illard B. Nixon, Ph.D ^ Manager, Analytical Chemistry 3. Timothy Z. Kendall, Supervisor 4. Kurt R. Drottar, Senior Biologist 5. Jon A. MacGregor, Scientist 6. Mark A. Mank, Laboratory Supervisor 001068 PROTOCOL PFOS: AN EARLY LIFE-STAGE TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephales promelas) U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1400 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue S t Paul, M innesota 55144 Wildlife International ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 June 10,1999 0010G 9 W il d l if e In t e r n a t io n a l , ltd -2- PFOS: AN EARLY LIFE-STAGE TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephales promelas) SPONSOR: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach TESTING FACILITY: V W ildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: Kurt Drottar Senior Aquatic Biologist LABORATORY MANAGEMENT: Henry 0 . Krueger, Ph.D. Director o f Aquatic Toxicology & Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: E xperim ental Start Date: ____ Experimental Termination Date: Project No.: Test Concentrations: Negative Control. 0 .1 6 .0 .3 1 .0 .6 3 .1 .3 .2 .5 and 5.0 mg a.i./L Test Substance No.: 4675______ Reference Substance No. (if applicable): 4526 PROTOCOL APPROVAL STUDY DIRECTOR DATE ORATORY MANAGEMENT Q-LaA SPONSOR'S REP`REESSEENNTTAATT]lVE DATE i&iii DATE 001070 PROTOCOL NO. : 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a t io n a l , ltd -3 - INTRODUCTION Wildlife International, Ltd. will conduct an early life-stage toxicity test w ith fathead minnow ([Pimephales promelas) for the Sponsor at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The study will be performed based on procedures in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1400 (1); A S IM Standard E l241-88 Standard Guide fo r Conducting Early Life Stage Toxicity Tests with Fish (2); and Standard Evaluation Procedure, FiskEarly Life-Stage Test (3). Raw data for all work performed at W ildlife Interna tional, Ltd. and a copy o f the final report will be filed by project number in archives located on the W ildlife International, Ltd. site, or at an alternative location to be specified in the final rep o rt PURPOSE The purpose o f this study is to determine the effects of a test substance on the time to hatch, hatching success, survival, and growth o f fathead minnows (Pimephales promelas) during early life stage development. EXPERIMENTAL DESIGN Fathead minnow embryos will be exposed to a series o f six test concentrations and a negative (Dilution water) control. Test concentrations will be chosen by the Study Director in conjunction w ith the Sponsor based on acute toxicity data. Each test concentration will be 50% o f the next higher treatment level The test will begin when groups o f newly-fertilized embryos are placed in incubation cups and exposed to test solution. Incubation cups, each containing 20 embryos, will be placed in each o f four replicate test chambers per treatment (total of 80 embryos per treatment and control). After hatching, larvae will be released from the incubation cups into the larger test chambers where exposure will continue and growth and mortality will be observed over a 28-day period. Observations o f the effects o f the test substance on hatching success, growth, and survival will be used to calculate the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC). The NOEC and LOEC will be used to calculate the maximum acceptable toxicant concentration (MATC). 001071 PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -4 - M ATERIALS AND M ETHODS Test Substance Information on the characterization o f test, control or reference substances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing Wildlife International, Ltd. m itten verification that the test substance has been characterized according to GLPs prior to its use in the study. If written verification o f GLP test substance characterization is not provided to Wildlife International, Ltd., it will be noted in the compliance statement o f the final report The attached farm IDENTIFICATION O F TEST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide information necessary for GLP compliance. The Sponsor is responsible for all information related to the test substance including the retention o f a reserve sample o f the lot or batch o f the test substance used in this study. The Sponsor also agrees to accept any unused test substance and/or test substance containers remaining at the a id o f the study. Preparation o f Test Concentrations The test substance will be administered to the test organism in water. This route o f administration was selected because it represents one possible route o f exposure to aquatic organisms. The test substance will be mixed directly with dilution water. Test Organism Fathead minnows (Pimephales promelas) will be used in this te st This species is representative of an im portant group o f organisms and was selected for use in the test based upon past use and ease of handling in die laboratory. Embryos used to start the test w ill be approximately 2 to 24-hours old and will be obtained from cultures maintained in the laboratory at W ildlife International, Ltd. or a commercial supplier. Embryos will be removed from spawning substrates using a gentle rolling motion with die index finger or with a clean razor blade and examined under a microscope to select healthy specimens in approx imately the same stage o f development. Embryos from a minimum o f three spawns will be combined and used to initiate testing. If more than 50% o f die eggs from a given substrate are fimgused or have not been fertilized, no embryos from that substrate will be used in the te st Q O lO V 2 PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -5- Ncwly-hatched larvae will be fed live brine shrimp nauplii (Artemia sp.) 3 times per day during the first 7 days post-hatch Thereafter, they will be fed live brine shrimp nauplii 3 times daily on weekdays and at least 2 times daily on weekends until the test is terminated. To ensure that the feeding rate per fish remains constant, the amount o f food supplied to each replicate test chamber will be adjusted on a weekly basis to account for losses due to mortality. Fish will not be fed for approximately 48 hours prior to the termination of the test to allow the fish to clear their digestive tracts before measurements o f weight are made. Specifications for acceptablelevels o f contaminants in fish diets have not been established. However, there are no known levels o f contaminants reasonably expected to be present in the diet that are considered to interfere with the purpose or conduct o f the te st Loading, defined as the total wet weight o f fish per liter o f test solution, will not exceed 0.5 grams o f fish per liter o f solution that passes through a chamber in 24 hours. Instantaneous loading will not exceed 5 grams o f fish per liter o f test solution present in the test chamber at any given time. Dilution W ater W ater used for the culturing and testing o f fathead minnows will be obtained from a well approximately 40 meters deep located on the W ildlife International, Ltd. site. The' water will be passed through a sand filter and pumped into a 37,800-L storage tank where the water will be aerated with spray nozzles. Prior to use the water will be filtered to 0.45 pm in order to remove fine particles and then passed through a UV sterilizer in order to remove microorganisms. W ater used for culturing and testing is characterized as moderately hard. Typical values for hardness, alkalinity, pH and specific conductance are approximately: Hardness, mg/L as C aC 03 Alkalinity, mg/L as C aC 03 pH Specific Conductance, pmhos/cm 145 190 . 8.1 330 Hardness, alkalinity, pH and specific conductance will be measured weekly to monitor the consistency o f the well water. Means and ranges o f the measured parameters for die four-week period preceding the test will be provided in the final report. Analyses will be performed at least once annually to determine the concentrations o f selected organic and inorganic constituents o f the well water and results o f the most recent GLP compliant analyses will be summarized in the final report PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd 6- - Test Apparatus A continuous-flow diluter will be used to provide each concentration o f the test substance and a negative (dilution water) control. A peristaltic pump will be used to deliver the test substance to mixing chambers where the test substance will be mixed w ith dilution water. The flow o f dilution water into each mixing chamber will be controlled using rotameters. The rotameters will be calibrated prior to test initiation and at approximately weekly intervals thereafter. A fter mixing, test solutions will be split to each replicate chamber. The proportion o f water split to each replicate w ill be checked prior to the test and at approximately weekly intervals thereafter to ensure that these flow rates vary by no more than 10% o f the mean flow rate o f the four replicates. The diluter will be adjusted so that each test chamber receives at least 5 volume additions o f test solution every 24 hours. Peristaltic pumps will be calibrated before each study and at approximately weekly intervals thereafter. The delivery o f test substance to test chambers will begin at least 48 hours prior to the test in order to establish equilibrium concentrations o f the test substance. The general operation of the diluter will be checked visually at least two times per day during the test and at least once at the beginning and end o f the test. Embryo incubation cups will be constructed from glass cylinders approximately SOmm in diameter, w ith 425-pm nylon or Teflon screen attached to the bottom using silicone sealant. Test chambers will be 9-L glass aquaria filled with approximately 7 L o f water. Test chambers will be positioned in a temperature- controlled environmental chamber to m aintain a temperature o f 25 1C. Test chambers will be labelled with project number, replicate and test concentration. Environmental Conditions Ambient room light will be used to illuminate the test systems. Fluorescent tubes that emit wavelengths similar to natural sunlight (e.g., Colortone 50) will be controlled by an automatic timer to provide a photoperiod o f 16 hours o f light and 8 hours o f darkness. A 30-minute transition o f low light intensity will be provided when lights go on and o ff to avoid sudden changes in light intensity. Light intensity will be measured at test initiation, at weekly intervals during the test, and at die end o f the test, with a SPER Scientific Ltd. light meter or equivalent The test will be conducted at a target temperature o f 25 2C. Temperature will be monitored and recorded continuously during the entire test in one control replicate using a Fulscope ER/C Recorder (1900 J Series Model A) or equivalent Recorder measurements will be verified with a liquid-in-glass thermometer prior to test initiation and at least weekly thereafter. The temperature in each test chamber also will be PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 C01074 Wild life In ter n a tio n a l, ltd -7 - measured using a Iiquid-in-giass thermometer at the beginning o f the test, at weekly intervals during the test, and at the end o f the test. Dissolved oxygen will be measured in alternate replicates o f each treatment and control group at the beginning o f the test, daily during the first 7 days o f the test, at weekly intervals during the test and at test termination using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter, or equivalent. In the event that dissolved oxygen levels fall below 60% saturation, dissolved oxygen measurements will be made daily in every test chamber and appropriate actions will be taken after consultation with the Sponsor. Measurements o f pH will be made in alternate replicates o f each treatment and control group at test initiation, at weekly intervals during the test and at test termination using a Fisher Accumet Model 915 pH meter, or equivalent. If a treatment level reaches 100% mortality, temperature, dissolved oxygen and pH measurements will be taken at that time, then discontinued. Hardness, alkalinity, and specific conductance will be measured in the dilution water and in the highest treatment level at test initiation, at weekly intervals during the test and at test term inatioa Hardness and alkalinity measurements will be made by titration using procedures based on methods in Standard Methodsfo r the Examination o f Water and Wastewater (4). Specific conductance will be measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter, or equivalent. Procedures Prior to test initiation, embryo incubation cups will be impartially placed in glass beakers containing dilution water within 3C o f the test temperature. In order to control bias, groups o f one, two or three embryos will be indiscriminately distributed among the cups until each contains 20 embryos. N o other potential sources o f bias are expected to affect the results o f fire study. Two incubation cups will be placed in each duplicate test chamber to achieve a total o f 40 embryos per replicate and 80 embryos per treatm ent Incubation cups will be attached to a rocker arm and suspended in the w ater column o f the test chambers. The reciprocating motion of the rocker arm (approximately 2 rpm) will facilitate circulation o f test solution around the embryos during the incubation period. During the first day o f exposure, embryos will be observed twice for mortality and eggs with fungus. Thereafter, until hatching is complete, embryo mortality and the removal o f dead embryos and/or embryos with fungus will be performed once daily. A fter hatch, larvae will be counted and released into the test chamber. The test will be repeated if the percentage o f embryos in the control group that hatches successfully is less than 66%, or if the post-hatch survival in any control chamber is less than 70%. 001075 PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l, ltd 8- - When hatching has reached >90% in the control group, larvae will be released to their respective test chambers. Any unhatched embryos will be kept in the egg cups until they have hatched or until death o f the embryo occurs. After the initial release o f the larvae, the exposure will continue for at least 28 days. Biological M easurements Data on time to hatch, hatching success, survival, and growth (wet weight, dry weight and total length) will be collected during the test. Due to high natural mortality that arises from fungal infections during embryonic development, all fungused embryos that are removed during the first 24 hours o f incubation will be considered natural mortality (D.A. Benoit, personal communication). Daily observations o f fiy mortality, unusual behavior, and overall appearance will be made during the 28-day post hatch growth period. A t test termination, all surviving fish will be retained for measurements o f wet weight, dry weight, and total length. Sampling for Analytical Measurements Prior to exposure water samples will be collected from two replicate test chambers o f each treatment group and the negative control. W ater samples also will be collected from two alternating replicate test chambers at the beginning o f the test, on Day 4 o f the test, at approximately weekly intervals during the test, and at the end o f the test to determine concentrations o f the test substance. In the event that 100% mortality occurs in any treatment, then sampling o f that treatment will terminate following the next sampling interval. Samples will be collected at mid-depth from each test chamber and analyzed immediately. The sample scheme is summarized below: PROPOSED NUMBERS OF VERIFICATION SAMPLES Experimental Group Pretest* DayO Day 4 Day 7 Day 14 Day 21 Day 28 End Control 2 222 2 2 2 2 Level 1-Low Concentration 2 2 2 2 2 2 2 2 Level 2 2 222 2 2 2 2 Level 3 2 222 2 2 22 Level 4 Level 5 2 2 22 2 2 22 2 222 2 2 22 Level 6-High Concentration 2 222 2 2 22 Totals 14 14 14 14 14 14 14 14 "Pre-test samples will be collected after conditioning o f the diluter. More than one pre-test sampling interval may be required, depending upon tire Sponsors needs, and additional sampling and results will be documented in the raw data and included in the final report. Total Number o f Verification Samples - 112 001076 PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l, ltd -9 - The above numbers of samples represent those collected from the test and do not include quality control (QC) samples such as matrix blanks and fortifications prepared and analyzed during the analytical chemistry phase o f the study. At the discretion o f the Study Director, water samples also will be collected from at least one appropriate chamber whenever a malfunction is detected in any part o f the test substance delivery system. Analytical Measurements v . Chemical analysis of the samples will be performed by W ildlife International, Ltd. The analytical method used will be based upon methodology provided by the Sponsor and identified in Appendix II. The methodology used to analyze the test samples will be documented in the raw data and summarized in die final report Data Analyses The endpoints used to determine the statistical significance o f toxicant-related effects on fathead minnows include hatching success, survival of juvenile fish, total length, wet weight and dry weight o f surviving fish. The data sets will be evaluated for normality and homogeneity o f variances. Data transformations will be employed, if necessary, to correct the data for non-normality or heterogeneous variances. When the data meet the conditions o f normality and homogeneity o f variances, tests for significant differences will use 2 X 2 contingency tables and analysis o f variance (ANOVA) procedure w ith an appropriate means test (e.g, Dunnett's test, Duncan's test, Tukey's test, etc.). When the conditions o f normality and homogeneity of variances are not met, and no transformations are successful in correcting the condition, then nonparametric statistical procedures will be used. The results o f the statistical analyses will aid in the determination of the NOEC and LOEC. The maximum acceptable toxicant concentration will be calculated as the geometric mean o f the NOEC and LOEC. RECORDS TO BE M AINTAINED Records to be maintained for data generated at W ildlife International, Ltd. will include but not be limited to: 1. A copy o f the signed protocol. 001077 PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l, ltd - 10 - 2. Identification and characterization of the test substance, if provided by the Sponsor. 3. Dates o f initiation and termination o f the te st 4. History o f the test organism. 5. W eight and length measurements. 6. Results o f rangefinding tests, when applicable. 7. Stock solution calculation and preparation. 8. Observations made dining the te st 9. W ater chemistry calculations (e.g., hardness and alkalinity). 10. If applicable, the methods used to analyze test substance concentrations and the results o f analytical m easurem ents. 11. Statistical calculations. 12. Test conditions and physical/chemical measurements. 13. Calculation and preparation o f test concentrations. 14. Copy o f final report. FINAL REPO RT A final report o f the results o f the study will be prepared by W ildlife International, Ltd. The report will include, but not be limited to, the following, when applicable: 1. Name and address o f the facility performing the study. 2. Dates upon which the study was initiated and completed. It is the responsibility o f the Sponsor to provide the final date that data are recorded for chemistry pathology and/or supporting evaluations that may be generated at other laboratories. 3. A statement o f compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives and procedures as stated in the approved protocol, including arty changes in the original protocol. 5. The test substance identification, including name, chemical abstract number or code number, strength, purity, composition, and other characteristics provided by the Sponsor. 6. Stability and solubility o f h e test substance under the conditions o f administration, if provided by the Sponsor. 7. A description o f the methods used to conduct the test. G 010V 8 PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -11- 8. A description o f the test organism s, including the source o f the test organisms, scientific name, age, life stage, means and ranges o f weights and lengths, observed diseases and treatments. 9. A description o f the preparation o f the test solutions, the methods used to allocate organisms to test chambers and begin the test, the number o f organisms and chambers per treatment, and the duration o f the test. 10. A description o f circumstances that may have affected the quality or integrity o f the data. 11. The name o f the Study Director and the names o f other scientists, professionals, and supervisory personnel involved in the study. 12. A description o f the transformations, calculations, and operations performed on the data, a summary and analysis o f the biological and analytical chemistry data, and a statement o f the conclusions drawn from the analyses. 13. The signed and dated reports o f each o f the individual scientists or other professionals involved in the study, if applicable. 14. Statistical methods employed for analyzing the data. 15. The location where raw data and final report are to be stored. 16. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made and the dates o f any findings reported to the Study Director and M anagement . 17. If it is necessary to make corrections or additions to a final report after it has been accepted, such changes will be made in the form o f an amendment issued by the Study Director. The amendment will clearly identify the part o f the final report that is being amended and the reasons for the alteration. Amendments will be signed and dated by the Study Director. CHANGING OF PROTOCOL Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part o f the protocol and will be attached to the final protocol. Any other changes will be in the form o f written deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be indicated in the final report GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFRPart 160 and/or Part 792); OECD Principles o f Good Laboratory Practice (ENV/MC/CHEM (98) 17); PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 001079 W il d l if e In te r n a tio n a l, ltd -12- and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). Each study conducted by W ildlife International, Ltd. is routinely examined by the W ildlife International, Ltd. Quality Assurance U nit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement o f compliance with Good Laboratory Practices will be prepared for all portions o f the study conducted by W ildlife International, Ltd. The Sponsor will be responsible for compliance with Good Laboratory Practices for procedures performed by other laboratories (e.g., residue analyses or pathology). Ravydata for all work performed at W ildlife Intranational, Ltd. and a copy o f the final report will be filed by project number in archives located on the W ildlife International Ltd. site, or at an alternative location to be specified in the final report y PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 o o io s o 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -13- REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 8S0- Ecological Effects Test Guidelines (draft), OPPTS Number 850.1400: Fish Early-Life Stage Toxicity Test. 2 ASTM Standard E1241-92. 1977. Standard Guidefo r Conducting Early Life-Stage Toxicity Tests-with Fishes. American Society for Testing and Materials. 3 United States Environmental Protection Agency. 1986. Standard Evaluation Procedure, Fish Early Life-Stage Test. Office o f Pesticide Programs, Hazard Evaluation Division. EPA 540/9-86138. 4 APHA, AWWA, WPCF. 1985. Standard Methods fo r the Examination ow Water and Wastewater. 16* Edition, American Public Health Association American Water Works Association Water Pollution Control Federation, New York. PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 001081 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -14- APPENDKI ENITFICATION OF TEST SUBSTANCE BY SPONSOR To be Completedby Sponsor I. Test Substance Identity (name to be used in the report): PFOS (Perfluorooctane Sulfonic Acid Potassium Salt Reference Standard (if applicable): Analytical Standard: N/A________________________________ Internal Standard: 1.1.2.2H.H.H.H Perfluorooctane Sulfonic Acid Test Substance Sample Code or Batch Number: Lot 217____________________________________ Test Substance Purity (% Active Ingredient): 98.9______ Expiration Date: 2008____________ E Test Substance Characterization Yes EL Test Substance Storage Conditions Please indicate tie recommendedstorage conditions atWildlife International, Ltd Amhient__________________________________________________________ Has the stability ofthe testsubstanceunderthese storage conditions been determined in accordancewith GLP Standards? _____ Yes Otherpertinent stability information: x __No x No IV. Test Concentrations: Adjusttest concentrationto 100% a i ______x____ basedupon the purity (%) given above. Da not adjusttest concentrationto 100% __________ ai. Tg^mematerialASIS. V. Toxicity Information: Mammalian: RatLD50 251 m e/ke MouseLD50 N/A Aquatic: Invertebrate Toxidly (EC/LC50) Fish Toxicity (LC50) Daphnia maena: 27 me/L__________ R ainbow Trout: 11 me/L Daphniam aena: 50 me/L__________ Fathead M innow- 38 me/L Other Toxicity Information (incliiHing findings o f rbmnic and snhrhmnic teste)- Please see MSDS________________________________________________________________ PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 001032 3M LAB REQUEST NO. U2723 W ild life Internatio nal, ltd -15- APPENDEX n Analytical Method Provided by Sponsor Samples will be analyzed based upon procedures provided by the Sponsor in the following analytical methods: 1. Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determination o f Perfluorooctane Sulfonic Acid Potassium Salt (PFOS) In Freshwater, Saltwater and Algal Medium A copy o f die above method will be maintained in the raw data. The actual methodology used to analyze the test samples will be documented in the raw data and summarized in the final report. PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 O O IO S3 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd. PROJECT N O .: 454A -108 Page 1 o f 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A N EARLY LIFE-STAGE TOXICITY TEST W ITH THE FATHEAD M INNO W (Pim ephales prom elas) PR O TO CO L N O .: 4 5 4 /0 6 1099/FA T -E L S/SU B 454 A M E N D M E N T N O .: 1 SPO NSO R: 3M Corporation PR O JEC T N O . : 454A -108 EFFECTIVE DATE: August 2, 1999 AMENDMENT: Page2 Add: Proposed Dates: Experimental Start Date: 8 /5/99 Experimental Termination D ate: 9/6/99 REASO N: The above inform ation w as not known when the protocol w as signed by the Study D irector. a4S4\J 08\am endl 0010S4 Iw 7rl W i / J i / i f l ) ft-Pr-QQ