REFERENCES This study was conducted at EnviroSystems Division, Resource Analysts, Incorporated, Hampton, NH, USA at the request of the 3M Company.

OTHER Last changed: 5/ 3/ 00 ENV/ JM/ RD( 2002) 17/ FINAL 154 Robust Study Report Reference No. 26   Acute toxicity to Fish TEST SUBSTANCE Identity: Perfluorooctanesulfonate, Ammonium salt; may also be referred to as PFOS NH4+ salt or FC 93. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, ammonium salt, CAS # 29081 56 9)

Remarks: Test sample was taken from 3M production lot # 1. The test sample is a mixture of the test substance in isopropanol and water ( 25% test substance, 20% isopropanol, 55% water). No calculations were made to adjust for the actual concentration of the test substance in the test sample.

METHOD Method: Not noted. Type: Static acute GLP: No Year completed: 1974 Species: Pimephales promelas Supplier: Not noted. Analytical monitoring: pH and DO content Exposure period: 96 hours Statistical methods: Plotted LC50 Test fish age: Not noted. Length and weight: Average length = 2 inches, Average weight = 1.5 g Loading: Not noted. Pretreatment: Not noted Test Conditions Dilution water: carbon filtered city of St. Paul, MN water Dilution water chemistry: Not noted.

Stock and test solution preparation: Not noted. Concentrations dosing rate: Once Stability of the test chemical solutions: Not noted. Exposure vessels: Not noted. Number of replicates: One. Number of concentrations: five plus a negative control Water chemistry during the study: Dissolved oxygen range ( 0   96 hours): 5.0 5.9 mg/ L ( control exposure) 4.2 5.0 mg/ L ( 100 mg/ L exposure) pH range ( 0   96 hours): 7.0 7.1 ( control exposure) 7.0 7.2 7.0 7.1 ( 100 mg/ L exposure) Test temperature range ( 0   96 hours): 21   22oC ( 70 72oF)

RESULTS Nominal concentrations: Negative control, 10, 25, 50, 75, 100 mg/ L ENV/ JM/ RD( 2002) 17/ FINAL 155 Element value: 96 hour LC50 = 85 mg/ L ( C. I. not determined) Mortality of controls: None Remarks: 95% confidence limits were not calculated for this material. Additionally, testing was conducted on the mixture of the test substance in 20% isopropanol and 55% water. The value reported applies to that mixture and not the test substance. No attempt was made to determine the impact of the presence of the organic solvent or what portion of the toxicity can be contributed to the Perfluorooctanesulfonate, ammonium salt.

CONCLUSIONS The test sample containing 25% Perfluorooctanesulfonate, ammonium salt exhibited a 96 hour LC50 for fathead minnow of 85 mg/ L.

Submitter: 3M Company, Environmental Laboratory P. O. Box 33331 St. Paul, MN 55133, USA DATA QUALITY Reliability: Klimisch ranking 3.

REFERENCES This study was conducted by the 3M Company, Environmental Laboratory, 7/ 29/ 74 to 8/ 2/ 74.

OTHER Last changed: 5/ 3/ 00 ENV/ JM/ RD( 2002) 17/ FINAL 156 Robust Study Report Reference No. 27   Acute toxicity to Fish TEST SUBSTANCE Identity: Perfluorooctanesulfonate, Ammonium salt; may also be referred to as PFOS NH4+ salt or FC 93. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, ammonium salt, CAS # 29081 56 9)

Remarks: Test sample was taken from 3M production lot # 1. The test sample is a mixture of the test substance in isopropanol and water ( 25% test substance, 20% isopropanol, 55% water). No calculations were made to adjust for the actual concentration of the test substance in the test sample.

METHOD Method: Not noted. Type: Static acute GLP: No Year completed: 1974 Species: Pimephales promelas Supplier: Not noted. Analytical monitoring: pH and DO content Exposure period: 96 hours Statistical methods: Plotted LC50. Test fish age: Not noted. Length and weight: Average length = 2 inches, Average weight = 1.5 g Loading: Not noted. Pretreatment: Not noted Test Conditions Dilution water: Carbon filtered city of St. Paul, MN water Dilution water chemistry: Not noted. Stock and test solution preparation: Not noted. Concentrations dosing rate: Once Stability of the test chemical solutions: Not noted. Exposure vessels: Not noted. Number of replicates: One. Number of concentrations: five plus a negative control Water chemistry during the study: Dissolved oxygen range ( 0   96 hours): 4.5 5.7 mg/ L ( control exposure) 3.8 5.0 mg/ L ( 125 mg/ L exposure) Not recorded at highest conc. ( 150 mg/ L) due to 100% mortality. pH range ( 0   96 hours): 7.0 7.0 ( control exposure) 7.0 7.0 ( 125 mg/ L exposure) Not recorded at highest conc. ( 150 mg/ L) due to 100% mortality. Test temperature range ( 0   96 hours): 20   21oC ( 69 70 oF) ENV/ JM/ RD( 2002) 17/ FINAL 157 RESULTS Nominal concentrations: Negative control, 50, 75, 100, 125, 150 mg/ L Element value: 96 hour LC50 = 100 mg/ L ( C. I. not determined) Mortality of controls: None Remarks: 95% confidence limits were not calculated for this material. Additionally, testing was conducted on the mixture of the test substance in 20% isopropanol and 55% water. The value reported applies to that mixture and not the test substance. No attempt was made to determine the impact of the presence of the organic solvent or what portion of the toxicity can be contributed to the Perfluorooctanesulfonate, ammonium salt.

CONCLUSIONS The test sample containing 25% Perfluorooctanesulfonate, ammonium salt exhibited a 96 hour LC50 for fathead minnow of 100 mg/ L Submitter: 3M Company, Environmental Laboratory P. O. Box 33331 St. Paul, MN 55133, USA DATA QUALITY Reliability: Klimisch ranking 3.

REFERENCES This study was conducted by the 3M Company, Environmental Laboratory, 10/ 15/ 74 to 10/ 19/ 74.

OTHER Last changed: 5/ 3/ 00 ENV/ JM/ RD( 2002) 17/ FINAL 158 Robust Study Report Reference No. 28   Acute toxicity of P3025 Developmental Material to Fathead minnow (

 )

TEST SUBSTANCE Identity: Perfluorooctylsulfonate, didecyldimethylammonium salt; may also be referred to as Fluoroalkyl ammonium derivative. [ 1 Decaminium, N decyl N, N dimethyl, salt with 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro 1 octanesulfonic acid ( 1: 1), CAS # 251099 16 8]

Remarks: The 3M production lot number was Lot 1. The test sample is L 14394 referred to by the test laboratory as P3025. The sample was labeled F 11615, Lot 1. The test sample is a mixture of the test substance in water ( approximately 30 40% test substance, 60 70% water, and 0 5% of residual perfluorochemicals). All values reported relate to this mixture. The test sample appears to be a 2 phase dispersion ( clear liquid with opaque solid) which rapidly separates after agitation. No calculations were made to adjust for the actual concentration of the test substance in the test sample.

METHOD Method: OECD 203 Type: Static acute GLP: No Year completed: 1996 Species: Pimephales promelas Supplier: Not noted. Analytical monitoring: DO, pH, temperature, and conductivity were monitored daily. Exposure period: 96 hours Statistical methods: LL50 values calculated using the Trimmed Spearman Karber method. The NOEL was calculated using Fisher's Exact tests. Test fish age: Not given. Length and weight: Average length = 11.3 mm, Average weight = 7.8 mg Loading: 0.26 g/ L Pretreatment: None Test Conditions Dilution water: Dechlorinated City of Duluth, MN tap water. Water was aerated for 24 hours prior to use in the test. Dilution water chemistry: Hardness: 48 mg/ L as CaCO3 pH: 8.08 Lighting: Cool white fluorescent bulbs. Photoperiod of 16 hours light, 8 hours dark used. No transition period noted. Stock and test solution preparation: Water accommodated fractions. Test solutions were prepared individually for each test replicate concentration by mass addition of vigorously shaken test substance in 4 L of dilution water. The solutions were vigorously stirred for 21 hours ( vortex 1/ 2 to 1/ 3 solution depth). The aqueous phase was siphoned from the vessel at mid depth.

Concentrations dosing rate: Once Stability of the test chemical solutions: Not noted. Exposure vessels: 4 L glass jars containing 3 L of test solution. The jars were sealed with Teflonlined lids fitted with stoppers to accommodate oxygen flushing of headspace. Number of replicates: two ENV/ JM/ RD( 2002) 17/ FINAL 159 Number of fish per replicate: ten Number of concentrations: three plus a negative control Water chemistry during the study: Dissolved oxygen range: ( 0   96 hours): 9.1   14.6 mg/ L ( control exposure) 8.7   18.2 mg/ L ( 700 mg/ L exposure) pH range: ( 0   96 hours): 7.80   8.08 ( control exposure) 7.78   7.99 ( 700 mg/ L exposure) Test temperature range ( 0   96 hours): 20.8   20.9oC Conductivity range ( 0   96 hours): 128   142 µ mhos/ cm ( control exposure) 118   154 µ mhos/ cm ( 700 mg/ L exposure) Remarks: Oxygen was added to the headspace in the jars before sealing initially and at each observation period. The dissolved oxygen concentrations were super saturated in the test vessels, particularly in the 700 mg/ L exposure concentration.

RESULTS Nominal loading concentrations: Negative control, 400, 700, 1,000 mg/ L. Element value: ( 95% confidence interval is given in brackets) 24 hour LL50 = 618 ( 568  673) mg/ L 48 hour LL50 = 607 ( 554  664) mg/ L 72 hour LL50 = 595 ( 551  643) mg/ L 96 hour LL50 = 562 ( 523  604) mg/ L 96 hour NOEL = < 490 mg/ L All element values based on nominal concentrations.

Biological observations after 96 hours: No mortality or abnormal behavior observed in the negative control during the test. Mortality was observed in the remaining exposure concentrations. Surfacing was observed in half of the fish at the 700 mg/ L exposure concentration at 24 hours, and 2 fish were quiescent at 96 hours. No abnormal behavior was observed in the 400 mg/ L exposure concentration.

Cumulative percent mortality:

Nominal Loading Test Concentration, mg/ L 24 hours 48 hours 72 hours 96 hours Neg. Control 0 0 0 0 490 10 15 15 25 700 75 75 80 90 1,000 100 100 100 100 Lowest concentration causing 100% mortality: 1,000 mg/ L Mortality of controls: None Remarks: Values reported are for the test sample. No calculations were made to adjust for the concentration of the test substance in the test sample.

CONCLUSIONS The test sample 96 hour LL50 for fathead minnow was determined to be 562 mg/ L with a 95% confidence interval of 523   604 mg/ L. The 96 hour no observed effects level ( NOEL) was < 490 mg/ L. ENV/ JM/ RD( 2002) 17/ FINAL 160 Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133, USA DATA QUALITY Reliability: Klimisch ranking 2. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. Additionally, data is for a mixture and toxicity cannot be positively attributed to didecyldimethylammonium Perfluorooctylsulfonate salt alone. Also, supersaturation of the test solutions with oxygen could also have contributed to the toxicity.

REFERENCES This study was conducted at AScI Corporation, Environmental Testing Division, Duluth, MN, at the request of the 3M Company.

OTHER Last changed: 5/ 24/ 00 ENV/ JM/ RD( 2002) 17/ FINAL 161 Robust Study Report Reference No. 29   Acute toxicity of P3025 Developmental Material

TEST SUBSTANCE Identity: Perfluorooctylsulfonate, didecyldimethylammonium salt; may also be referred to as Fluoroalkyl ammonium derivative. [ 1 Decaminium, N decyl N, N dimethyl, salt with 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro 1 octanesulfonic acid ( 1: 1), CAS # 251099 16 8]

Remarks: The 3M production lot number was Lot 1. The test sample is L 14394 referred to by the test laboratory as P3025. The sample was labeled F 11615, Lot 1. The test sample is a mixture of the test substance in water ( approximately 30 40% test substance, 60 70% water, and 0 5% of residual perfluorochemicals). All values reported relate to this mixture. The test sample appears to be a 2 phase dispersion ( clear liquid with opaque solid) which rapidly separates after agitation. No calculations were made to adjust for the actual concentration of the test substance in the test sample.

METHOD Method: OECD 202 Test type: Static acute GLP: No Year Completed: 1996 Species: Daphnia magna Analytical monitoring: DO, pH, temperature and conductivity were monitored daily. Statistical methods: EL50 values calculated using Trimmed Spearman Karber method. NOEL value calculated using Steel's Many One Rank test. Test daphnid source: Obtained from cultures maintained by AScI Corporation, Duluth, MN. Test daphnid age at study initiation: < 24 hours Test Conditions: Dilution water: Dechlorinated City of Duluth, MN tap water. Water was aerated for 24 hours prior to use in the test. Dilution water chemistry: Hardness: 44 mg/ L as CaCO3 pH: 8.04 Lighting: Cool white fluorescent bulbs. Photoperiod of 16 hours light, 8 hours dark. No transition period noted. Stock and test solutions preparation: Water accommodated fractions. Test solutions were prepared individually for each concentration by mass addition of vigorously shaken test substance in 1 L of dilution water. The solutions were vigorously stirred for 23 hours ( vortex 1/ 2 to 1/ 3 solution depth). The aqueous phase was siphoned from the vessel at mid depth after settling for 1 hour. Exposure vessels: 250 mL borosilicate glass beakers containing 200 mL of test solution. The solutions were kept covered during the test. Number of replicates: Four Number of daphnids per replicate: Five Number of concentrations: Five plus a negative control Water chemistry during the study: Dissolved oxygen range ( 0   48 hours): 8.6   9.1 mg/ L ( control exposure) 8.0   8.8 mg/ L ( 50 mg/ L exposure) ENV/ JM/ RD( 2002) 17/ FINAL 162 pH range ( 0   48 hours): 8.04   8.11 ( control exposure) 7.92   8.00 ( 50 mg/ L exposure) Test temperature range ( 0   48 hours): 20.9   21.0oC Conductivity range ( 0   48 hours): 142   155 µ mhos/ cm ( control exposure) 120   124 µ mhos/ cm ( 50 mg/ L exposure) Element basis: mortality and immobilization RESULTS Nominal loading concentrations: Negative control, 3.13, 6.25, 12.5, 25, 50 mg/ L Element value: ( 95% confidence interval is given in brackets) 24 hour EL50 = 27.0 ( 18.7 39.0) mg/ L 48 hour EL50 = 11.3 ( 9.6 13.2) mg/ L 48 hour NOEL = 6.25 mg/ L All element values based on nominal concentrations.

Statistical Evaluation: The EL50 values and 95% confidence intervals were calculated by the Trimmed Spearman Karber method. The NOEL was calculated using Steel's Many One Rank test using the TOXSTAT statistical software Version 3.2, University of Wyoming.

Biological observations: Daphnids in the negative control, and the 3.13 and 6.25 mg/ L treatments appeared healthy and normal throughout the test with no mortality, immobility or overt clinical signs of toxicity. The effects noted in this study were mortality; no immobilization was noted at any test concentration. The number of surviving organisms were determined visually and recorded initially and after 24 and 48 hours.

Cumulative percent mortality:

Nominal Loading Test Concentration mg/ L 24 hours 48 hours Negative Control 0 0 3.13 0 0 6.25 0 0 12.5 5 70 25 50 95 50 75 100 Control response: satisfactory Remarks: Values reported are for the test sample. No calculations were made to adjust for the concentration of the test substance in the test sample.

CONCLUSIONS The test substance 48 hour EL50 for Daphnia magna was determined to be 11.3 mg/ L with a 95% confidence interval of 9.6 13.2 mg/ L. The 48 hour no observed effect level ( NOEL) was 6.25 mg/ L.

Submitter: 3M Company, Environmental Laboratory P. O. Box 33331 St. Paul, MN, 55133, USA. ENV/ JM/ RD( 2002) 17/ FINAL 163 DATA QUALITY Reliability: Klimisch ranking 2. The study lacks analytical measurement of test substance concentrations in the test solutions and sample purity is not sufficiently characterized. Additionally, data is for a mixture and toxicity cannot be positively attributed to didecyldimethylammonium Perfluorooctylsulfonate salt alone.

REFERENCES This study was conducted at ASci Corporation, Environmental Testing Division, Duluth, MN, at the request of the 3M Company.

OTHER Last changed: 5/ 24/ 00 ENV/ JM/ RD( 2002) 17/ FINAL 164 Robust Study Report Reference No. 30  Acute toxicity of PFOS to Rainbow trout in saltwater TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance is a white powder of uncharacterized purity.

METHOD Method: Standard procedures for Testing Acute Lethality of Liquid Effluents ( Environment Canada, 1980) Type: Acute static, renewal after 48 hours GLP: No Year completed: 1985 Species: Rainbow Trout, Salmo gairdneri Fish source: Rainbow Springs, Thamesford Fish age at test initiation: not noted Fish acclimation to salt water: fish gradually acclimated to increasing salinity; held at 30 parts per thousand salinity 8 days prior to test initiation. Exposure period: 96 hours Analytical monitoring: Dissolved oxygen, pH, conductivity Statistical methods: Not noted. Element values were calculated for each replicate series, but not combined for the whole study. A cumulative mortality concentration plot was used to estimate the LC50.

Test conditions: Dilution water: Mississauga dechlorinated tap water amended with calcium, magnesium, sodium, and chloride to obtain 30 parts per thousand salinity Dilution water chemistry ( initial): pH: 7.4  8.0 D. O.: 8.6   9.0 mg/ L Conductivity: > 20,000 µ mhos/ cm Stock solution preparation: 1000 mg/ L Exposure vessels: Not noted; solution volume 35 L Number of replicates: 2 tests   run 4 days apart, not replicated Number of organisms/ vessel: 6 Loading: 0.75 g/ L Number of concentrations: 4 plus a blank control Water chemistry during the studies: Dissolved oxygen ranges 8.1   10.3 ( control) 8.8   10.1 ( 30 mg/ L) pH ranges 7.6   8.2 ( control) 7.3   8.0 ( 30 mg/ L) Test temperature ( 0   48 hours): 15 ° C Photoperiod: 12 hours light, 12 hours dark Element basis: mortality ENV/ JM/ RD( 2002) 17/ FINAL 165 RESULTS Nominal concentrations: 5, 10, 20, 30 mg/ L Element values ( 95% confidence interval) calculated per replicate: 96 hour LC50 = 13.7 ( 10.7   17.7) mg/ L 96 hour LC50 = 13.7 ( 10.7   17.8) mg/ L Mortality of controls: 17% ( 1/ 6 in both studies)

DATA QUALITY Reliability: Klimisch ranking = 2. This study satisfied all criteria for quality testing at the time performed, but actual concentrations were not measured. Results were based on nominal concentrations. Additionally, sample purity was not adequately characterized.

REFERENCES This study was conducted by Beak Consultants Limited, Mississauga, Ontario, Canada for Panarctic Oils Ltd, Calgary, Alberta, Canada.

OTHER Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 Last changed: 7/ 19/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 166 Robust Study Report Reference No. 31  Acute toxicity of PFOS to Rainbow trout in freshwater TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance is a white powder of uncharacterized purity.

METHOD Method: Standard procedures for Testing Acute Lethality of Liquid Effluents ( Environment Canada, 1980) Type: Acute, renewal after 48 hours GLP: No Year completed: 1985 Species: Rainbow Trout Fish source: Rainbow Springs, Thamesford Fish age at test initiation: not noted Exposure period: 96 hours Analytical monitoring: Dissolved oxygen, pH, conductivity Statistical methods: Not noted. Element values were calculated for each replicate series, but not combined for the whole study. A cumulative mortality concentration plot was used to estimate the LC50.

Test conditions: Dilution water: Mississauga dechlorinated tap water Dilution water chemistry ( initial): pH: 7.5  8.5 D. O.: 9.0   10.4 mg/ L Stock solution preparation: 1000 mg/ L; noted as cloudy Exposure vessels: Not noted; solution volume 35 L Number of replicates: 2 tests   run one week apart, not replicated Number of organisms/ vessel: 6 Loading: 0.72 g/ L Number of concentrations: 5 plus a blank control, 4 plus a blank control Water chemistry during the studies: Dissolved oxygen ranges 8.2   10.4 ( control) 8.0   9.5 ( 30 mg/ L) pH ranges 7.4   8.3 ( control) 7.5   8.7 ( 30 mg/ L) Conductivity range: 270   380 µ mhos/ cm Test temperature ( 0   48 hours): 15 ° C Element basis: mortality RESULTS Element values ( 95% confidence interval) calculated per replicate: 96 hour LC50 = 7.8 ( 6.2   9.8) mg/ L ENV/ JM/ RD( 2002) 17/ FINAL 167 96 hour LC50 = 9.9 ( 7.5   13.4) mg/ L Mortality of controls: None DATA QUALITY Reliability: Klimisch ranking = 2. This study satisfied all criteria for quality testing at the time performed, but actual concentrations were not measured. Results were based on nominal concentrations. Additionally, sample purity was not adequately characterized.

REFERENCES This study was conducted by Beak Consultants Limited, Mississauga, Ontario, Canada for Panarctic Oils Ltd, Calgary, Alberta, Canada.

OTHER Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 Last changed: 7/ 19/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 168 Robust Study Report Reference No. 32  Acute toxicity of PFOS to

sp.

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance is a white powder of uncharacterized purity.

METHOD Method: Draft International Standards Organization ( Vanhaecke and Persoone, 1981) Type: Acute static GLP: No Year completed: 1985 Species: Artemia sp. Artemia source: Salt lake Brine Shrimp Inc. Artemia age at test initiation: naupuli < 24 hours old. Exposure period: 48 hours Analytical monitoring: Dissolved oxygen, pH, conductivity Statistical methods: Not noted. Element values were calculated for each replicate series, but not combined for the whole study. A cumulative mortality concentration plot was used to estimate the LC50. Test conditions: Dilution water: 30 parts per thousand NaCl solution Dilution water chemistry ( initial): pH: 8.0   8.2 D. O.: > 6 mg/ L Stock solution preparation: 1000 mg/ L; noted as cloudy Exposure vessels: Not noted; solution volume 10 mL Number of replicates: 3 Number of organisms/ replicate: 10 Number of concentrations: 6 plus a blank control Water chemistry during the study: Dissolved oxygen ranges ( test and control): > 6.0 mg/ L pH ( test and control) 8.0   8.2 Test temperature range ( 0   48 hours): 21  21 ° C Element basis: mortality RESULTS Nominal concentrations: 1, 2, 3, 5, 10, 20 mg/ L Element values ( 95% confidence interval) calculated per replicate: 48 hour EC50 = 9.4 ( 7.4   12.1) mg/ L 48 hour EC50 = 9.4 ( 7.3   12.2) mg/ L 48 hour EC50 = 8.9 ( 6.7   11.9) mg/ L Mortality of controls: None ENV/ JM/ RD( 2002) 17/ FINAL 169 DATA QUALITY Reliability: Klimisch ranking = 2. This study satisfied all criteria for quality testing at the time performed, but actual concentrations were not measured. Results were based on nominal concentrations. Additionally, sample purity was not adequately characterized.

REFERENCES This study was conducted by Beak Consultants Limited, Mississauga, Ontario, Canada for Panarctic Oils Ltd, Calgary, Alberta, Canada.

OTHER Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 Last changed: 6/ 12/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 170 Robust Study Report Reference No. 33  Acute toxicity of PFOS to

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance is a white powder of uncharacterized purity. The following summary is abbreviated due to the fact that this study has been superceded by a more recent test.

METHOD Method: International Standards Organization ( 1982) Type: Acute static GLP: No Year completed: 1985 Species: Daphnia magna RESULTS Nominal concentrations: 10, 20, 30, 50, 100 mg/ L Number of replicates: 2 Element values ( 95% confidence interval) calculated per replicate 48 hour EC50 = 58 ( 46   72) mg/ L 48 hour EC50 = 67 ( 48   92) mg/ L DATA QUALITY Reliability: Klimisch ranking = 2. This study satisfied all criteria for quality testing at the time performed, but actual concentrations were not measured. Results were based on nominal concentrations. Additionally, sample purity was not adequately characterized.

REFERENCES This study was conducted by Beak Consultants Limited, Mississauga, Ontario, Canada for Panarctic Oils Ltd, Calgary, Alberta, Canada.

OTHER Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 Last changed: 5/ 26/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 171 Robust Study Report Reference No. 34  Perfluorooctanesulfonate, Potassium salt ( PFOS): An acute oral toxicity study with the Honey bee TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks field: The 3M production lot number was 217. The test substance is a white powder. Sample was stored AT 16 20oC prior to testing. Purity determined to be 86.9% by LC/ MS, 1H HMR, 19F NMR and elemental analyses techniques.

METHOD Method: OECD Guideline 213, EPPO Guideline 170 Test type: Acute Oral GLP: Yes Year Completed: 2001 Species: Apis mellifera L. Analytical monitoring: None   nominal concentrations Test honey bee source: Obtained from colony number 32 belonging to the Central Science Laboratory ( CSL), Sand Hutton, York, UK, National Bee Unit. Test honey bee age at study initiation: Young adult Test honey bee type: Worker honey bees, free of acarine, nosema and amoeba. Varroacide treatment: None within the 4 weeks prior to test initiation.

Test conditions Humidity: 65% + 5% Temperature: 25 + 2oC Lighting: Conducted in darkness Stock and test solutions preparation: Test substance: Initial stock solution prepared in analytical grade acetone to a final concentration of 47.8 µ g PFOS/ µ L ( nominal concentration). Final test concentrations prepared from dilutions of this solution with 50% w/ v sucrose. Resulting acetone concentration was 5%. Reference toxicant: Primary stock solution of dimethoate was prepared in deionized water containing 1 g/ L Triton X 100 to a final concentration of 3.0 µ g/ µ L. Secondary stock solutions were made by diluting the primary stock solution in deionized water containing 1 g/ L Triton X 100. Final test concentrations prepared from dilutions of these solutions with 50% w/ v sucrose. Stability of the test chemical solution: A dispersion test was carried out on an 86 µ g PFOS/ µ L acetone solution before the toxicity study was performed. The homogeneity of the mixture was assessed after 2 hours. The test item formed a clear solution on mixing; after 2 hours at room temperature, slight sediment was noted. For the toxicity test, all solutions were re mixed prior to use. The contract laboratory considered the solutions of the test doses to be homogenous for the purpose of administration. Exposure vessels: Clean, well ventilated, inverted petri dishes, measuring approximately 9 cm in diameter. Feeding: During the first four hours of the test, bees provided with 50% w/ v aqueous sucrose solutions containi ng the appropriate PFOS dose. After 4 hours, dosed sucrose removed, and bees provided with 50% w/ v aqueous sucrose solutions, continuously available through the end ENV/ JM/ RD( 2002) 17/ FINAL 172 of the exposure period. Number of replicates: Three Number of bees per replicate: Ten Negative control: 50% w/ v sucrose Solvent control: 50% w/ v sucrose plus 5% acetone Reference substance: Dimethoate Reference substance control: Triton X 100 Number of concentrations: five plus a negative and a solvent control Dose administration: The bees were anaesthetized with carbon dioxide immediately before dosing and gently tipped out onto filter paper and counted into the petri dish cage ( drones were discarded). Each group of 10 bees was offered 0.2 mL of a given test concentration or control solution. The dose was measured into a small, pre weighed, glass feeder within the cage using a variable volume pipette. This volume of solution is equivalent to 20 µ L per bee. Dose frequency: Once, for 4 hours of exposure Dose calculation: Feeders were weighed after removal from the cages to determine the dose consumed per bee. Element basis: Mortality RESULTS Nominal concentrations: Negative control ( sucrose only), acetone + sucrose control, 0.205, 0.450, 0.991, 2.17, 4.78 µ g/ bee Element value and 95% confidence interval: 24 hour LD50 = 0.72 ( 0.60   0.85) µ g/ bee 48 hour LD50 = 0.46 ( 0.32   0.55) µ g/ bee 72 hour LD50 = 0.40 ( 0.33   0.48) µ g/ bee 72 hour NOEL = 0.21 µ g/ bee All element values based on nominal concentrations Statistical Evaluation: Probit mortality plotted against the logarithm of dose using the contract laboratory Probit 1 package. A least squares regression ( Finney 1971) was fitted to these. The NOELs were estimated using Student's t test ( p< 0.05)

Biological observations: There was significant mortality at all doses above a mean intake of 0.21 µ g/ bee with a steep dose response between mean intakes of 0.45 and 2.2 µ g/ bee.

Cumulative percent mortality: Nominal Test Conc., µ g/ bee 4 hours 24 hours 48 hours 72 hours Negative Control 0 0 0 0 Solvent Control 0 3.3 3.3 3.3 0.205 0 0 6.7 10 0.450 0 20 50 60 0.991 0 70 93 97 2.17 6.7 100 100 100 4.78 30 100 100 100 ENV/ JM/ RD( 2002) 17/ FINAL 173 Sub lethal Effects   Percent Knockdown ( K) or Stumbling ( S): Nominal Test Conc., µ g/ bee 4 hours 24 hours 48 hours 72 hours Negative Control 0 0 0 0 Solvent Control 0 0 0 0 0.205 0 0 0 0 0.450 0 3.3 ( S) 3.3 ( K) 3.3 ( K) 0.991 0 0 0 0 2.17 0 0 0 0 4.78 10 ( K) 0 0 0 Control response: satisfactory Reference toxicant response: satisfactory   dimethoate 72 hour LD50 = 0.11 µ g/ bee CONCLUSIONS The potassium perfluorooctanesulfonate 72 hour oral LD50 for the honey bee was determined to be 0.40 µ g/ bee with a 95% confidence interval of 0.33   0.48. The 72 hour no observed effect level was 0.21 µ g/ bee. The dose response was steep between a mean uptake of 0.45 and 2.2 µ g/ bee.

Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 1 REFERENCES This study was conducted at Central Science Laboratory, Sand Hutton, York, UK, under contract by Wildlife International, Ltd, Easton, MD at the request of the 3M Company, Lab Request Number U2723, 2001.

OTHER Last changed: 5/ 1/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 174 Robust Study Report Reference No. 35  Perfluorooctanesulfonate, Potassium salt ( PFOS): An acute contact toxicity study with the Honey bee TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks field: The 3M production lot number was 217. The test substance is a white powder. Sample was stored AT 16 20oC prior to testing. Purity determined to be 86.9% by LC/ MS, 1H HMR, 19F NMR and elemental analyses techniques.

METHOD Method: USEPA OPPTS 850.3020 ( draft), OECD Guideline 214, EPPO Guideline 170 Test type: Acute Contact GLP: Yes Year Completed: 2001 Species: Apis mellifera L. Analytical monitoring: None   nominal concentrations Test honey bee source: Obtained from colony number 32 belonging to the Central Science Laboratory ( CSL), Sand Hutton, York, UK, National Bee Unit. Test honey bee age at study initiation: Young adult Test honey bee type: Worker honey bees, free of acarine, nosema and amoeba. Varroacide treatment: None within the 4 weeks prior to test initiation.

Test conditions Humidity: 65% + 5% Temperature: 25 + 2oC Lighting: Conducted in darkness Stock and test solutions preparation: Test substance: Stock solution prepared in analytical grade acetone to a final concentration of 90.4 µ g PFOS/ µ L ( nominal concentration). Final test concentrations prepared in acetone from dilutions of this solution. Reference toxicant: Stock solution of dimethoate was prepared in deionized water containing 1 g/ L Triton X 100 to a final concentration of 3.0 µ g/ µ L. Final test concentrations prepared from dilutions of this solution. Stability of the test chemical solution: A dispersion test was carried out on an 86 µ g PFOS/ µ L acetone solution before the toxicity study was performed. The homogeneity of the mixture was assessed after 2 hours. The test item formed a clear solution on mixing; after 2 hours at room temperature, slight sediment was noted. For the toxicity test, all solutions were re mixed prior to use. The contract laboratory considered the solutions of the test doses to be homogenous for the purpose of administration. Exposure vessels: Clean, well ventilated, inverted petri dishes, measuring approximately 9 cm in diameter. Feeding: 50% w/ v aqueous sucrose solution, continuously available Number of replicates: Three Number of bees per replicate: Ten Negative control: Undosed Solvent control: Acetone Reference substance: Dimethoate ENV/ JM/ RD( 2002) 17/ FINAL 175 Reference substance control: Triton X 100 Number of concentrations: five plus a negative and a solvent control Dose administration: The bees were anaesthetized with carbon dioxide immediately before dosing and gently tipped out onto filter paper and counted into the petri dish cage ( drones were discarded). Each bee was dosed on the thorax with a 1 µ L drop of a given test item concentration or 1 µ L acetone before being placed into the test chamber. Dose frequency: Once Element basis: Mortality RESULTS Nominal concentrations: Negative control, acetone control ( 1.0 µ / bee), 1.93, 4.24, 9.30, 20.5, 45, µ g PFOS/ bee Element value and 95% confidence interval: 24 hour LD50 = 38.9 ( 28.2   71.2) µ g/ bee 48 hour LD50 = 10.4 ( 8.2   13.0) µ g/ bee 72 hour LD50 = 6.0 ( 4.7   7.6) µ g/ bee 96 hour LD50 = 4.78 ( 3.8   5.8) µ g/ bee 96 hour NOEL = 1.93 µ g/ bee All element values based on nominal concentrations Statistical Evaluation: Probit mortality plotted against the logarithm of dose using the contract laboratory Probit 1 package. A least squares regression ( Finney 1971) was fitted to these. The NOELs were estimated using Student's t test ( p< 0.05)

Biological observations: There was significant mortality at all doses above 1.93 µ g/ bee with a steep dose response between 4.24 and 9.30 µ g/ bee Cumulative percent mortality: Nominal Test Conc., µ g/ bee 4 hours 24 hours 48 hours 72 hours 96 hours Negative Control 0 0 3.3 3.3 3.3 Solvent Control 3.3 3.3 3.3 3.3 3.3 1.93 0 6.7 6.7 13 13 4.24 0 0 13 37 37 9.30 0 6.7 40 63 90 20.5 0 40 93 97 100 45.0 0 50 93 100 100 Sub lethal Effects   Percent Knockdown ( K) or Stumbling ( S): Nominal Test Conc., µ g/ bee 4 hours 24 hours 48 hours 72 hours 96 hours Negative Control 0 0 0 0 0 Solvent Control 0 0 0 0 0 1.93 3.3 ( K) 0 0 0 0 4.24 0 0 0 0 0 9.30 0 0 0 0 3.3 ( K) 20.5 0 3.3 ( K) 0 3.3 ( K) 0 45.0 0 3.3 ( K) 3.3 ( K) 0 0 ENV/ JM/ RD( 2002) 17/ FINAL 176 Control response: satisfactory Reference toxicant response: satisfactory   dimethoate 96 hour LD50 = 0.19 µ g/ bee CONCLUSIONS The potassium perfluorooctanesulfonate 96 hour contact LD50 for the honey bee was determined to be 4.78 µ g/ bee with a 95% confidence interval of 3.8   5.8. The 96 hour no observed effect level was 1.93 µ g/ bee. The dose response was steep between 4.24 and 9.30 µ g/ bee.

Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 1 REFERENCES This study was cond ucted at Central Science Laboratory, Sand Hutton, York, UK, under contract by Wildlife International, Ltd, Easton, MD at the request of the 3M Company, Lab Request Number U2723, 2001.

OTHER Last changed: 5/ 1/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 177 Robust Study Report Reference No. 36  PFOS: A 96 hour toxicity test with the freshwater alga (

 )

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: Sample from 3M production lot number 217. The test substance is a white powder. Purity determined to be 86.9% by LC/ MS, 1H HMR, 19F NMR and elemental analyses techniques.

METHOD Method: OPPTS 850.5400 Test: Acute static GLP: Yes Year completed: 2001 Species: Anabaena flos aquae Source: Originally from UTEX   The Culture Collection of Algae at the university of Texas at Austin, and maintained in culture medium at Wildlife International Ltd., Easton, MD Analytical monitoring: PFOS measured at 0, 72, 96 hours Element basis: Reported three ways: number of cells/ ml, area under the growth curve and growth rate Exposure period: 96 hours Start date: 1/ 28/ 00 End date: 6/ 5/ 00 Test organisms laboratory culture: Algae cultures had been actively growing in algal culture medium for at least two weeks prior to test initiation. Stock nutrient solutions were prepared by adding reagent grade chemicals to reverse osmosis purified well water. Solutions were then diluted in purified well water to prepare final growth media. Test Conditions: Freshwater Algal medium Compound Nominal Concentration Units MgCl2.6H2O 12.16 mg/ L CaCl2.2H2O 4.40 mg/ L H3BO3 0.1856 mg/ L MnCl2.4H2O 0.416 mg/ L ZnCl2 3.28 µ g/ L FeCl3.6H20 0.1598 mg/ L CoCl2.6H20 1.428 µ g/ L Na2MoO4.2H20 7.26 µ g/ L CuCl2.2H2O 0.012 µ g/ L Na2EDTA. 2H20 0.300 mg/ L NaNO3 25.5 mg/ L MgSO4.7H2O 14.7 mg/ L K2HPO4 1.044 mg/ L NaHCO3 15.0 mg/ L Dilution water source: The pH of the medium was adjusted to 7.5 + 0.1 and it was sterilized by ENV/ JM/ RD( 2002) 17/ FINAL 178 filtration ( 0.22 µ m) prior to use. Test solution preparations: Individual test solutions were prepared in algal medium at each of the six nominal concentrations. The solutions were stirred with magnetic stir plates for approximately 18 hours. The final test solutions appeared clear and colorless. Exposure vessels: Sterile 250 mL glass Erlenmeyer flasks plugged with foam stoppers containing 100 mL of test solution. Agitation: Shaken continuously at 100 rpm Number of replicates: six ( including 3 for analysis of exposure concentration). Initial algal cell loading: 1.0 X 104 cells/ mL Cell counts method: hemacytometer and microscope Number of concentrations: six plus a negative control plus an abiotic control at the highest concentration tested Water chemistry: pH range ( 0   96 hours) 7.4   7.6 ( control exposure) 7.4   7.4 ( 329 mg/ L exposure) Test temperature range ( 0   96 hours) 22.8   23.8 º C Light levels: ( 0   96 hours) 1990   2310 lux from cool white fluorescent lighting Photoperiod: 24 hours light Method of calculating mean measured concentrations: arithmetic mean obtained using results obtained at 0 hours, 72 hours and 96 hours RESULTS Nominal concentrations: Negative control, 37.9, 58.6, 88.8, 139, 216, 331 mg/ L plus 331 abiotic replicate Measured concentrations: < LOQ, 37.9, 63.9, 93.8, 143, 235, 329 mg/ L; abiotic replicate = 349 mg/ L Element values ( 95% confidence interval): 24 hour EC50 ( cell density) = 105 mg/ L ( C. I. not calculable) 24 hour EbC50 ( area under curve) = 90 ( 40  150) mg/ L 24 hour ErC50 ( growth rate) = 94 ( 33  145) mg/ L 48 hour EC50 ( cell density) = 117 mg/ L ( C. I. not calculable) 48 hour EbC50 ( area under curve) = 103 mg/ L ( C. I. not calculable) 48 hour ErC50 ( growth rate) = 128 mg/ L ( C. I. not calculable) 72 hour EC10 ( cell density) = 43 ( 34   84) mg/ L 72 hour EbC10 ( area under curve) = < 38 mg/ L ( C. I. not calculable) 72 hour ErC10 ( growth rate) = 82 ( 49  116) mg/ L 72 hour EC50 ( cell density) = 120 ( 92  139) mg/ L 72 hour EbC50 ( area under curve) = 116 ( 49  142) mg/ L 72 hour ErC50 ( growth rate) = 174 ( 146  208) mg/ L 72 hour EC90 ( cell density) = 224 ( 193  275) mg/ L 72 hour EbC90 ( area under curve) = 204 ( 134  226) mg/ L 72 hour ErC90 ( growth rate) = 275 ( 162  330) mg/ L 96 hour EC10 ( cell density) = 82 ( 29  123) mg/ L 96 hour EbC10 ( area under curve) = 56 ( 26  107) mg/ L 96 hour ErC10 ( growth rate) = 109 ( 84  125) mg/ L 96 hour EC50 ( cell density) = 131 ( 106  142) mg/ L 96 hour EbC50 ( area under curve) = 124 ( 104  138) mg/ L 96 hour ErC50 ( growth rate) = 176 ( 169  181) mg/ L 96 hour EC90 ( cell density) = 213 ( 203  219) mg/ L ENV/ JM/ RD( 2002) 17/ FINAL 179 96 hour EbC90 ( area under curve) = 209 ( 197  218) mg/ L 96 hour ErC90 ( growth rate) = 225 ( 220  235) mg/ L 72 hour NOAEC ( cell density, area under curve): 37.9 mg/ L 72 hour NOAEC ( growth rate): 93.8 mg/ L 96 hour NOAEC ( cell density, growth rate): 93.8 mg/ L 96 hour NOAEC ( area under curve): 63.9 mg/ L All element values based on mean measured concentrations Statistical methods: Cell densities, area under the growth curve values, growth rates and percent inhibition values were calculated using " The SAS System for Windows", Release 6.12. The EC10, EC50, and EC90 values and 95% confidence limits were calculated by linear interpolation with treatment response and exposure concentration data using TOXSTAT Version 3.5. Cell densities, areas under the growth curve and growth rates at 72 and 96 hours were evaluated for normality and homogeneity of variances using the Shapiro Wilk's test and Levene's test, respectively. Where the data were normally distributed with equal variances, the treatment groups were compared to the control using Dunnett's test. In the one instance where data were not normally distributed, the non parametric Kruskal Wallis test was used. Results of the statistical analyses were used to determine the NOAEC values.

Analytical Methodology: Analyses of test solutions were performed at Wildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection ( HPLC/ MS). When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ ( limit of quantitation) was 4.80 mg/ L in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 103%. Samples collected at test initiation had measured values from 100 to 112% of nominal. The measured values for the samples taken at 72 hours were 99.0  110% of nominal. The measured values for the samples taken at 96 hours were 99.0  109% of nominal. For the abiotic replicate, the measured value for the sample taken at 72 hours was 103% of nominal and for the sample taken at 96 hours, 107% of nominal.

Summary of analytical chemistry data: Nominal Test Concentration, mg/ L Measured Values at 0, 72, and 96 hours, Respectively, mg/ L Mean Measured Concentration, mg/ L Percent of Nominal Negative Control All < LOQ < LOQ  37.9 37.9, 38.2, 37.6 37.9 100 58.6 65.6, 62.7, 63.4 63.9 109 88.8 94.3, 97.4, 89.8 93.8 106 139 142, 146, 142 143 103 216 230, 238, 236 235 109 331 331, 328, 329 329 99.4 331 ( abiotic) Not analyzed, 342, 356 349 105 ENV/ JM/ RD( 2002) 17/ FINAL 180 Biological observations after 96 hours: Mean Measured Concentration, mg/ L Mean Number of Cells per mL Percent Inhibition via Density Percent Inhibition via Area Under the Curve Percent Inhibition via Growth Rate Negative Control 569,167    37.9 605,000  6.3 3.0  1.3 63.9 585,833  2.9 13  0.97 93.8 492,500 13 24* 3.6 143 228,833 60* 66* 22* 235 2,333 100* 99* 100* 329 8,500 99* 100* 96* * Indicates a significant difference from the negative control using the appropriate statistical test ( p < 0.05)

Control response: satisfactory Observations: After 96 hours of exposure, there were no signs of aggregation or adherence of the algae to the flasks in the negative control or any treatment group. In addition, there were no noticeable changes in cell morphology when compared to the negative control.

Reversibility of Growth Inhibition: Aliquots of the 235 and 329 mg/ L test solutions were diluted with algal medium and cultured for nine days after the exposure phase of the study concluded. Based on the increase in growth observed by Day 9 of the recovery phase, the effect on algal growth was algistatic at a concentration of 235 mg/ L. However, no algal cells were detected during the recovery phase in the 329 mg/ L treatment, indicating that PFOS was algicidal at that concentration.

CONCLUSIONS The potassium perfluorooctanesulfonate 96 hour EC50 and 95% confidence interval for Anabaena flosaquae was determined using three calculation methods. By cell density, it was 131 ( 106   142) mg/ L, by area under the growth curve it was 124 ( 104   138) mg/ L and by growth rate 176 ( 169   181) mg/ L. The 96 hour NOAEC values were determined to be 63.9 mg/ L using the area under the growth curve, and 93.8 mg/ L with the cell density and growth rate calculation method. No signs of cell aggregation or adherence were noted in any of the test solutions or the controls. PFOS was determined to be algistatic at a concentration of 235 mg/ L and algicidal at 329 mg/ L.

Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking = 1 REFERENCES This study was conducted at Wildlife International Ltd., Easton, MD at the request of the 3M Company, Lab Request number U2723.

OTHER Last changed: 7/ 19/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 181 Robust Study Report Reference No. 37  PFOS: A 7 day toxicity test with Duckweed (

G3)

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: Sample obtained from 3M production lot number 217. The test substance is a white powder. Sample was stored under ambient conditions prior to testing. Purity determined to be 86.9% by LC/ MS, 1H HMR, 19F NMR and elemental analyses techniques.

METHOD Method: OPPTS 850.4400 Test: Static acute GLP: Yes Year completed: 2001 Species: Lemna gibba G3 Source: Originally from The United States Department of Agriculture. Maintained in culture medium at Wildlife International Ltd., Easton, MD Analytical monitoring: Test concentrations measured at 0, 3, 5, and 7 days Element basis: Number of fronds Exposure period: 7 days Start date: 3/ 3/ 00 End date: 3/ 10/ 00 Test organisms laboratory culture: Duckweed cultures had been actively growing in freshwater medium ( 20X AAP) for at least two weeks prior to test initiation. Stock nutrient solutions were prepared by adding reagent grade chemicals to reverse osmosis purified well water. Test Conditions: Test temperature range: 24.2   25.2 º C Light levels: 5000 + 750 lux from continuous warm white fluorescent lighting Growth medium: USEPA OPPTS 850.4400 20X AAP, 1996 Compound Nominal Concentration Units MgCl2 .6H20 243.2 mg/ L CaCl2 .2H20 88.0 mg/ L H3BO3 3.712 mg/ L MnCl2 .4H20 8.32 mg/ L ZnCl2 65.6 µ g/ L FeCl3 .6H20 3.196 mg/ L CoCl2 .6H20 28.56 µ g/ L Na2Mo04.2H20 145.2 µ g/ L CuCl2 .2H20 0.240 µ g/ L Na2EDTA. 2H20 6.00 mg/ L NaNO3 510 mg/ L MgSO4 .7H20 294 mg/ L K2HPO4 20.88 mg/ L NaHCO3 300 mg/ L ENV/ JM/ RD( 2002) 17/ FINAL 182 The pH of the medium was adjusted to 7.5 + 0.1 using 10% HCl. Dilution water source: Wildlife International Ltd. well water purified by reverse osmosis. The test medium was prepared by adding the appropriate volumes of stock nutrient solutions to purified well water. The pH of the medium was adjusted to 7.5 + 0.1 using 10% HCl and the medium was sterilized by filtration ( 0.22 µ m) prior to use. Stock and test solution preparation: A primary stock solution was prepared in duckweed medium at a concentration of 351 mg/ L. The primary stock solution was stirred with a magnetic stir plate for approximately 24 hours. After mixing, the primary stock solution was proportionally diluted with duckweed medium to prepare the five additional test concentrations. All final test solutions appeared clear and colorless. Exposure vessels: 250 mL plastic beakers containing 100 mL test solution, each covered with a disposable petri dish lid. Agitation: None Number of replicates: three plus 2 additional replicates for analytical sampling on Days 3 and 5 Initial loading: 5 plants/ replicate, 15 fronds/ replicate Number of concentrations: six plus a negative control plus abiotic controls at the highest concentration tested Water chemistry: pH range ( 0   96 hours) 7.9   8.9 ( control exposure) 8.4   8.7 ( 230 mg/ L exposure) Method of calculating mean measured concentrations: arithmetic mean obtained using results obtained at Days 0, 3, 5, and 7.

RESULTS Nominal concentrations: Negative control, 11, 22, 43.9, 87.9, 176, and 351 mg/ L plus 351 mg/ L abiotic control. Measured concentrations: < LOQ, 7.74, 15.1, 31.9, 62.5, 147, 230 mg/ L; abiotic control = 231 mg/ L Element value and 95% confidence interval ( based on frond number): 3 day IC 10: 101 mg/ L ( C. I. not calculable) 3 day IC 50: > 230 mg/ L ( C. I. not calculable) 3 day IC 90: > 230 mg/ L ( C. I. not calculable) 5 day IC 10: 30.7 mg/ L ( 13.3   142 mg/ L) 5 day IC 50: 182 mg/ L ( 89.1   240 mg/ L) 5 day IC 90: > 230 mg/ L ( C. I. not calculable) 7 day IC10: 22.1 mg/ L ( 13.3   26.0 mg/ L) 7 day IC 50: 108 mg/ L ( 45.7   144 mg/ L) 7 day IC 90: > 230 mg/ L ( C. I. not calculable) 7 day NOAEC ( number of fronds): 15.1 mg/ L All element values based on mean measured concentrations Statistical methods: Mean plant and frond numbers, percent inhibition values and the percentages of necrotic, chlorotic and dead fronds were calculated using " Microsoft Excel Version 5.0", while statistical analyses were conducted using " TOXSTAT Version 3.5". Percent inhibition values were calculated for each treatment group as the percent reduction in mean frond number relative to mean frond number in the control replicates. The IC 10, IC50, and IC 90 values and 95% confidence intervals were determined, when possible, using linear interpolation with frond number and exposure concentration data. The percentages of dead, chlorotic and necrotic fronds also were calculated relative to the total number of fronds in each test chamber. The frond number data was evaluated for normality and homogeneity of variances ( p = 0.05) ENV/ JM/ RD( 2002) 17/ FINAL 183 using the Shapiro Wilks' and Levene's tests, respectively. The data were normally distributed and the variances were homogeneous, thus statistically significant differences between the control and treatment groups were identified using ANOVA and Dunnett's test. Results of the statistical analyses, as well as an evaluation of the concentration response pattern and other observations of effects were used in the determination of the no  observed adverse effect concentration ( NOAEC).

Analytical Methodology: Analyses of test solutions were performed at Wildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection ( HPLC/ MS). Samples were centrifuged as necessary prior to analysis. When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ ( limit of quantitation) was 4.39 mg/ L in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 104%. Samples collected at test initiation had measured values from 64.2 to 82.6% of nominal. Measured values for samples taken at Day 3 ranged from 67.3 to 83.3% of nominal. Measured values for samples taken at Day 5 ranged from 65.4 to 85.4% of nominal. Samples collected at test termination ( Day 7) ranged from 63.9 to 83.8% of nominal. For the abiotic controls, measured values for samples taken at Day 3, Day 5, and Day 7 ranged from 64.2   66.9% of nominal.

Summary of analytical chemistry data: Nominal Test Concentration, mg/ L Measured Values at Days 0, 3, 5, and 7, Respectively, mg/ L Mean Measured Concentration, mg/ L Percent of Nominal Negative Control All < LOQ < LOQ 11 7.57, 8.35, 7.47, 7.55 7.74 70 22 15.2, 15.4, 14.6, 15.2 15.1 69 43.9 32.2, 31.9, 31.8, 31.7 31.9 73 87.9 63.5, 63.1, 61.5, 61.8 62.5 71 176 145, 146, 150, 147 147 84 351 226, 237, 232, 224 230 66 351 ( abiotic) not analyzed, 225, 235, 232 231 66 Biological observations after 7 Days: Counts Mean Measured Concentration, mg/ L Mean Number of Plants Mean Number of Fronds Percent Inhibition via Frond Number Negative Control 19 197  7.74 18 177 10 15.1 20 219  11 31.9 14 151* 24 62.5 11 134* 32 147 15 69* 65 230 17 37* 81 * Statistically significant difference ( p < 0.05) from the negative control using ANOVA and Dunnett's Test. ENV/ JM/ RD( 2002) 17/ FINAL 184 Effects Mean Measured Concentration, mg/ L Mean Dead Fronds, % Mean Chlorotic Fronds, % Mean Necrotic Fronds, % Negative Control 0 0 0 7.74 0 0 0 15.1 0 1.1 0 31.9 0 0 0.23 62.5 0 0.9 0.61 147 1.0 11 4.5 230 3.8 9.4 19 Control response: satisfactory. Plants appeared healthy and exhibited normal growth throughout the test with the exception of one necrotic frond observed on Day 3 and Day 5 of the test.

Observations: Duckweed exposed to 147 and 230 mg PFOS/ L exhibited a dose responsive increase in the incidence of dead, chlorotic or necrotic fronds during the test. By Day 7, all treatment groups > 31.9 gm/ L showed evidence of sublethal effects, including root destruction and/ or a cupping of the plant downward on the water surface.

CONCLUSIONS The potassium perfluorooctanesulfonate 7 Day IC 50 and 95% confidence interval for duckweed was determined to be 108 ( 45.7   144) mg/ L. The 7 Day NOAEC, based on the inhibition of frond production and evidence of sub lethal effects, was 15.1 mg/ L.

Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking = 1 REFERENCES This study was conducted at Wildlife International Ltd., Easton, MD at the request of the 3M Company.

OTHER Last changed: 7/ 19/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 185 Robust Study Report Reference No. 38  PFOS: A 96 hour toxicity test with freshwater diatom ( !

)

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: Sample obtained from 3M production lot number 217. The test substance is a white powder. Sample was stored under ambient conditions prior to testing. Purity determined to be 86.9% by LC/ MS, 1H HMR, 19F NMR and elemental analyses techniques.

METHOD Method: OPPTS 850.5400 Test: Acute static GLP: Yes Year completed: 2001 Species: Navicula pelliculosa Source: Originally from The Culture Collection of Algae at the University of Texas at Austin, maintained in culture medium at Wildlife International Ltd., Easton, MD Analytical monitoring: PFOS measured at 0, 72, 96 hours Element basis: Reported three ways: number of cells/ ml, area under the growth curve and growth rate Exposure period: 96 hours Start date: 2/ 25/ 00 End date: 2/ 29/ 00 Test organisms laboratory culture: Algae cultures had been actively growing in freshwater algal culture medium with silica and selenium for at least two weeks prior to test initiation. Stock nutrient solutions were prepared by adding reagent grade chemicals to reverse osmosis purified well water. Test Conditions: Growth medium Compound Nominal Concentration Units MgCl2 .6H20 12.16 mg/ L CaCl2 .2H20 4.40 mg/ L H3BO3 0.1856 mg/ L MnCl2 .4H20 0.416 mg/ L ZnCl2 3.28 µ g/ L FeCl3 .6H20 0.1598 mg/ L CoCl2 .6H20 1.428 µ g/ L Na2Mo04.2H20 7.26 µ g/ L CuCl2 .2H20 0.012 µ g/ L Na2EDTA. 2H20 0.300 mg/ L NaNO3 25.50 mg/ L MgSO4 .7H20 14.70 mg/ L K2HPO4 1.044 mg/ L NaHCO3 15.0 mg/ L Na2SiO3 .9H20 20.0 mg/ L Na2SeO3 .5H20 0.010 mg/ L ENV/ JM/ RD( 2002) 17/ FINAL 186 Dilution water source: Wildlife International Ltd. well water purified by reverse osmosis. The test medium was prepared by adding the appropriate volumes of stock nutrient solutions to purified well water. The pH of the medium was adjusted to 7.5 + 0.1 using 10% HCl and 0.1 N NaOH. The medium was sterilized by filtration ( 0.22 µ m) prior to use. Test solution preparation: A primary stock solution was not prepared for this study. Individual test solutions were prepared in algal medium at each of the seven nominal concentrations. The individual test solutions were stirred with a magnetic stir plate for approximately 24 hours. All final test solutions appeared clear and colorless. Exposure vessels: Sterile 250 mL plastic Erlenmeyer flasks plugged with foam stoppers containing 100 mL of test solution. Agitation: Shaken continuously at ~ 100 rpm Number of replicates: three. Initial algal cell loading: 1.0 X 104 cells/ mL Number of concentrations: seven plus a negative control plus an abiotic control at the highest concentration tested Water chemistry: pH range ( 0   96 hours) 7.5   8.6 ( control exposure) 7.5   7.7 ( 335 mg/ L exposure) Test temperature range ( 0   96 hours) 23.1   24.6 º C Light levels: ( 0   96 hours) 3910   4510 lux from continuous cool white fluorescent lighting Method of calculating mean measured concentrations: arithmetic mean obtained using results obtained at 0 hours, 72 hours and 96 hours RESULTS Nominal concentrations: Negative control, 61.5, 81.3, 110, 147, 198, 264, 347 mg/ L plus 347 mg/ L abiotic control. Measured concentrations: < LOQ, 62.3, 83.2, 111, 150, 206, 266, 335 mg/ L; abiotic control = 339 mg/ L Element value ( 95% confidence interval): 24 hour EC50 ( cell density) = 281 ( 214  312) mg/ L 24 hour EbC50 ( area under curve) = 262 ( 205  308) mg/ L 24 hour ErC50 ( growth rate) = 279 ( 212  306) mg/ L 48 hour EC50 ( cell density) = 261 ( 219  306) mg/ L 48 hour EbC50 ( area under curve) = 259 ( 227  303) mg/ L 48 hour ErC50 ( growth rate) = 294 ( 271  307) mg/ L 72 hour EC10 ( cell density) = < 62.3 ( C. I. not calculable) mg/ L 72 hour EbC10 ( area under curve) = < 62.3 ( C. I. not calculable) mg/ L 72 hour ErC10 ( growth rate) = 221 ( 190  252) mg/ L 72 hour EC50 ( cell density) = 242 ( 200  276) mg/ L 72 hour EbC50 ( area under curve) = 246 ( 210  277) mg/ L 72 hour ErC50 ( growth rate) = 295 ( 288  305) mg/ L 72 hour EC90 ( cell density) = 317 ( 306  326) mg/ L 72 hour EbC90 ( area under curve) = 318 ( 307  325) mg/ L 72 hour ErC90 ( growth rate) = 335 ( 323  335) mg/ L 96 hour EC10 ( cell density) = < 62.3 ( C. I. not calculable) mg/ L 96 hour EbC10 ( area under curve) = < 62.3 ( C. I. not calculable) mg/ L 96 hour ErC10 ( growth rate) = 243 ( 209  295) mg/ L 96 hour EC50 ( cell density) = 263 ( 217  299) mg/ L ENV/ JM/ RD( 2002) 17/ FINAL 187 96 hour EbC50 ( area under curve) = 252 ( 220  285) mg/ L 96 hour ErC50 ( growth rate) = 305 ( 295  316) mg/ L 96 hour EC90 ( cell density) = 322 ( 310  328) mg/ L 96 hour EbC90 ( area under curve) = 319 ( 308  326) mg/ L 96 hour ErC90 ( growth rate) = > 335 mg/ L ( C. I. not calculable) 72 hour NOAEC ( cell density, area under the curve): < 62.3 mg/ L 72 hour NOAEC ( growth rate): 206 mg/ L 96 hour NOAEC ( cell density): 150 mg/ L 96 hour NOAEC ( area under the curve): < 62.3 mg/ L 96 hour NOAEC ( growth rate): 206 mg/ L All element values based on mean measured concentrations Statistical methods: Cell densities, area under the growth curve values, growth rates and percent inhibition values were calculated using " The SAS System for Windows", Release 6.12. These values were then analyzed by linear interpolation using TOXSTAT Version 3.5 to estimate the EC10, EC50, and EC90 values and 95% confidence limits. Cell densities, areas under the growth curve and growth rates at 72 and 96 hours were also evaluated fo r normality and homogeneity of variances using the Shapiro Wilkes's test and Levene's test, respectively. The treatment groups were then compared to the control using Dunnett's test. Results of the statistical analyses were used to determine the NOAEC values.

Analytical Methodology: Analyses of test solutions were performed at Wildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection ( HPLC/ MS). When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ ( limit of quantitation) was 4.39 mg/ L in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 108%. Samples collected at test initiation had measured values from 96.2 to 106% of nominal. Measured values for samples taken at 72 hours ranged from 98.5 to 106% of nominal. Measured values for samples taken at 96 hours ranged from 94.8 to 101% of nominal. For the abiotic controls, the measured value for the sample taken at 72 hours was 98.2% of nominal and for the sample taken at 96 hours, 96.8% of nominal.

Summary of analytical chemistry data: Nominal Test Concentration, mg/ L Measured Values at 0, 72, and 96 hours, Respectively, mg/ L Mean Measured Concentration, mg/ L Percent of Nominal Negative Control All < LOQ < LOQ 61.5 62.3, 63.6, 61.1 62.3 101 81.3 83.8, 84.7, 81.0 83.2 102 110 109, 113, 110 111 101 147 147, 154, 149 150 102 198 209, 209, 199 206 104 264 271, 268, 258 266 101 347 334, 342, 329 335 96.5 347 ( abiotic) Not analyzed, 341, 336 339 97.7 ENV/ JM/ RD( 2002) 17/ FINAL 188 Biological observations after 96 hours: Mean Measured Concentration, mg/ L Mean Number of Cells per mL Percent Inhibition via Density Percent Inhibition via Area Under the Curve Percent Inhibition via Growth Rate Negative Control 2,726,667    62.3 2,366,667 13 24* 2.5 83.2 2,366,667 13 22* 2.7 111 2,373,333 13 24* 2.5 150 2,473,333 9.3 16 1.7 206 2,093,333 23* 24* 4.7 266 1,330,000 51* 58* 13* 335 35,333 99* 99* 79*

* Indicates a significant difference from the negative control using Dunnett's test ( p < 0.05)

Control response: satisfactory Observations: After 96 hours of exposure, there were no signs of aggregation or adherence of the algae to the flasks in the negative control or any test treatment group. In addition, there were no noticeable changes in cell color or morphology when compared to the negative control, although at 72 and 96 hours of exposure a few cells in the 335 mg/ L treatment group appeared small in comparison to the control.

Reversibility of Growth Inhibition: The 335 mg/ L treatment group was maximally inhibited after 96 hours. The treatment group was diluted to a concentration of the test substance that would not inhibit growth and exposed for 7 days. Based on the growth observed in the recovery phase, the effect on algal growth was found to be algistatic.

CONCLUSIONS The potassium perfluorooctanesulfonate 96 hour EC50 and 95% confidence interval for Navicula pelliculosa was determined using three calculation methods. B y cell density, it was 263 ( 217  299) mg/ L, by area under the growth curve it was 252 ( 220  285) mg/ L and by growth rate 305 ( 295  316) mg/ L. The 96 hour NOAEC was determined by Dunnett's procedure ( p < 0.05) to be 150 mg/ L using cell density, < 62.3 mg/ L when using area under the curve and 206 mg/ L by growth rate. No signs of cell aggregation or adherence were noted in any of the test solutions or the controls. This test substance was determined to be algistatic.

Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking = 1 REFERENCES This study was conducted at Wildlife International Ltd., Easton, MD at the request of the 3M Company, Lab Request number U2723.

OTHER Last changed: 6/ 19/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 189 Robust Study Report Reference No. 39  PFOS: A 96 hour toxicity test with the marine diatom (  "

   )

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: Sample from 3M production lot number 217. The test substance is a white powder. Purity determined to be 86.9% by LC/ MS, 1H HMR, 19F NMR and elemental analyses techniques.

METHOD Method: OPPTS 850.5400 Test: Acute static GLP: Yes Year completed: 2001 Species: Skeletonema costatum Source: Originally from The Culture Collection of Algae and Protozoa, Dunstaffnage marine Laboratory at Oban Argyll, Scotland, and maintained in culture medium at Wildlife International Ltd., Easton, MD Analytical monitoring: PFOS measured at 0, 72, 96 hours Element basis: Reported three ways: number of cells/ ml, area under the growth curve and growth rate Exposure period: 96 hours Start date: 5/ 19/ 00 End date: 5/ 23/ 00 Analytical monitoring: Test concentrations measured at 0, 72, and 96 hours. Test organisms laboratory culture: Algae cultures had been actively growing in saltwater algal culture medium for at least two weeks prior to test initiation. Stock nutrient solutions were prepared by adding reagent grade chemicals to reverse osmosis purified well water. Solutions were then diluted in artificial saltwater to prepare final growth media.

Test Conditions: Algal saltwater medium Compound Nominal Concentration Units FeCl3 .6H20 0.72 mg/ L MnCl2 .4H2O 2.16 mg/ L ZnSO4 .7H2O 0.675 mg/ L CuSO4 .5H2O 2.36 µ g/ L CoCl2 .6H20 6.06 µ g/ L H3BO3 17.1 mg/ L Na2EDTA. 2H20 15.0 mg/ L K3PO4 3.0 mg/ L NaNO3 50.0 mg/ L Na2SiO3 .9H20 20.0 mg/ L Thiamine Hydrochloride 0.25 mg/ L Biotin 0.05 µ g/ L B12 0.5 µ g/ L ENV/ JM/ RD( 2002) 17/ FINAL 190 Dilution water source: The stock nutrient solutions were prepared by adding the appropriate volumes reagent grade chemicals to Wildlife International Ltd. well water purified by reverse osmosis. The algal medium was prepared by adding appropriate volumes of the stock nutrient solutions to artificial saltwater at 30 ppt salinity. The pH of the medium was 8.1 and it was sterilized by filtration ( 0.22 µ m) prior to use. Test solution preparation: A single test solution ( 3.46 mg/ L) was prepared for this study in algal saltwater medium. The solution was sonicated for approximately 30 minutes and was stirred with a magnetic stir plate for approximately 43 hours. The final test solution appeared clear and colorless. Exposure vessels: Sterile 250 mL glass Erlenmeyer flasks plugged with foam stoppers containing 100 mL of test solution. Agitation: Shaken continuously at 100 rpm Number of replicates: six. Initial algal cell loading: 7.7 X 104 cells/ mL Number of concentrations: one plus a negative control plus an abiotic control at the highest concentration tested Water chemistry: pH range ( 0   96 hours) 8.0   8.4 ( control exposure) 8.0   8.4 ( 3.20 mg/ L exposure) Test temperature range ( 0   96 hours) 20.2   21.4 º C Light levels: ( 0   96 hours) 3880   4710 lux from cool white fluorescent lighting Photoperiod: 14 hours light and 10 hours dark Method of calculating mean measured concentrations: arithmetic mean obtained using results obtained at 0 hours, 72 hours and 96 hours RESULTS Nominal concentrations: Negative control, 3.46 mg/ L plus 3.46 mg/ L abiotic control. This is apparently the highest concentration of PFOS attainable in this saltwater algal media. Measured concentrations: < LOQ, 3.20 mg/ L; abiotic control = 3.18 mg/ L Element value ( 95% confidence interval): 72 and 96 hour EC10 via cell density, area under the curve and growth rate: > 3.20 mg/ L ( C. I. not calculable) 24, 48, 72, and 96 hour EC50 via cell density, area under the curve and growth rate: > 3.20 mg/ L ( C. I. not calculable) 72 and 96 hour EC90 via cell density, area under the curve and growth rate: > 3.20 mg/ L ( C. I. not calculable) 72 hour NOAEC ( cell density, area under the curve, growth rate): 3.20 mg/ L 96 hour NOAEC ( cell density, area under the curve, growth rate): 3.20 mg/ L All element values based on mean measured concentrations Statistical methods: Cell densities, area under the growth curve values, growth rates and percent inhibition values were calculated using " The SAS System for Windows", Release 6.12. The EC10, EC50, and EC90 values and 95% confidence limits could not be calculated using statistical methods. Cell densities, areas under the growth curve and growth rates at 72 and 96 hours were evaluated for normality using the Shapiro Wilk's test and for equality of variance using an F test. The treatment groups were then compared to the control using ANOVA and a 2 sample t test. ENV/ JM/ RD( 2002) 17/ FINAL 191 Results of the statistical analyses were used to determine the NOAEC values.

Analytical Methodology: Analyses of test solutions were performed at Wildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection ( HPLC/ MS). The 72 and 96 hour samples were centrifuged approximately 10 minutes at approximately 2000 rpm prior to analysis. When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ ( limit of quantitation) was 0.480 mg/ L in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 108%. Samples collected at test initiation had measured values from 96.2 to 88.5% of nominal. The measured value for the sample taken at 72 hours was 92.2% of nominal. The measured value for the sample taken at 96 hours was 91.2% of nominal. For the abiotic control, the measured value for the sample taken at 72 hours was 97.1% of nominal and for the sample taken at 96 hours, 86.8% of nominal.

Summary of analytical chemistry data: Nominal Test Concentration, mg/ L Measured Values at 0, 72, and 96 hours, Respectively, mg/ L Mean Measured Concentration, mg/ L Percent of Nominal Negative Control All < LOQ < LOQ 3.46 3.26, 3.19, 3.15 3.20 92.5 3.46 ( abiotic) Not analyzed, 3.36, 3.00 3.18 91.9 Biological observations after 96 hours: Mean Measured Concentration, mg/ L Mean Number of Cells per mL Percent Inhibition via Density Percent Inhibition via Area Under the Curve Percent Inhibition via Growth Rate Negative Control 2,481,667    3.20 2,601,667  4.8  7.3  1.3 Control response: satisfactory Observations: After 96 hours of exposure, there were no signs of aggregation or adherence of the algae to the flasks in the treatment group. However there were signs of adherence to the test chamber in the negative control group. There were no noticeable changes in cell morphology when compared to the negative control.

Reversibility of Growth Inhibition: After 96 hours of exposure, there was no significant inhibition of growth in the highest concentration tested ( 3.20 mg/ L). Therefore, a recovery phase was not conducted.

CONCLUSIONS A single concentration of potassium perfluorooctanesulfonate was evaluated for toxicity to Skeletonema costatum. This mean measured concentration, 3.20 mg/ L, was the highest concentration attainable in this algal media. The 96 hour EC50 and 95% confidence interval for Skeletonema costatum, as determined by cell density, area under the growth curve, and by growth rate was found to be > 3.20 mg/ L. The 96 hour NOAEC was determined by ANOVA and a 2 sample t test to be 3.20 mg/ L calculated using cell density, area under the curve and growth rate. No signs of cell aggregation or adherence were noted in any of the test solutions or the controls. ENV/ JM/ RD( 2002) 17/ FINAL 192 Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking = 1 REFERENCES This study was conducted at Wildlife International Ltd., Easton, MD at the request of the 3M Company, Lab Request number U2723.

OTHER Last changed: 7/ 19/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 193 Robust Study Report Reference No. 40  PFOS: A frog embryo teratogenesis assay          ( FETAX)

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS, U2723 or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: Sample obtained from 3M production lot number 217. The test substance is a white powder. Purity determined to be 86.9% by LC/ MS, 1H HMR, 19F NMR and elemental analyses techniques.

METHOD Method: ASTM E1439 91 Test type: Static renewal GLP: In life phase   no; stock solution preparation and measurement of test concentrations  yes Year completed: 2001 Number of studies: 3 Study 1 Study 2 Study 3 Start date: 5/ 15/ 00 5/ 22/ 00 5/ 22/ 00 End date: 5/ 19/ 00 5/ 26/ 00 5/ 26/ 00 ( Study 2 and 3 set up concurrently with common stock solutions) Analytical monitoring: PFOS measured at 0 and 96 hours Species: Xenopus laevis Source: Breeding colonies at the University of Maryland Wye Research and Education Center ( UMD/ WREC), Queenstown, Maryland. Test organisms laboratory culture: Mating pairs were bred in the dark in 23.5 + 0.5 º C UMD/ WREC non chlorinated well water at ~ 70 day intervals by injecting 400 and 800 I. U. of human chorionic gonadotropin ( HCG) in the dorsal lymph sac of the males and females, respectively. Amplexus occurred 4  6 hours after injecting HCG; egg deposition occurred 9 12 hours following HCG injection. Age at test initiation: Embryos; normal stage 8 blastula to normal stage 11 gastrula Loading: 25 embryos/ 10 mL Pretreatment: Embryos de jelled in a 2% L  cysteine solution, then rinsed and re suspended in FETAX solution prior to introduction to test chambers. Element basis: mortality, malformations ( via the atlas of Bantle et al., 1991), growth Exposure period: 96 hours Test Conditions ( all 3 studies): Dilution water: ASTM ( 1998) FETAX solution Test temperature: 24.0 + 0.2 º C Light levels: 60 85 foot candle fluorescent lights Photoperiod: 12 hour light: 12 hour dark Stock and test solution preparation: A primary stock solution was prepared in FETAX medium ( supplied by UM WREC) by Wildlife International, Ltd. at 48 mg PFOS/ L. The primary stock solution was mixed by sonication and stirring. After mixing, the primary stock solution was proportionally diluted with FETAX medium to prepare the six test concentrations. The six test concentration solutions were delivered to UM WREC prior to the start of each study. Reference substance: 6 aminonicotinamide Stock and reference substance solution preparation: as outlined in the ASTM ( 1998) protocol Exposure vessels: Covered 60 mm glass Petri dishes containing 10 mL test solution ENV/ JM/ RD( 2002) 17/ FINAL 194 Number of replicates: controls   4, treatments   2 Number of embryos per replicate: 25 Number of concentrations: six plus a negative control plus an abiotic control at the highest concentration tested, plus two reference substance concentrations. Renewal frequency: every 24 hours Stability of the test chemical solutions: Extremely stable Water chemistry during all 3 studies: pH range ( 0   96 hours) 7.1   7.7 ( control exposure) 7.0   7.6 ( 24 mg/ L nominal exposure) Dissolved oxygen range ( 0 96 hours) 7.3   8.4 mg/ L ( control exposure) 7.0   8.5 mg/ L ( 24 mg/ L nominal exposure) Method of calculating mean measured concentrations: arithmetic mean RESULTS Nominal PFOS concentrations: Negative control, 1.82, 3.07, 5.19, 8.64, 14.4 and 24.0 mg/ L plus 24.0 mg/ L abiotic control. Nominal 6 aminonicotinamide concentrations: 5.5 and 2500 mg/ L Mean measured PFOS concentrations: Study 1: < LOQ, 2.00, 2.83, 4.73, 7.90, 14.7, 24.6 mg/ L; abiotic control = 23.7 mg/ L Study 2: < LOQ, 1.91, 3.04, 4.82, 7.97, 13.3, 23.1 mg/ L; abiotic control = 23.9 mg/ L Study 3: < LOQ, 1.93, 3.27, 5.25, 8.26, 14.0, 23.9 mg/ L; abiotic control = 24.1 mg/ L PFOS element values and 95% confidence intervals, mg/ L Study Number 96 Hr LC50 96 Hr EC50 Minimum conc. to Inhibit Growth ( MCIG) Teratogenic Index ( TI)

1 13.8 ( 12.4  15.3) 12.1 ( 10.0   14.6) Not calculable 1.1 2 17.6 ( 15.5   20.0) 17.6 ( 13.5   22.9) 7.97 1.0 3 15.3 ( 13.1   17.8) 16.8 ( 12.4   22.8) 8.26 0.9 All element values based on mean measured concentrations Statistical methods: The Trimmed Spearman Karber statistical procedure was used to determine the 96 hour LC50 for mortality and 96 hour EC50 for malformations. The MCIG was determined by Bonferroni's T Test. All statistical tests were performed using Toxstat ( WEST and Gulley, 1994). A minimum probability level of 0.05 was used. The teratogenic index ( TI) was calculated by dividing the LC50 by the EC50.

Analytical Methodology: Analyses of test solutions were performed at Wildlife International Ltd., Easton, MD using high performance liquid chromatography with mass spectrometric detection ( HPLC/ MS). When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the ENV/ JM/ RD( 2002) 17/ FINAL 195 basis of individual isomeric components. The LOQ ( limit of quantitation) was 0.240 mg/ L in these studies. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 97.2%. Samples collected at test initiation had measured values from 112 to 141% of nominal in the first study, and in the second and third studies, from 95.8 to 117% of nominal. Measured values for samples taken at 96 hours ranged from 54.7 to 98.6% of nominal in the first study and 80.7 to 112% of nominal in the second and third studies. The samples from the abiotic 24.0 mg/ L treatment group was comparable to samples from the 24.0 mg/ L treatment group with the embryos present.

Summary of analytical chemistry data: Study 1 Nominal Test Concentration, mg/ L Measured Values at 0 and 96 hours Respectively, mg/ L Mean Measured Concentration, mg/ L Percent of Nominal Negative Control All < LOQ < LOQ 1.82 2.58, 1.42 2.00 110 3.07 3.94, 1.72 2.83 92.2 5.19 6.62, 2.84 4.73 91.1 8.64 10.7, 5.09 7.90 91.4 14.4 18.5, 10.8 14.7 102 24.0 26.9, 22.3 24.6 103 24.0 ( abiotic) not analyzed, 23.7 23.7 98.6 Study 2 Nominal Test Concentration, mg/ L Measured Values at 0 and 96 hours Respectively, mg/ L Mean Measured Concentration, mg/ L Percent of Nominal Negative Control All < LOQ < LOQ 1.82 1.77, 2.04 1.91 105 3.07 3.59, 2.49 3.04 99.0 5.19 5.45, 4.18 4.82 92.9 8.64 8.43, 7.51 7.97 92.2 14.4 14.5, 12.1 13.3 92.4 24.0 23.0, 23.1 23.1 96.3 24.0 ( abiotic) not analyzed, 23.9 23.9 99.6 Study 3 Nominal Test Concentration, mg/ L Measured Values at 0 and 96 hours Respectively, mg/ L Mean Measured Concentration, mg/ L Percent of Nominal Negative Control All < LOQ < LOQ 1.82 1.77, 2.08 1.93 106 3.07 3.59, 2.94 3.27 107 5.19 5.45, 5.05 5.25 101 8.64 8.43, 8.09 8.26 95.6 14.4 14.5, 13.5 14.0 97.2 24.0 23.0, 24.7 23.9 99.6 24.0 ( abiotic) not analyzed, 24.1 24.1 100 ENV/ JM/ RD( 2002) 17/ FINAL 196 Biological observations after 96 hours:

Mortality and Malformations Test 1 Test 2 Test 3 Nominal Concentration*, mg/ L Percent Mortality Percent Malformations Percent Mortality Percent Malformations Percent Mortality Percent Malformations Negative Control 1.0 4.0 1.0 4.0 0 2.0 1.82 2.0 8.2 0 8.0 0 6.0 3.07 4.0 15 10 4.4 0 4.0 5.19 10 22 8.0 11 0 6.0 8.64 12 25 10 20 14 14 14.4 38 65 30 37 44 39 24.0 100  70 67 78 73 * Nominal concentrations used for ease of comparison table Malformations: The most common types of malformations noted were improper gut coiling, edema, notochord abnormalities and facial abnormalities.

Growth   Mean length ( mm) after 96 hours Exposure Nominal Concentration*, mg/ L Test 1 Test 2 Test 3 Negative Control 8.59 8.88 9.47 1.82 8.29 8.45 9.10 3.07 8.80 8.57 9.28 5.19 8.51 8.72 9.28 8.64 8.71 7.93** 8.51** 14.4 8.08 7.51** 8.11** 24.0  ( total mortality) 7.39** 7.80** * Nominal concentrations used for ease of comparison table ** Significantly different at alpha = 0.05 ( Bonferroni T Test)

Control response: satisfactory.

Reference substance response: satisfactory at low concentration ( 5.5 mg/ L). Did not meet ASTM ( 1998) criteria for high concentration ( 2,500 mg/ L). However, results obtained at high concentration were consistent and not at variance with previous experience in this testing laboratory.

Observations: Majority of embryo mortality appeared to be caused by the gut coiling through the body wall at the two highest test concentrations.

CONCLUSIONS The potassium perfluorooctanesulfonate 96 hour LC50 range for FETAX was determined to be 13.8   17.6 mg/ L. The 96 hour EC50 range was 12.1   17.6 mg/ L. The range for Minimum Concentration to Inhibit Growth ( MCIG) was 7.97 to > 14.7 mg/ L. The Teratogenic Index ( TI) was found to be 0.9   1.1. This TI range indicates that potassium perfluorooctanesulfonate has a low potential to be a developmental hazard. ENV/ JM/ RD( 2002) 17/ FINAL 197 Submitter: 3M Company, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking = 2 Although these were well conducted studies, the in life phases were not conducted in accordance with Good Laboratory Practices.

REFERENCES These studies were conducted at the University of Maryland Wye Research and Education Center ( UMWREC in Queenstown, Maryland and at Wildlife International Ltd., Easton, MD at the request of the 3M Company. Lab Request number U2723 OTHER Last changed: 7/ 19/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 198 Robust Study Report Reference No. 41  Perfluorooctanesulfonate, Potassium salt ( PFOS): A flowthrough bioconcentration test with the Bluegill (

         )

TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: Sample from 3M production lot number 217. The test substance is a white powder. Purity determined to be 86.9% by LC/ MS, 1H HMR, 19F NMR and elemental analyses techniques.

METHOD:

Method/ guideline followed: US EPA OPPTS 850.1730 and OECD 305 Type: Flow through exposure with flow through depuration phase. GLP ( Y/ N): Yes Year: 2001 Species: Bluegill ( Lepomis macrochirus) Supplier: Osage Catfisheries, Inc., Osage Beach, Missouri Length and weight at test termination: Mean length = 62 mm, range 56 66 mm Mean weight = 2.70 g, range 2.03   3.32 g Loading: 0.48 g fish/ L/ day ( based on initial loading of 90 fish per tank, using mean fish weight at the end of the study and volume of water that passed through test chamber in 24 hours). Fish age: Approximately 7 months at test initiation Analytical monitoring: Concentration of PFOS in water and fish. Pretreatment: None Number of concentrations: Two plus a negative control Test concentrations ( mean measured): Negative control, 0.086 and 0.87 mg/ L Uptake period: 62 days ( 0.086 mg/ L exposure) 35 days ( 0.87 mg/ L exposure   this exposure ended after 35 days due to fish mortality) Depuration period: 56 days ( 0.086 mg/ L exposure None ( 0.087 mg/ L exposure) Test conditions: Dilution water: Moderately   hard well water Dilution water chemistry: Specific conductance: 313 ( 310   315 µ mhos/ cm) Hardness: 130 ( 128   132 mg/ L) Alkalinity: 178 ( 176   178) pH: 8.1 ( 8.0   8.2) Measured during the 4  week period immediately preceding the test.

Stock and test solution preparation: Two stock solutions were prepared at 10 and 100 mg a. i./ L. Stock solutions stirred with an electric top down mixer to aid in the solubilization of the test substance. After mixing, the stocks appeared clear and colorless. Stocks were prepared at approximately weekly intervals during the uptake phase. Stocks injected into the diluter mixing chambers at a rate of 3.5 mL/ minute where they were mixed with dilution water at a rate of 350 mL/ minute to achieve the desired test concentrations. All final test solutions appeared clear and colorless. Diluter flow rate: Approx. 6.3 volume additions per 24 hours ENV/ JM/ RD( 2002) 17/ FINAL 199 Exposure vessels: 104 L stainless steel aquaria filled with approximately 80 L solution. Number of replicates: None   one vessel per concentration Number of fish per vessel: 90 Diet: Flake food, Ziegler Brothers, Inc., Gardners, PA Water chemistry ranges during the study: Neg. Control 0.086 mg/ L 0.87 mg/ L Dissolved oxygen, mg/ L: 6.8   8.6 6.8   8.6 6.4   8.2 Temperature, ° C: 21.8   22.0 21.7   22.0 21.7   21.9 pH: 7.9   8.2 7.9   8.2 7.9   8.2 Photoperiod: 16 hours light and 8 hours dark with a 30 minute transition period. Light intensity: 278 lux at surface of the negative control vessel at test initiation Collection of tissue samples: Fish were collected from test chambers by random selection at 12 time points during the 62 day uptake phase. They were euthanized, blotted dry, weighed and measured. Fish then rinsed with dilution water, blotted dry again and dissected into edible and non edible tissue fractions. The fractions were individually weighed. The head, fins and viscera were considered to be non edible tissue. The remaining tissue, including skin was considered to be edible tissue.

Statistical methods: Whole fish concentrations were calculated based on the sum of the edible and nonedible parts. Steady state bioconcentration BCF values calculated from the tissue concentrations at apparent steady state divided by the mean water concentration. Tissue concentrations were considered to be at apparent steady state if 3 or more consecutive sets of tissue concentrations were not significantly different ( p > 0.05). Tissue concentrations were evaluated for normality and homogeneity of variance suing the Shapiro Wilk's test and Bartlett's test, respectively. If the data did not meet the assumptions, data was transformed in an attempt to correct the data. Mean tissue concentrations were then compared using ANOVA and Dunnett's test.

The kinetic bioconcentration factor ( BCFK), uptake rate ( k1) and depuration rate ( k2) were calculated for the edible, nonedible and whole fish exposed to 0.086 mg/ L PFOS using BIOFAC computer software. BIOFAC is a nonlinear parameter estimate routine which estimates rate constants from a set of sequential time concentration data. These rate constants were then used to calculate a BCFK ( BCFK = K / K 1 2).

RESULTS Nominal concentrations: Negative control, 0.1 and 1.0 mg/ L Mean measured concentrations: < 0.05, 0.086 and 0.87 mg/ L Bioconcentration factors ( BCF):

0.086 mg/ L apparent steady state BCF Edible Non edible Whole Fish 484 1124 856 0.87 mg/ L ( study ended prior to achieving steady state) BCF: Edible Non edible Whole Fish 136 386 278 ENV/ JM/ RD( 2002) 17/ FINAL 200 BIOFAC Estimates ( using 0.086 mg/ L exposure) Edible Non edible Whole Fish BCFK: 1866 4312 3614 Time to reach 50% clearance: 146 days 133 days 152 days PFOS Concentrations in Tissues of Bluegill Exposed to 0.086 mg/ L Values are from 4 individual fish at each sample period. Uptake Day Edible Tissue, mg/ kg Non edible Tissue, mg/ kg Whole Fish Conc., mg/ kg 0 ( 4 hours) 0.167, 0.155, 0.144, 0.182 0.415, 0.519, 0.417, 0.497 0.293, 0.351, 0.286, 0.363 1 0.734, 0.726, 0.631, 0.806 1.68, 1.85, 1.72, 2.07 1.26, 1.34, 1.29, 1.53 3 1.73, 2.07, 2.03, 2.11 4.59, 5.50, 5.47, 5.97 3.21, 4.04, 4.18, 4.38 7 3.73, 4.25, 4.73, 6.25 10.2, 10.6, 11.9, 15.2 7.33, 7.66, 8.73, 11.4 14 11.4, 9.07, 13.7, 12.6 27.3, 23.2, 35.3, 32.6 20.2, 16.9, 26.0, 24.6 21 11.7, 12.0, 12.9, 10.6 33.3, 22.7, 24.6, 24.4 23.3, 18.4, 19.8, 18.5 28 18.3, 13.7, 23.9, 23.1 49.4, 40.7, 65.3, 57.9 35.3, 29.2, 45.4, 44.1 35 22.6, 27.7, 23.8, 20.6 67.1, 73.3, 62.0, 59.1 46.3, 53.8, 46.6, 40.9 42 27.6, 25.3, 21.2, 27.6 64.0, 68.1, 54.4, 79.6 50.1, 49.4, 40.9, 56.3 49 33.3, 36.2, 39.0, 30.6 85.0, 95.1, 93.1, 77.7 62.8, 69.6, 70.8, 57.4 56 48.3, 38.9, 44.1, 38.3 122, 94.2, 73.2, 106 90.6, 71.6, 63.3, 74.8 62 42.4, 66.2, 42.2, 39.2 101, 112, 105, 96.4 77.0, 92.7, 79.6, 73.1 Depuration Day 14 48.5, 31.8, 31.6, 42.0 124, 79.4, 81.8, 113 90.3, 60.4, 61.6, 85.3 28 26.0, 33.3, 38.7, 55.8 85.7, 95.1, 85.7, 94.8 58.2, 70.1, 68.1, 81.1 42 24.1, 31.2, 30.0, 33.0 71.7, 80.6, 78.3, 82.1 51.4, 61.4, 61.0, 62.2 56 21.1, 37.6, 32.9, 31.2 57.7, 80.3, 85.4, 84.4 41.6, 66.5, 65.8, 62.1 PFOS Concentrations in Tissues of Bluegill Exposed to 0.87 mg/ L Values are from 4 individual fish at each sample period. Uptake Day Edible Tissue, mg/ kg Non edible Tissue, mg/ kg Whole Fish Conc., mg/ kg 0 ( 4 hours) 1.46, 1.48, 1.19, 1.39 3.52, 4.37, 4.22, 4.06 2.71, 3.08, 2.84, 2.89 1 4.68, 6.59, 5.56, 5.64 11.1, 14.2, 13.3, 12.1 8.00, 10.9, 10.2, 9.47 3 17.3, 15.8, 19.0, 20.8 39.3, 42.0, 43.8, 51.8 30.5, 30.7, 34.5, 39.1 7 42.0, 44.0, 57.7, 46.8 100, 102, 102, 120 74.9, 77.0, 85.3, 89.8 14 87.1, 81.6, 90.7, 73.3 177, 207, 245, 214 141, 157, 180, 158 21 79.4, 117, 104, 102 201, 278, 246, 229 146, 210, 185, 172 28( 1) 102, 131, 107, 133 289, 372, 320, 361 205, 267, 232, 263 ( 1) Sampling of fish stopped after Uptake Day 28 due to mortality.

Test organism mortality: Negative control: None during the uptake phase ( 62 days) or depuration phase ( 35 days) 0.086 mg/ L exposure: One fish died after 49 days and one after 59 days of exposure in the uptake phase, none during the depuration phase. 0.87 mg/ L exposure: Mortality first noted on Day 9 and continued through Day 35 of the uptake phase at which time all of the fish had either died or had been sampled Analytical methodology: Analyses of test solutions and fish tissues were performed at Wildlife ENV/ JM/ RD( 2002) 17/ FINAL 201 International, Ltd. Water samples were diluted and analyzed by HPLC with single quadrupole mass spectrometric detection. Tissue samples were homogenized, extracted, diluted and analyzed by HPLC with triple quadrupole mass spectrometric detection. When determining the concentration of the test substance in the samples, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ was 0.05 mg/ L for water in this study. For tissue samples, the LOQ was calculated on an individual basis for each sample since each entire submitted sample, of differing weight, was extracted without an adjustment to constant weight.

Recovery was excellent in both water and fish tissues, ranging from 84.9 to 122% of fortification levels. Analytical results were not corrected for procedural recovery.

CONCLUSIONS PFOS bioconcentrated in the tissues of bluegill sunfish during this study. Apparent steady state was attained on Day 49 for the fish exposed to 0.086 mg a. i./ L. Although Day 49, 56 and 62 tissue residues were not statistically significantly different, PFOS concentrations appeared to be still increasing during this time. Apparent steady state BCF values for edible, non edible and whole fish tissues were calculated to be 484, 1124, and 859, respectively.

PFOS depurated slowly. The BIOFAC estimates for the time to reach 50% clearance for edible, nonedible and whole fish tissues were 146, 133 and 152 days, respectively.

DATA QUALITY Reliability: Klimisch ranking = 1 REFERENCES This study was conducted at Wildlife International, Ltd., Easton, MD at the request of the 3M Company, Lab Request number U2723.

OTHER Last changed: 7/ 19/ 01 ENV/ JM/ RD( 2002) 17/ FINAL 202 Robust Study Report Reference No. 42  Perfluorooctanesulfonate, Potassium salt ( PFOS): 96 Hour Static Acute Toxicity Test with the Rainbow Trout ( #               "    ) in freshwater TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3) Remarks: The test substance is a white powder. Sample was taken from 3M lot number 217. Sample was stored under ambient conditions prior to testing. Purity determined to be 86.9% by LC/ MS, 1 H HMR, 19 F NMR and elemental analyses techniques.

METHOD Method: OPPTS 850.1075 and OECD 203 Type: Static acute GLP: Yes Date completed: Study completed 2001, report completed 2002 Species: Oncorhynchus mykiss Supplier: Thomas Fish Company, Anderson, CA Analytical monitoring: PFOS measured at 0, 48, 96 hours Exposure period: 96 hours Statistical methods: LC50 values calculated, when possible, by probit analysis, moving average method or binomial probability with non linear interpolation using the computer software of C. E. Stephan. Test fish age: juveniles Average Total Length and weight: 3.6 ( 3.4   4.0) cm, 0.34 ( 0.25 0.47) g Loading: 0.23 g fish/ L Pretreatment: None Test conditions: Dilution water: 0.45 µ m filtered moderately hard well water Dilution water chemistry ( during the 4 week period immediately preceding the test): hardness: 130 ( 128 132) mg/ L as CaCO3 alkalinity: 177 ( 176 178) mg/ L as CaCO3 pH: 8.3 ( 8.2   8.4) TOC: Not given Conductivity: 311 ( 310 315) µ mhos/ cm Stock and test solution preparation: Primary stock prepared in dilution water at 150 mg/ L and mixed for ~ 23 hours prior to use. After mixing, primary stock solution was proportionally diluted with dilution water to prepare the five test concentrations.

Concentrations dosing rate: Once Stability of the test chemical solutions: Extremely stable Exposure vessels: 25L polyethylene aquaria containing approximately 15L of test solution; water depth approx 17.5 cm. Number of replicates: two Number of fish per replicate: ten ENV/ JM/ RD( 2002) 17/ FINAL 203 Number of concentrations: five plus a negative control Water chemistry during the study: Dissolved oxygen range ( 0   96 hours) : 9.4   10.7 mg/ L ( control exposure) 9.2   10.8 mg/ L ( 50 mg/ L exposure) pH range ( 0   96 hours) 8.1   8.4 ( control exposure) 8.2   8.4 ( 50 mg/ L exposure) Test temperature range ( 0   96 hours) 12.1   12.6 º C ( control exposure) 11.8   12.9 º C ( 50 mg/ L exposure)  Method of calculating mean measured concentrations: arithmetic mean RESULTS Nominal concentrations: Bk control, 3.1, 6.3, 13, 25, 50 ( exposure), 50 ( abiotic) mg/ L Measured concentrations: < LOQ, 3.0, 6.3, 13, 25, 50, 52 mg/ L Element value: 24 hour LC50 = > 50 mg/ L ( C. I. not calculable) 48 hour LC50 = > 50 mg/ L ( C. I. not calculable) 72 hour LC50 = > 50 mg/ L ( C. I. not calculable) 96 hour LC50 = 22 ( 18  27) mg/ L All element values based on mean measured concentrations Statistical Evaluation of Mortality: Element values and confidence limits for 24, 48, and 72 hours could not be calculated due to lack of mortality. Probit Analysis was used to calculate the 96 hour LC50.

Analytical Methodology: Analyses of test solutions were performed at Wildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection ( HPLC/ MS). When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for perfluorooctanesulfonate was used.

No attempt was made to quantify on the basis of individual isomeric components. The LOQ ( limit of quantitation) was 0.200 mg/ L in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 101. Samples collected at test initiation had measured values from 93.2 to 103% of nominal. Measured values for the biotic samples taken at 48 hours ranged from 93.6 to 103% of nominal, while abiotic samples ranged from 105 to 106% of nominal. Measured values for biotic samples taken at 96 hours ranged from 91.4 to 105% of nominal, while the abiotic samples were 102% of nominal. ENV/ JM/ RD( 2002) 17/ FINAL 204 Summary of analytical chemistry data:

Nominal Test Concentration, Mg/ L Measured Duplicate Values at 0, 48, and 96 hours, Respectively, mg/ L Mean Measured Concentration, mg/ L Percent of Nominal Negative Control All < LOQ < LOQ  3.1 3.15, 3.02, 2.90, 3.01,2.83, 2.97 3.0 97 6.3 6.22, 6.21, 6.16, 6.43, 6.15, 6.60 6.3 100 13 13.2, 12.1, 12.7, 12.3, 13.1, 12.6 13 100 25 25.0, 25.7, 24.3, 25.7, 25.7, 26.2 25 100 50 49.7, 49.8, 51.1, 51.5, 49.6, 50.8 50 100 50 ( abiotic) ( 1) 53.1, 52.6, 50.9, 51.0 52 104 ( 1) Samples taken at 48 and 96 hours only Biological observations after 96 hours:

Fish in the negative control and the 3.0 and 6.3 mg/ L exposure concentration appeared normal with no mortalities or overt signs of toxicity. All surviving fish in the 13 and 25 mg/ L exposures appeared normal with no overt signs of toxicity after 96 hours.

Cumulative percent mortality:

Mean Measured Test Conc., mg/ L 24 hours 48 hours 72 hours 96 hours Neg. Control 0 0 0 0 3.0 0 0 0 0 6.3 0 0 0 0 13 0 0 0 20 25 0 0 0 50 50 0 5 35 100 Lowest concentration causing 100% mortality: 50 mg/ L Mortality of controls: None CONCLUSIONS The potassium perfluorooctanesulfonate 96 hour LC50 for rainbow trout was determined to be 22 mg/ L with a 95% confidence interval of 18   27 mg/ L. The 96 hour no mortality and no effects concentration was 6.3 mg/ L.

Submitter: 3M, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 ENV/ JM/ RD( 2002) 17/ FINAL 205 DATA QUALITY Reliability: Klimisch ranking = 1 REFERENCES This study was conducted at Wildlife International Ltd., Easton, MD at the request of 3M.

OTHER Last changed: 1/ 24/ 02 ENV/ JM/ RD( 2002) 17/ FINAL 206 Robust Study Report Reference No. 43  Perfluorooctanesulfonate, Potassium salt ( PFOS): 96 Hour Semi Static Acute Toxicity Test with the Sheepshead Minnow (

   ) in saltwater TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The test substance is a white powder. Sample was taken from 3M lot number 217. Sample was stored under ambient conditions prior to testing. Purity determined to be 86.9% by LC/ MS, 1 H HMR, 19 F NMR and elemental analyses techniques.

METHOD Method: OPPTS 850.1075 Type: Static renewal GLP: Yes Date completed: Study completed 2001, report completed 2002 Species: Cyprinodon variegatus Supplier: Aquatic BioSystems, Inc., Fort Collins, CO Analytical monitoring: PFOS measured at initiation, prior to and after renewal at 24, 48, and 72 hours and at test termination ( 96 hours) Exposure period: 96 hours Statistical methods: The use of a single test concentration ( at water solubility) precluded the statistical calculation of LC50 values. Test fish age: Juveniles Average Total Length and weight: 3.0 ( 2.4   3.5) cm, 0.44 ( 0.21 0.66) g Loading: 0.29 g fish/ L Pretreatment: None Test conditions:

Dilution water: Natural seawater, filtered and diluted to a salinity of approximately 20 parts per thousand with well water Dilution water chemistry ( during the 4 week period immediately preceding the test):

Salinity: 20 ( 20   20) parts per thousand pH: 8.2 ( 8.1   8.3)

Stock and test solution preparation: Primary stock prepared in methanol at 40 mg/ L, sonicated for approximately 20 minutes and inverted to mix prior to use. After mixing, primary stock solution was proportionally diluted with dilution water to prepare the one test concentration. Each solution was stirred with a stainless steel whisk for approximately one minute. All test solutions appeared clear and colorless.

Solvent: Methanol Solvent concentration ( treatment and solvent control groups): 0.5 mL/ L ENV/ JM/ RD( 2002) 17/ FINAL 207 Concentrations dosing rate: Daily static renewal Stability of the test chemical solutions: Extremely stable Exposure vessels: 25L polyethylene aquaria containing approximately 15L of test solution; water depth approximately 17.1 cm.

Number of replicates: three ( biotic), two ( abiotic) Number of fish per replicate: ten Number of concentrations: One plus a negative and a solvent control, and an abiotic solution.

Water chemistry during the study: Dissolved oxygen range ( 0   96 hours): 2.8   7.4 mg/ L ( negative control exposure) 1.7   7.6 mg/ L ( solvent control exposure) 1.6   7.6 mg/ L ( 15 mg/ L exposure) pH range ( 0   96 hours) 7.9   8.3 ( negative control exposure) 7.9   8.3 ( solvent control exposure) 7.9   8.3 ( 15 mg/ L exposure) Test temperature range ( 0   96 hours) 21.9   22.6 º C ( negative control exposure) 22.1   22.9 º C ( solvent control exposure) 22.2   23.1 º C ( 15 mg/ L exposure)  Method of calculating mean measured concentrations: arithmetic mean RESULTS Nominal concentrations: Bk control, solvent control, 20 mg/ L ( biotic), 20 mg/ L ( abiotic)

Measured concentrations: < LOQ, < LOQ, 15, 13 mg/ L Element value: 24 hour LC50 = > 15 mg/ L ( C. I. not calculable) 48 hour LC50 = > 15 mg/ L ( C. I. not calculable) 72 hour LC50 = > 15 mg/ L ( C. I. not calculable) 96 hour LC50 = > 15 mg/ L ( C. I. not calculable) All element values based on mean measured concentrations Statistical Evaluation of Mortality: Element values and confidence interval could not be calculated due to lack of mortality.

Analytical Methodology: Analyses of test solutions were performed at Wildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection ( HPLC/ MS). When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ ( limit of quantitation) was 5.00 mg/ L in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 99.2. Samples collected in exposure vessels at test initiation had measured values from 78.6 to 82.0% of nominal. New samples collected at Hours 24, 48 and 72 had measured concentrations from 74.9 to 81.2 and 86.9 to 92.9, and 82.9 to 87.1% of ENV/ JM/ RD( 2002) 17/ FINAL 208 nominal, respectively. Old samples collected at Hours 24, 48, and 72 had measured concentrations ranging from 66.6 to 76.2, 56.4 to 66.9, and 75.8 to 89.9% of nominal, respectively. Mean measured concentrations of PFOS in samples collected at test termination were 55.6 to 68.4% of nominal. The measured concentrations of PFOS from the abiotic treatment group were slightly lower than those from the exposure treatment group. This may have been due to increased deposition of test substance at the limit of solubility, which could have resulted from the absence of the natural mixing action that was provided by the movement of the fish in the exposure treatment group.

Summary of analytical chemistry data:

Nominal Test Concentration, mg/ L Measured Replicate Values of old and new Test Solutions Respectively, mg/ L ( 1) Mean Measured Concentration, mg/ L Percent of Nominal Negative Control All < LOQ < LOQ

Solvent Control All < LOQ < LOQ

20 ( 2) 16.4, 15.7, 16.0; 15.2, 13.3, 15.2; 16.2, 15.7, 15.0; 13.4, 11.3, 12.7; 17.4, 18.2, 18.6; 15.2, 18.0, 16.2; 17.0, 16.6, 17.4; 13.7, 11.1, 13.4 15 75 20 ( 3)

( abiotic) 10.9, 9.22; 16.1, 16.6; 9.62, 9.69; 16.6, 17.5; 9.01, 9.25; 17.7, 15.9; 9.84, 9.22 13 65 ( 1) Replicate samples are listed in this order: Day 0 ( new), 24 hours ( old), 24 hours ( new), 48 hours ( old), 48 hours ( new), 72 hours ( old), 72 hours ( new), 96 hours ( old) ( 2) Triplicate samples ( 3) Day 0 not measured, Duplicate samples Biological observations after 96 hours: Fish in the negative control and solvent control appeared healthy and normal throughout the exposure period. No mortalities appeared in the 15 mg/ L treatment group during the study. However, upon transfer to the new test solution at approximately 48 and 72 hours, some fish were observed swimming erratically and turning a dark color. The fish appeared normal within approximately two hours after transfer, although one fish still appeared to be discolored at test termination.

Cumulative percent mortality:

Mean Measured Test Conc., mg/ L 24 hours 48 hours 72 hours 96 hours Negative Control 0 0 0 0 Solvent control 0 0 0 0 15 0 0 0 0 Lowest concentration causing 100% mortality: none   mortality limit apparently greater than solubility limit. Mortality of controls: None ENV/ JM/ RD( 2002) 17/ FINAL 209 CONCLUSIONS The potassium perfluorooctanesulfonate 96 hour LC50 for sheepshead minnow was determined to be > 15 mg/ L, the limit of solubility in this study. The 96 hour no mortality concentration was 15 mg/ L and no observed effects concentration was < 15 mg/ L mg/ L ( 1 fish out of 30 was discolored at 96 hours).

Submitter: 3M, Environmental Laboratory, P. O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking = 1 REFERENCES This study was conducted at Wildlife International Ltd., Easton, MD at the request of 3M.

OTHER Last changed: 1/ 24/ 02 ENV/ JM/ RD( 2002) 17/ FINAL 210 Robust Study Report Number 44. Bioconcentration test of Salt ( Na, K, Li) of perfluoroalkyl ( C= 4 12) sulfonic acid [ This test was performed using Perfluorooctane sulfonic acid, potassium salt ( Test substance number K 1520)] in carp TEST SUBSTANCE Identity: K 1520. Perfluorooctane sulfonic acid, potassium salt. Lot number A37626B Supplier: Kishida Chemical Co., Ltd. Remarks: Test substance number K 1520. The test substance is a white powder. Purity determined to be 100%

METHOD Method/ guideline followed: Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body" stipulated in the " Test Method for New Chemical Substance" July 13, 1974, Revised October 8, 1998, No. 5, Planning and Coordination Bureau, Environmental Agency; No. 615, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare: and No. 392, Basic Industries Bureau, Ministry of International Trade and Industry, Japan), and Bioconcentration : Flow through Fish test ( Guideline 305, June 14, 1996)" in the OECD Guidelines for Testing of Chemicals. Type: Flow through system GLP ( Y/ N): Yes Year: October 13, 2000   February 16, 2001 Species: Carp ( Cyprinus carpio) Supplier: Fukuokaken yabegawa fisherman's cooperative association ( Address: 193 1 Yamauchi, Yameshi Fukuoka 834 0012, Japan) Length and weight at test termination: Mean length = 6.4   9.6 Mean weight = No weight recorded Loading: 2 and 20 ug/ L respectively Fish Age: Yearling fish Analytical monitoring: High performance liquid chromatography Mass spectrometry Pretreatment: The fish were checked visually in the receiving and those demonstrating any abnormality were removed. The fish were reared for 8 days in a flow thorough system following an external disinfection. After rearing, the fish were medicated to eliminate parasites and transferred to an acclimatizing aquarium. After the second external disinfection, they were acclimatized. The fish demonstrating any abnormality during this period were removed and the remainder of the fish were reared for 15 days in a flow through system at temperatures of 25 ± 2o C. The fish were transferred to test tanks and reared at the same temperature in the flow through system for another 27 days. The fish were starved for 24 hours before sampling. Number of concentrations: Two plus a negative control Test concentration: ( mean measured): Negative control, 2 and 20 ug/ L Uptake period: 58 days Depuration period: 37 days Test conditions: Dilution water: Groundwater from the premises of Kurume Laboratory Dilution water chemistry: Specific conductance: Not recorded Hardness: 111 mg/ L Alkalinity: 96.1 pH: 7.6 to 7.8 Dissolved Oxygen: 7.9 to 8.1 ENV/ JM/ RD( 2002) 17/ FINAL 211 Temperature: 25.0 to 25.8 C Stock and test solution preparation: Based on preliminary test results for the 96 hour LC50 value and analytical detection limits, test concentrations of the test substance were decided as follows. The control was set as a blank test. Level 1 was 20 ug/ L and Level 2 was 2 ug/ L. The test substance was dissolved with ion exchanged water to prepare 16 and 1.6 mg/ L stock solutions. Diluter flow rate: 2 mL/ min for stock solution and 1600 mL/ min for dilution water; 2307 liters/ day for test water were supplied. Exposure Vessels: 100 liter tank Number of replicates: None Number of fish per vessel: 40 Diet: Nippon Formula Feed Mfg. Co., Ltd.

Water chemistry ranges during the study: Neg. Control 2 ug/ L 20 ug/ L Dissolved Oxygen: 8.0   8.1 mg/ L 8.0   8.1 mg/ L 7.9   8.1 mg/ L Temperature C: 25.1   25.4 C 25.5   25.8 C 25.0   25.4 C pH: 7.6   7.8 7.6   7.8 7.6   7.8 Photoperiod: Artificial light of white fluorescent lamp ( 14 hrs./ day) Light intensity: Artificial light of white fluorescent lamp Collection of tissue samples: Analysis of test fish was performed six times at each level in duration of exposure. Four fish were taken out at each sampling time and divided into two groups, and then both were analyzed individually. Analysis of control fish was performed before the experimental starting and after the experimental completion. Six fish were taken out at each sampling time and divided into three groups, and then both were analyzed individually. Because the stored sample taken out from one fish was too small for the measurement of lipid content, a group of two fish was employed. The fish were separated into parts; tegument, head viscera except liver, liver and remaining matter were weighted separately. The tegument consisted of the skin except head, scales, fin, alimentary canal or gills. The viscera consisted of internal organs except alimentary canal. Statistical methods: Steady state bioconcentation BCF values calculated from the tissue concentrations at apparent steady state divided by the mean water concentration. Tissue concentrations were considered to be at apparent steady state if 3 or more consecutive sets of tissue concentrations were not significantly different.

RESULTS Nominal concentrations: Negative control, Mean measured concentrations: 2 and 20 ug/ L Bioconcentration factors ( BCF):

PFOS Concentration in test water: Conc. After 1 Day After 7 Days After14 Days After 21 Days After 28 Days After 43 Days After 58 Days Average ( STD) 20 ug/ L 15.1 14.5 15.7 16.5 17.7 15.4 16.8 16 + 1.12 2 ug/ L 1.78 1.76 1.87 1.93 1.89 1.92 2.01 1.88 + 0.087 PFOS BCFs of Carp Exposed to 2 and 20 ug/ L: Conc. After 7 Days After14 Days After 21 Days After 28 Days After 43 Days After 58 Days 20 ug/ L 260 440 300 690 750 720 ENV/ JM/ RD( 2002) 17/ FINAL 212 2 ug/ L 240 520 410 860 890 1300 Analysis in parts of test fish Level 1 ( 20 ug/ L) BCF Parts Tegument Head Viscera Liver Remainder Parts T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 Concentration ( ng/ g) 22800 1670 0 23400 17500 36000 45300 37800 32000 6260 5380 BCF 1400 1000 1400 1100 2200 2700 2300 1900 380 320 Level 2 ( 2 ug/ L) BCF Parts Tegument Head Viscera Liver Remainder Parts T1 T2 T1 T2 T1 T2 T1 T2 T1 T2 Concentration ( ng/ g) 5490 4750 5600 4730 9900 7410 9190 7650 1810 1390 BCF 2800 2400 2900 2400 5100 3800 4700 3900 930 720 Time to reach 50% clearance: 49 days at level 1 and 152 days at level 2 Test organism mortality: Negative control: None documented. Level 1 ( 20 ug/ L): None documented. Level 2 ( 2 ug/ L): None documented.

Analytical methodology: Analysis of PFOS in the test water and carp was performed using high performance liquid chromatography mass spectrometry ( HPLC MS) analysis. The test water of each level was analyzed once before first analysis of test fish and at the same time as the analysis of the test fish. Steady state was reached when three successive analyses of BCFs made on samples taken at intervals of at least 48 hours were within + 20% of each other. When BCFs were less that 100, it was evaluated that a stead state had been reached after 28 days.

Recoveries in water and fish tissues were 89.3% and 90.6% respectively. Analytical results were corrected for procedural recovery.

CONCLUSIONS In this study, PFOS bioconcentrated in the tissues of carp. Test concentrations of 2 and 20 ug/ L were used. The fish were exposed for 58 days to makeup for excessive mortality. Bioconcentration factors were calculated to be 720 for 20 ug/ L and 200  1500 for 2 ug/ L.

REFERENCES Kurume Laboratory ( 2001). Chemicals Evaluation and Research Institute, Japan. Test number: 51520 ENV/ JM/ RD( 2002) 17/ FINAL 213 Annex 3. Application of Equilibrium Partitioning Models to Determining Effect Concentrations for PFOS Salts in Soil and Sediment The review of the complete set of ecotoxicity test reports presented in section 2.2.1, 2.3.1 and 2.3.2 has highlighted the absence of data describing the toxicity of PFOS salts to sediment and soil dwelling organisms and terrestrial plants. Since tests with aquatic invertebrates and plants have demonstrated toxicity of PFOS salts, and there is generally a correlation between toxicity to aquatic organisms living in the water column and toxicity to sediment and soil dwelling organisms. There is as a consequence a need to consider the possibility of deriving effect concentrations for PFOS in soil and sediment to fill the data gaps based on data that already exist for aquatic organisms. One approach to deriving such data is to use equilibrium partitioning models.

Equilibrium partitioning models are used in two ways:

  To calculate soil/ sediment pore water concentration from overall soil/ sediment concentration   To calculate PNECsoil/ sediment from PNECwater.

In both cases, the equilibrium constant for distribution between water and soil/ sediment, Kpsoil/ sediment, is needed. For many substances, Kpsoil/ sediment is replaced by Koc, which is equivalent to assuming that only the organic components of soil or sediment are important in affecting the equilibrium. However, for an anionic surfactant, such as PFOS, it is very likely that interaction with the inorganic substrate will also be important ( Salloum et al, 2000), as has been demonstrated in a recent study with PFOS ( Ellefson). Therefore Kp values for an appropriate soil or sediment would have to be used. It should also be noted that salinity affects the solubility of PFOS; the high ionic content of interstitial water could also affect the adsorption behaviour of PFOS. Furthermore, it is uncertain over what time scale equilibrium would be achieved ( Ellefson), and as a consequence the extent of adsorption may be dependent upon the concentration of PFOS in the aqueous phase.

Whilst for all substances extrapolation of PNEC from aquatic data to the terrestrial or sediment compartment is subject to uncertainty, that uncertainty is compounded when the mode of uptake is different for organisms present in different environmental compartments. The mode of toxic action and the mechanism of uptake of surfactants are complex. Therefore the use of equilibrium partition models to obtain PNECsoil or PNECsediment is subject to considerable doubt.

Should any laboratory soil or sediment organism test results become available then there is a further complication in extrapolating from these results to PNEC values appropriate to these compartments. The normal method of extrapolation is to apply a correction that takes into account the organic matter content of the matrix. For substances that adsorb preferentially to inorganic matter it would be necessary to correct for both the type of inorganic matrix, the composition of the aqueous phase and the concentration of the substance. This is unlikely to be straightforward.

A search of the open literature was carried out in order to assess the environmental fate and behaviour properties of substances with similar chemistry and properties to PFOS. Published papers are relevant to the consideration of equilibrium partitioning as a relevant model for PFOS environmental fate. Several papers were located and the abstracts are summarised below. It is beyond the scope of this work to investigate the papers in detail but it can be noted that the technical quality of the work, as with all open literature, has already been peer reviewed ( although not subject to GLP audit). The findings of these papers do not preclude the possibility of applying equilibrium partitioning to PFOS but do highlight the technical difficulties that might be encountered. ENV/ JM/ RD( 2002) 17/ FINAL 214 Conclusion It is concluded that on the basis of the presently available data for PFOS that equilibrium partitioning theory cannot be applied to determine either concentrations in interstitial water of soil and sediments or PNEC values for soil and sediments for the following reasons:

  the nature of the adsorption process cannot be assumed to be linearly dependent upon concentration;   the adsorption is likely to be highly dependent upon soil composition, particularly the inorganic component; and   the rate at which equilibrium might be achieved is unknown REFERENCES Salloum, M. J., Dudas, M. J., McGill, W. B. and Murphy, S. M. ( 2000). Surfactant sorption to soil and geologic samples with varying mineralogical and chemical properties. Environ. Toxicol. Chem. 19( 10), 2436 2442.

Soil Adsorption/ Desorption Study of Potassium Perfluorooctanesulfonate ( PFOS), Mark E. Ellefson, 3M Laboratory Report No: E00 13 11 ENV/ JM/ RD( 2002) 17/ FINAL 215 Review of open literature Report title Summary of abstract Author( s) Journal Terrestrial risk assessments for linear alkylbenzenesulfonate ( LAS) in sludgeamended soils Reviews investigations of factors affecting fate of LAS in the terrestrial environment. Refers to precipitating effect of calcium/ magnesium ions in addition to primary sorption. Sorbed form is noted as being different to the commercially available form ( for which data were available). DeWolfe, Watze; Feijtel, Tom Chemosphere, vol 36, no. 6, pp 1319 1343, 1998 Effect of sediment organic carbon on the toxicity of a surfactant to Hyalella azteca Investigation of the effects of sediment organic carbon levels on the sorption and toxicity to Hyalella azteca of anionic surfactants, specifically ABS ( alkylbenzene sulfonate). Studies indicated that higher levels of organic carbon led to higher apparent sorption coefficients. A higher sediment concentration was required to elicit the toxic response at higher organic carbon levels. Cano, M. L.; Dyer, S. C.; Decarvalho, A. J. Environ. Toxicol. Chem., vol 15, no. 8, pp 1411 1417, 1996 Anionic surfactant transport characteristics in unsaturated soil Investigations showed that for an alkyl ether sulfate and a linear alkylbenzenesulfonate, soilsurfactant interactions were reversible and that equilibrium conditions were quickly achieved. Allred, Barry; Brown, Glenn O. Soil Sci., vol 161, no. 7, pp 415 425. 1996 ENV/ JM/ RD( 2002) 17/ FINAL 216 Annex 4. Summary of the Lowest Acceptable Effect Concentrations Endpoint Species Protocol Result ( mg/ l)

Freshwater fish  acute toxicity Pimephales promelas ( Fathead minnow) Not given 96 hour LC50 4.7 Freshwater fish  chronic toxicity Pimephales promelas ( Fathead minnow) OECD 210 & OPPTS 850.1400 42 day NOECsurv/ growth 0.30 Invertebrate  acute toxicity ( Freshwater) Daphnia magna ( Water flea) ASTM 1981 & OECD 1981 48 hour EC50 27 Invertebrate  acute toxicity ( Salt water) Mysidopsis bahia ( Mysid shrimp) OPPTS 850.1035 96 hour LC50 3.6 Invertebrate  chronic toxicity ( Freshwater) Daphnia magna ( Water flea) ASTM 1981 & OECD 1981 ( Semi static) 28 day NOECrepro 7 Invertebrate  chronic toxicity ( Salt water) Mysidopsis  bahia ( Mysid shrimp) OPPTS 850.1350 35 day NOECrepro/ growth 0.25 Aquatic plants   growth inhibition of freshwater algae  Short term exposure Selenastrum capricornutum ( now

     "

) OECD 201, OPPTS 850.5400 & ASTM 1218 90E 96 hour EC50 cell density 96 hour EbC50 area under the curve 96 hour ErC50 growth rate 96 hour NOECgrowth rate, cell density,

area under the growth curve 72 hour EC50 cell density 72 hour EbC50 area under the curve 72 hour ErC50 growth rate 72 hour NOECgrowth rate, cell density,

area under the growth curve 71 71 126 44 70 74 120 70 Aquatic plants   growth inhibition of Saltwater algae   Short term exposure Skeletonema costatum OPPTS 850.5400 96 hour EC50 growth rate 96 hour NOECgrowth rate > 3.2 > 3.2 Aquatic plants   growth inhibition of freshwater algae  Longer term exposure Selenastrum capricornutum ( now

     "

) OECD 201, US EPA 600/ 9 78 018 & ASTM E 35.23 14 day EC50 cell density 14 day NOECcell density 14 day EC10 cell density 95 < 26 16 Freshwater higher plants   growth inhibition Lemna gibba OPPTS 850.4400 7 day IC50 108 Amphibians   embryo survival, growth and development Xenopus laevis ( African clawed frog) ASTM E1439 91 96 hour LC50 96 hour EC50 malformations Minimum concentration to inhibit growth 13.8 12.1 7.97 Sewage treatment organisms Activated sludge OECD 209 3 hour IC50 respiration inhibition > 905 ENV/ JM/ RD( 2002) 17/ FINAL 217 Endpoint Species Protocol Result ( mg/ kg of food)

Dietary toxicity to birds Anas platyrhynchos ( Mallard duck) OECD 205, OPPTS 850.2200 & FIFRA E 71 2 LC50 NOECmortality NOECbody weight 628 146 37 Colinus virginianus ( Northern Bobwhite quail) OECD 205, OPPTS 850.2200 & FIFRA E 71 2 LC50 NOECmortality NOECbody weight 220 73 73 Endpoint Species Protocol Result ( µ g/ bee)

Oral toxicity to bees Apis mellifera ( Honey bee) OECD 213, EPPO 170 72 hour LD50 72 hour NOEL 0.40 0.21 Contact toxicity to bees OECD 214, EPPO 170, OPPTS 850.3020 ( Contact) 96 hour LD50 96 hour NOEL 4.78 1.93 ENV/ JM/ RD( 2002) 17/ FINAL 218 Annex 5. Robust Summaries for Physical Chemical Properties and Environmental Fate Studies VAPOR PRESSURE Title: Impinger Studies of Volatility of FC 95 and FC 143 TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC 95. ( 1 Octanesulfonic acid, 1, 1, 2, 2, 3, 3, 4, 4, 5, 5, 6, 6, 7, 7, 8, 8, 8 heptadecafluoro, potassium salt, CAS # 2795 39 3)

Remarks: The 3M production lot number was not noted. The test sample is a white powder of uncharacterized purity.

METHOD Method: Internal GLP: No Year study performed: 1993 Remarks: Details outlined in the results section. As described below, there are a number of methodological concerns with this study. In addition, the document submitted is a combination of two reports. The first report contained Parts I and II, and a second report contained Experiment # 3. In addition, the first report contains portions of two versions. As neither of the first report versions is dated, it is unknown which is the final version. Part I Procedure: A mixture of 10 ppm FC 95 ( PFOS) and 10 ppm FC 143 ( PFOA) was prepared in water or water/ isopropanol solutions of the polar organic compounds listed in the table below. Aliquots of each solution were analyzed in triplicate for fluorochemical content by LC thermospray mass spectrometry both before and after bubbling 280 liters of air through them. The flow rate of the air passing through the stock solutions was 1 L/ min. The solutions were kept in an ice bath during bubbling. The concentrations before and after bubbling were compared after adjusting for volume lost during bubbling. Part II Procedure: Air was passed through an apparatus containing dry test material and then through glass wool at room temperature to a chain of impingers. A 50: 50 propanol: water solution containing 500 ppm ammonium acetate was used in the impingers to catch any volatilized PFOS. All impingers were in ice water. Experiment # 3 Procedure: The same study as in Part II was conducted in duplicate at 90 ° C.

RESULTS Vapor Pressure Value: Part II 8.7 x 10 8 torr, Experiment # 3 1.2 x 10 7 torr Temperature ° C: 90 ° C in experiment # 3 Decomposition: not stated ENV/ JM/ RD( 2002) 17/ FINAL 219 Part I Analysis before and after passing 280 liters of air through various stock solutions.

Results: Solution Original PFOS Conc., ppm % PFOS retained 500 ppm Tetrabutylammonium hydroxide 10.0 90 500 ppm Ammonium acetate 10.0 71 503 ppm Laurylpyridinium chloride 10.0 90 500 ppm N Alkyldimethylbenzylammonium chloride 10.0 100 500 ppm Cetyltrimethylammonium bromide 10.0 95 505 ppm Tallowtrimethylammonium chloride 10.0 93 500 ppm Dicocodimethylammonium chloride 10.0 84 Water/ 1 Propanol ( 50: 50) 10.0 89 Water/ 1 Propanol ( 50: 50) 0 0 500 ppm Ammonium acetate in water/ 1 propanol ( 50: 50) 0.10 96 500 ppm Ammonium acetate in water/ 1 propanol ( 50: 50) 0.20 101 500 ppm Ammonium acetate in water/ 1 propanol ( 50: 50) 0.40 90 500 ppm Ammonium acetate in water/ 1 propanol ( 50: 50) 0.80 95 500 ppm Ammonium acetate in water/ 1 propanol ( 50: 50) 2.0 96 Remarks: These findings appear to suggest a small loss of PFOS. However, in comments dated 12/ 7/ 93, Dr. Edwin Tucker of the Chemistry Department at the University of Oklahoma indicates that it is very unlikely that these flourochemicals were removed by bubbling air through water due to their vapor pressures, which are very low. Tucker thought more likely mechanisms for loss from the solution phase were concentration of the surfactants in foam and loss from the bubbled solutions as foam or microdroplets

Part II Analysis of impinger ammonium acetate solutions.

Results: No test material was found to be present in either the first or second impinger. This indicates that any test material transported from the solids to air and then into the ammonium acetate solutions in the impingers is below the detection limit. The calculated maximum pressure was 8.7 x 10 8 torr.

Remarks: In the report, the maximum vapor pressure calculations section contains errors. Equation 1 should use the value 0.625: g, not 0.625: g/ mL. In addition, the " maximum" vapor pressure calculated was erroneously called " minimum" vapor pressure in the text below both equations 5 and 7. Dr. Edwin Tucker of the Chemistry Department at the University of Oklahoma states that the experimental conditions do not provide firm evidence that the number is reasonable. There is no evidence that vapor pressure equilibrium was attained between the solid and the flowing gas. ENV/ JM/ RD( 2002) 17/ FINAL 220 Experiment # 3 Measuring Vapor Pressure at 90 ° C Results: The report concludes that PFOS has a measurable vapor pressure at this temperature. The minimum vapor pressure for the test material was purported to be 1.2 x 10 7 torr at 90 ° C. It was considered a minimum because the impinger trains may not have caught all of the fluorochemical that had been volatilized.