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AR226-2815 SPONSOR APME Avenue E. van Niewenhuyse 4 BOX 3 B-1160 Brussels Belgium TESTITEM AMMONIUM PERFLUOROOCTANOATE (APFO) STUDY TITLE ALGAL INHIBITION TEST DATA REQUIREMENT Directive 92/69/EEC C.3,31st July 1992 OECD Guideline No. 201, 7th June 1984 US EPA/OPPTS 850.5400 Guidelines, April 1996 STUDYDIRECTOR Jacques L'Haridon EXPERIMENTAL COMPLETION DATE 7 March 2003 DATE OF ISSUE J(o'3ajwA^ <eo^ TEST FACILITY CIT BP 563 - 27005 Evreux - France LABORATORY STUDY NUMBER 23685 EAA IFM Recherche S.N.C. ou Copilolde 6AS.092 788 060 465 R.C.S. EVIBU* VAT:FR197880604A5 Agrfaneni Cridil lmp61Recherche Page 1 of 53 Safety <6 Health Research Laboratories B.P.563 27005 Evreux Cedex T6l.: +33 (0)2 32 29 26 26 FCDC : +33 (0)2 32 67 87 05 E-mail: aT@citox.coni Web site: www.citox.coni Fronce Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 2 of 53 STATEMENT OF CONFIDENTIALITY OR NO CONFIDENTIALITY CLAIMS (To be completed by the Sponsor) Company Sanitized. Does not contain TSCA CBI % CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 3 of 53 STATEMENT OF TIIE STUDY DIRECTOR AND CIT SCIENTIFIC MANAGEMENT The study was performed in compliance with the principles of Good Laboratory Practice as described in: . OECD Principles on Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98) 17. . Decret N 98-1312 du 31 decembre 1998 concemant les Bonnes Pratiques de Laboratoire, (Journal Officiel du l^onvier 1999) Miniature de 1'Economie, des Finances et de llndustrie. . Commission Directive 1999/1 I/EC of 8 March 1999 adapting to technical progress the Principles of Good Laboratory Practice as specified in Council Directive 87/18/EEC on the hannomsation of laws, regulations and administrative provisions relating to the application of the Principles of Good Laboratory Practice and the verification of their applications for tests on chemical substances (OJ No. L 77 of 23.3.1999). . US Environmental Protection Agency, Federal Register, 40 CFR Part 792; Toxic Substances Control Act; Good Laboratory Practice Standards, August 17, 1989 (and subsequent amendments). . Japanese Ministry of International Trade and Industry, Good Laboratory Practice Standards, Basic Industries Bureau, KanHogyo No. 39, March 31,1984. . Japanese Ministry of Health and Welfare, Good Laboratory Practice Standards, Pharmaceutical Affairs Bureau, YakuHatsu No. 229 and Environmental Agency, 59 KiKyoku No. 85, March 31,1984. I declare that this report constitutes a true and faithful record of the procedures undertaken and the results obtained in the performance of the study. This study was performed at CIT, BP 563,27005 Evreux, France. Ecotoxicology J.L'Haridon Study completion date: 4 (, Doctor of Ecotoxicology Study Director J&wi.ia. ry IDOU v GroultDate:H \_ R. Doctor of Chemistry Scientific Management ^ ^' CJ C\^>^[ OTHER SCIENTIST INVOLVED IN TfflS STUDY For Pharmacy: X. Monciaux Doctor of Pharmacy Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME STATEMENT OF QUALITY ASSURANCE UNIT 4 of 53 Type of inspections Study plan Study Report Inspections 24 July 2002 14 October 2002 30 July 2003 Dates Reported to Study Directory) 26 July 2002 25 October 2002 22 August 2003 Reported to Management (*) 29 July 2002 28 October 2002 8 December 2003 In addition to the above-mentioned inspections, at about the same time as the study described in this report, "process-based" and routine facility inspections of critical procedures relevant to this study type were also made by the Quality Assurance Unit. The findings of these inspections were reported to the Study Director and to CTT Management. The inspections were performed in compliance with CTT Quality Assurance Unit procedures and Principles of Good Laboratory Practice. The reported methods and procedures were found to describe those used and the results to constitute an accurate and complete reflection of the study raw data. C. Galli-Kar Date: JC Jan. fag. Biol. Head of Quality Assurance Unit ^oC>l< (*) The dates indicated correspond to the dates of signature of audit reports by Study Director and Management. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME PAGE RESERVED FOR 40 CFR 158.34 (c) (I) CERTIFICATION (To be completed by the Sponsor) 5 of 53 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 6 of 53 CONTENTS STATEMENT OF CONFIDENTIALITY OR NO CONFIDENTIALITY CLAIMS 2 STATEMENT OF THE STUDY DIRECTOR AND CIT SCIENTIFIC MANAGEMENT 3 OTHER SCIENTIST INVOLVED IN THIS STUDY 3 STATEMENT OF QUALITY ASSURANCE UNIT 4 PAGE RESERVED FOR 40 CFR 158.34 (c) (1) CERTIFICATION 5 SUMMARY 9 1. INTRODUCTION 13 2. MATERIALS AND METHODS 13 2.1 TEST ITEM 13 2.1.1 Identification 13 2.1.2 Preparation of the test solutions 14 2.2 TEST SYSTEM 15 2.2.1 Algae 15 2.2.2 Environmental conditions during culture 15 2.2.3 Environmental conditions during the test 16 2.3 TREATMENT 17 2.3.1 Study design 17 2.3.2 Time schedule 17 2.4 OBSERVATIONS 17 2.5 CHEMICAL ANALYSIS 18 2.5.1 Range-finding test 18 2.5.2 First and second tests 18 2.6 DATA EVALUATION 18 2.6.1 Determination of the ErC50 and the EbC50 18 2.6.2 Determination of the No Observed Effect Concentration (NOEC) 19 2.7 ARCHIVING 19 2.8 STUDY PLAN ADHERENCE 19 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 7 of 53 3. 3.1 3.1.1 3.1.2 3.1.3 3.2 3.2.1 3.2.2 3.2.3 3.3 3.3.1 3.3.2 4. RESULTS RANGE-FINDING TEST Water quality Chemical analysis Growth inhibition FIRST TEST Water quality Chemical analysis Growth inhibition SECOND TEST Water quality Growth inhibition and chemical analysis CONCLUSION 5. REFERENCES Table 1: Range-finding test - temperature and pH Table 2: Range-finding test - T72 hours - number of cells and calculation of the specific growth rate at each concentration Table 3: Table 4: Table 5: Range-finding test - T96 hours - number of cells and calculation of the specific growth rate at each concentration Range-finding test - Number of cells (104 cells x mL"1)at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 72 hours. Area = cell x mL"1 x hour Range-finding test - Number of cells (104 cells x mL"1) at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 96 hours. Area = cell x mL"1 x hour Table 6: First test - temperature and pH Table 7: First test - T72 hours - number of cells and calculation of the specific growth rate at each concentration Table 8: First test - T96 hours - number of cells and calculation of the specific growth rate at each concentration Table 9: First test - Number of cells (104 cells x mL'1) at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 72 hours. Area = cell x mL'' x hour Table 10: First test - Number of cells (104 cells x mL'1) at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 96 hours. Area = cell x mL"1 x hour - Table 11: Second test - temperature and pH Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 8 of 53 Table 12: Second test - T72 hours - number of cells and calculation of the specific growth rate at each concentration 35 Table 13: Second test - T96 hours - number of cells and calculation of the specific growth rate at each concentration 36 Table 14: Second test - Number of cells (104 cells x mL'') at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 72 hours. Area = cell x mL'1 x hour 37 Table 15: Second test - Number of cells (104 cells x mL"1) at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 96 hours. Area = cell x mL"1 x hour 38 APPENDICES 1. Analytical certificate and composition of the test item 2. Substances required for the preparation ofLC reconstituted water 3. Chemical analysis of test solutions 4. Comparison of areas under the growth curves 39 40 43 45 51 to 53 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 9 of 53 SUMMARY At the request of APME, Brussels, Belgium, the acute toxicity of the test item AMMONIUM PERFLUOROOCTANOATE (APFO) was evaluated in the algal strain Pseudokirchneriella subcapitata using a 96-hour static test based on: . Directive 92/69/EEC C.3,31 st July 1992, . OECD Guideline No. 201,7th June 1984, . US EPA/OPPTS 850.5400 Guidelines, April 1996, . Requirements of the Japanese Government under the revised Chemical Substance Law according to the Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry, 9th December 1986. The main criterion measured is the EC50 (Median Effective Concentration); a statistically derived concentration which can be expected to cause a reduction in algal growth of 50% in comparison with the control. This criterion is evaluated as: . The ErC50 - the concentration of test item resulting in 50% reduction of the specific growth rate with respect to the control. Specific growth rate can be defined as the rate of increase in the natural log of the cell number (In no. of cells/mL) over specific intervals (e.g. 0 to 24, 0 to 48, 0 to 72 and 0 to 96 hours). . The EbC50 - the concentration of test item resulting in 50% reduction of the biomass with respect to the control. Biomass, in this case, may be defined as the increase in the cell number per mL of solution x time in hours and therefore represents an area (cell.mL''.h). The No Observed Effect Concentration (NOEC), the highest concentration tested which does not induce a statistically significant reduction of the algal growth (based on specific growth rate or biomass), is also determined. Methods The test item was dissolved in reconstituted water (LC) with a hardness of approximately 34 mg/L as CaCOs. The study included three tests. Range-finding test Three replicate solutions of algae with an initial dilution of 1 x 104 cells/mL were exposed to a nominal concentration of lOOmg/L expressed as the APFO (solids) content of the test item, while four other groups of two replicate flasks were exposed to nominal concentrations of 0.01, 0.1, 1 and 10 mg/L expressed as the APFO (solids) content of the test item. A further group of six replicate algal solutions without test item was used as the control. The number of replicates of the control and the 100 mg/L group was higher compared to other groups to provide a limit test if there was no effect at this concentration. The growth of the cell culture in each flask was monitored by counting the number of cells at 24, 48, 72 and 96 hours. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 10 of 53 First test Seven concentrations of AMMONIUM PERFLUOROOCTANOATE (APFO) were used together with a control at: 0, 6.25, 12.5, 25.0, 50.0, 100, 200 and 400 mg/L expressed as the APFO (solids) content of the test item. Three replicates were exposed to each concentration and six replicates to dilution water only (to act as the control) for 96 hours. Observations of cell growth were recorded at 24, 48, 72 and 96 hours. Second test A second test was undertaken since the results of the range-finding test were not in accordance with those of the first test. This second test was considered as the reference test for determination of all toxicity parameters. Seven concentrations of AMMONIUM PERFLUOROOCTANOATE (APFO) were used together with a control at: 0, 6.25, 12.5, 25.0, 50.0, 100, 200 and 400 mg/L expressed as the APFO (solids) content of the test item. Three replicates were exposed to each concentration and six replicates to dilution water only (to act as the control) for 96 hours. Observations of cell growth were recorded at 24, 48, 72 and 96 hours. Environmental parameters were: . pH: 6.61 to 10.74, . temperature: 22.6C to 23.8C, . lighting: 6710 lux to 7620 lux. Chemical analysis Chemical analysis was undertaken to measure the concentration of the test item in each test solution of the second test, except for the control, at the beginning (TO hour) and the end (T96 hours) of the test. Results Range-finding test The following results are based on nominal concentrations. According to the results obtained in this test, the 72-hour and 96-hour EC50s were > 100 mg/L. After 72 and 96 hours, the growth rate was equivalent to that of the control at 100 mg/L (highest concentration tested) while there was evidence of effect on biomass at this concentration. A further test was therefore performed (limit test not applicable). Company Sanitized. Does not contain TSCA CBI n CIT/Study-No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 11 of 53 First test The following results are based on nominal concentrations. According to the results obtained in this test, the 72-hour and 96-hour EC50s were > 400 mg/L. The growth rate NOEC was 200 mg/L after 72 hours (using the two replicates giving valid results at this concentration) -and only 6.25 mg/L after 9& hours. However, after 96 hours, inhibition of the growth rate at 400 mg/L was low (14%). The biomass NOEC was 400 mg/L after 72 hours and 100 mg/L after 96 hours. The large differences between the growth rate NOECs (72 and 96 hours) and between the growth rate and biomass NOECs at 96 hours, were considered unusual, and it was considered possible that the low slope of the concentration-effect curve may have prevented determination of precise NOECs. The results were not considered conclusive and, consequently, a second test was performed to determine whether the results of the first test were reproducible, using the same range of concentrations. Second test The following results are based on nominal concentrations. The second test confirmed that EC50 values after 72 and 96 hours were > 400 mg/L. The growth rate NOEC was 200 mg/L after 72 hours and only 12.5 mg/L after 96 hours. However, inhibition of growth rate at 400 mg/L was once again low after 96 hours (15%). The biomass NOEC was 200 mg/L after 72 hours and 12.5 mg/L after 96 hours. The biomass NOEC at 96 hours was therefore equivalent to the growth rate NOEC at 96 hours in this second test. Then, the second test generally confirmed that the low slope of the concentration-effect curve made it difficult to determine precisely NOECs after 72 and 96 hours using statistical analysis, as observed in the first test. However, the second test was considered to provide more reliable data because there was not the large difference in the growth rate and biomass NOEC values that had been observed in the first test. Therefore, the second test was considered as the reference test for determination of all toxicity parameters and, consequently, all samples taken during this test were analyzed to measure actual concentrations of AMMONIUM PERFLUOROOCTANOATE (APFO) in test solutions. Measured concentrations in the solutions of the second test with and without algae were within 20% of the corresponding nominal values at the beginning (TO hour) and the end (T96 hours) of the test. Hence, the test item was stable in solution throughout the test and was not significantly bioaccumulated or adsorbed at the surface of the cells. Furthermore, the study results could be based on nominal concentrations. These study results were as follows: Growth rate (mg/L) (1) Biomass (mg/L) (1) Time(h) ErC50 NOEC 72 >400 200 96 >400 12.5 (1) concentrations expressed as the APFO (solids) content of the test item EbC50 >400 >400 NOEC 200 12.5 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONTUM PERFLUOROOCTANOATE (APFO)/ APME 12 of 53 Conclusion Under our experimental conditions, the 72-hour ErC50 and the 72-hour EbC50 of AMMONIUM PERFLUOROOCTANOATE (APFO) in a static test system were > 400 mg/L for Psevdokirchneriella subcapitata. The NOEC at 72 hours was 200 mg/L based on the specific growth rate and biomass data. The NOEC at 96 hours was 12.5 mg/L based on the specific growth rate and biomass data. These results are based on the APFO (solids) content of the test item. Company Sanitized. Does not contain TSCA CBI CIT/StudyNo. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 13 of 53 1. INTRODUCTION The. objective of this study was to assess the acute toxicity of AMMONIUM PERFLUOROOCTANOATE (APFO), to Pseudoldrchneriella subcapitata, in a 96-hour static test based on: . Directive 92/69/EEC C.3, 31 st July 1992, . OECD Guideline No. 201, 7th June 1984, . US EPA/OPPTS 850.5400 Guidelines, April 1996, . Requirements of the Japanese Government under the revised Chemical Substance Law according to the Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry, 9th December 1986. The criterion measured is the EC50 (Median Effective Concentration), a statistically derived concentration of the test item in water which can be expected to cause a reduction of algal growth of 50% in comparison with the control. This criterion is evaluated as; . The ErC50 - the concentration of test item resulting in 50% reduction of the specific growth rate with respect to the control. . The EbC50 - the concentration of test item resulting in 50% reduction of the biomass with respect to the control. The No Observed Effect Concentration (NOEC), the highest concentration tested which does not induce a statistically significant reduction of the algal growth (based on specific growth rate or biomass), is also determined. 2. MATERIALS AND METHODS 2.1 TEST ITEM 2.1.1 Identification . supplier: 3M . name: - Study plan: AMMONIUM PERFLUOROOCTANOATE (APFO) Both namescorrespon^)tnesame test item. . description at receipt: colorless liquid . description on the analytical certificate:! . containers: three drums ' . date of receipt: 28 January 2002 Data relating to the characterisation of the test item are documented in an analytical certificate and in the composition of the test item (presented in appendix 1) provided by the Sponsor. The test concentrations^were expressed as concentrations of APFO, based on the stated APFO content of the test itemi^TM'""^""^""^^"^"""" " Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 14 of 53 2.1.2 Preparation of the test solutions The stock solutions, for the three tests carried out, were prepared by dissolving the test item directly in LC reconstituted water (see 2.2.2). The conditions of preparation of the stock solutions are reported in the following table: Range-finding test Quantity of test item (as received) (mg) 510.2 Quantity of APFO (solids) (mg) 100 Concentration of the test item (mg/L)(l) 100 Duration of the agitation (minutes) 10 First test 2041 400 400 - 15 Second test 2041 400 400 15 (1) expressed as the APFO (solids) content of the test item The agitation was continued until the stock solutions were used to prepare the test solutions. Test solutions were prepared by further dilution of the stock solution with LC reconstituted water to provide a geometric series of concentrations (expressed as the APFO (solids) content of the test item): . 0, 0.01, 0.1,1, 10 and 100 mg/L for the range-finding test, . 0, 6.25,12.5, 25.0, 50.0, 100, 200 and 400 mg/L for the first and second tests. Glass test vessels (250 mL Erienmeyer flasks) containing algae and test solutions (or dilution water in the case of the controls) were filled directly from the test solution containers immediately after preparation and test solutions remained unchanged throughout the study. The pH of the test solutions remained within acceptable limits (between 6 and 9 and in the range 0.5 unit of the test water) after preparation and there was no adjustment ofpH before addition of the algae. For the test solution at 100 mg/L in the range-finding test and all test solutions of the first and second tests (except for the controls), a further group of vessels was prepared for possible chemical analysis by adding test solutions but no algal pre-culture. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 15 of 53 2.2 TEST SYSTEM 2.2.1 Algae Species: Psendokirchneriella subcapitata. Strain No.: CCAP 278/4. Reason for this choice: species commonly used in Europe for aquatic toxicity testing and recommended in OECD, EEC and EPA guidelines. Cultured at: Origin: err. Culture Collection of Algae and Protozoa, Institute of Ecology, Far Sawrey, Ambleside, Cumbria, LA22 OLP, UK. Freshwater Culture method: the algae are cultured under sterile conditions and maintained at exponential growth rate. 2.2.2 Environmental conditions during culture During the culture period the conditions were as follows: Water: reconstituted water (LC oligo medium, see appendix 2), recommended in the French guideline AFNOR T 90-304. A primary stock solution is made up every month from which a final culture medium is prepared. This solution is autoclaved at which point it has a stock life of one week. Before use, the pH is verified at 7.5 0.3 or adjusted until this pH is obtained. The hardness of this solution is approximately 34 mg/L as CaCC>3. This medium does not fully comply with the requirement of OECD guideline concerning P, N and chelators concentrations (in excess). However, it is considered as suitable for this type of study, according to the above-mentioned French guideline and experience of our laboratory. Culture period: Temperature: algae are cultivated for 7 days before harvesting for use in a new culture. between 21C and 25C in water. Algae are maintained at 2C during the acclimation period. Temperature is checked daily. Illumination: Aeration: Preculture loading: 24 hours per day of constant illumination maintained at approximately 2000 lux throughout the culture period. For lighting details see 2.2.3. the cultures are constantly aerated with air filtered using a 0.22 um porosity filter to maximize the C02 availability (and thereby the cell growth) while maintaining the sterility. new cultures are loaded at a concentration of 1 x 104 cells/mL. Each weekly culture is prepared under sterile conditions using autoclaved medium while week old cell cultures are checked for contamination before being transferred to fresh medium. Company Sanitized. Does not contain TSCA CBI CIT/Study-No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 16 of 53 2.2.3 Environmental conditions during the test Test water: Temperature: Illumination: Duration of test: reconstituted-LC (see 2.2.2). controlled daily in a flask containing test water but no algae, run alongside me test. Between 21C and 25C (regulated to be as close as possible to 23C at the beginning of the test). continuous. A set of 15 and 30 W Osram Fluora fluorescent tubes between "white" and "daylight" type (spectral range 400 to 700 nm) were set approximately 40 cm above the cultures. These emit light measured using a lux meter equipped with a spherical collector at a level corresponding to half the height of the cultures in their conical flasks (corresponding to an irradiance of 252 mW/m2 for 15 W and 476 mW/m2 for 30 W tubes x no. of bulbs = 3724 mW/m2). The light intensity at each position to be occupied by a culture flask is measured regularly. The color temperature of the fluorescent lamps is between 4400 K and 5000 K. 96 hours. Transfer: Culture homogeneity: pH: Hardness: Forced aeration: the number of cells in the week-old culture was counted and the quantity of algal pre-culture to be added to the fresh test medium calculated to give a test solution concentration of 1 x 104 cells/mL. The algal pre-cultures were then added where appropriate to the 100 mL test solutions and all the 250 mL conical flasks stoppered with sterile cotton wool wrapped with lint and transferred to the agitator. the solutions were agitated throughout the test. the pH values of the control and of all the test concentrations, except for the range-finding concentrations at 0.01, 0.1, 1 and 10 mg/L, were measured at the beginning of the tests and after 72 and 96 hours. water hardness was measured once in LC reconstituted water before the start of the test. was not used during the test but the cultures were constantly agitated. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 17 of 53 2.3 TREATMENT 2.3.1 Study design The study included three tests. The duration of each test was 96 hours. Range-finding test Nominal concentration (mg/L)(l) Control 0 Treated 0.01 Treated 0.1 Treated 1 Treated 10 Treated 100 (1) expressed as the APFO (solids) content of the test item Number cif replicates with algae without algae 6 0 2 0 2 0 2 0 2 0 3 1 The number of replicates of the control and the 100 mg/L group was higher compared to other groups to provide a limit test if there was no effect at this concentration. First and second tests Group 1 (control) Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Nonlinal concentration (mg/L)(l) 0 6.25 12.5 25.0 50.0 100 200 400 (1) expressed as the APFO (solids) content of the test item. Numberofrepli cates with algae 1without algae 6 0 3 1 3 1 3 1 3 1 3 1 3 1 3 1 2.3.2 Time schedule Experimental starting date (first day of treatment): 14 October 2002, Experimental completion date: 7 March 2003. 2.4 OBSERVATIONS The number of cells in solutions was calculated at TO as 1 x 104 cells/mL. For the remaining observation times (T24, T48, T72 and T9'6 hours), cell numbers were determined using a Malassez cell counter. An observation of less than 1 x 104 cells/mL is below the sensitivity of the Malassez cell counter. Possible observations of this type were considered as 1 x 104 cells/mL - i.e. the test item was taken to be algaestatic at this test concentration rather than algaecidal, as no further experiment was undertaken to determine whether algae exposed to algaestatic concentrations were capable of growth post-exposure. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 18 of 53 2.5 CHEMICAL ANALYSIS Each sample contained 5 mL. 2.5.1 Range-finding test At TO hour, a sample was taken from the test solution container at 100 mg/L nominal and stored at-20C. At T24, T48, T72 and T96 hours, samples were taken from each test solution replicate at 100 mg/L nominal, pooled (5 mL in total) and stored at -20C. Further samples were also taken at T24, T48, T72 and T96 hours from the test solution at 100 mg/L nominal which contained no algae and had been run alongside the test to determine the influence of adsorption (at the surface of algae cells) and/or bioaccumulation on the possible decrease in test item concentration throughout the test. Although samples were taken during the range-finding test, no chemical analysis was performed on these samples because the test item was found to be toxic at 100 mg/L and consequently, at least one further test was necessary. 2.5.2 First and second tests At TO hour, samples were taken from each test solution container, except for the control, and stored at -20C. At T24, T48, T72 and T96 hours, samples were taken from all test replicate groups at each concentration (except for the control), pooled by concentration (5 mL in total) and stored at -20C. Further samples were also taken at T24, T48, T72 and T96 hours from the solutions which contained no algae and had been run alongside the test to determine the influence of adsorption (at the surface of algae cells) and/or bioaccumulation on the possible decrease in test item concentration throughout the test. Chemical analysis was only performed on samples taken at the beginning (TO hour) and the end (T96 hours) of the second test (see 3.3.2). The analytical procedure is presented in appendix 3. 2.6 DATA EVALUATION 2.6.1 Determination of the ErC50 and the EbC50 Specific growth rate can be defined as the rate of increase in the natural log of the cell number (In no. of cells/mL) over specific intervals (e.g. 0 to 24, 0 to 48, 0 to 72, 0 to 96 hours) (see appendix 4 for details). The concentration causing a reduction of growth rate to 50% that of the control (ErC50) is obtained for each observation time (T24, T48, T72 and T96 hours) by calculation from the average specific growth rate at each concentration. Biomass, in this case may be defined as the'number of cells per mL of solution x time in hours and therefore represents an area (cells.mL'1.!!). The biomass at 72 or 96 hours was determined using the trapezoidal method from the number of cells observed at TO, T24, T48, T72 and T96 hours. The concentration causing a reduction of biomass at 72 or 96 hours to 50% that of the control (EbC50) is then calculated. When a partial inhibition (0% < inhibition < 100%) is observed at at least two concentrations, the EC50 is calculated according to Probit analysis (i.e. Finney's method, published by E. Weber, combined with Bliss's method). The confidence interval limits are calculated statistically according to Fieller's method. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 19 of 53 When a partial inhibition (0% < inhibition < 100%) is observed at only one concentration, the EC50 is estimated using Probit analysis or an interpolation procedure (depending on the data obtained). In this case, the. highest concentration causing no inhibition and the lowest concentration producing 100% inhibition are used as confidence limits. If at all concentrations, inhibition is 0% or 100%, an EC50 can not be calculated. It is estimated using the geometric mean of the highest concentration causing no inhibition and the lowest concentration producing 100% inhibition. 2.6.2 Determination of the No Observed Effect Concentration (NOEC) After checking of the normality of the data with Chi-square and Shapiro-Wilks tests (normal at p = 0.01) as well as the variance homogeneity (Bartlett test; p = 0.01), the NOEC is determined by ANOVA (the Bonferroni T-test or the Dunnett test; p = 0.05) using the individual replicates of the area under the curve and the specific growth rate. 2.7 ARCHIVING The following study materials are archived by CIT (BP 563, 27005 Evreux, France) for 5 years after the end of the in vivo phase of the study: . Study plan and possible amendments, . raw data, . correspondence, . fmal report and possible amendments. On completion of this period, the archived study materials will be returned to the Sponsor, or may be archived at CIT for a further period (at additional cost). The total duration of archiving (depending on regulations) will be the responsibility of the Sponsor. In addition, raw data not specific to the study including, but not limited to, records of environmental data and equipment calibration, will also be archived by CIT and retained for at least 30 years. 2.8 STUDY PLAN ADHERENCE The study was performed in accordance with the Study plan No. 23685 EAA and subsequent amendments, with the following deviations from the agreed Study plan: . after 72 hours, the pH varied by more than 1.5 units in the first and second tests, based on individual treatment values. The maximum variation was observed in the control for both tests (1.67 and 2.94 units between TO hour and T72 hours in the controls of the first and second tests, respectively). These pH variations were due to the high algal growth rate under our experimental conditions. . the temperature varied by more than 1C during the first test ( 1.35C). These minor deviations were, not considered to have compromised the validity or integrity of the study. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 20 of 53 3. RESULTS 3.1 RANGE-FINDING TEST 3.1.1 Water quality The test solution was a translucent colorless solution at all test item concentrations. The minimum and maximum parameters measured during the range-finding test were: . pH: 6.98 and 9.67 between TO and T72 hours; 6.98 and 10.35 between TO and T96 hours, . temperature: 23.6C and 24.3C, . lighting: 6930 lux and 7410 lux. The temperature and pH data are presented in table 1. 3.1.2 Chemical analysis No chemical analysis was performed since the test item was found to be toxic at 100 mg/L and consequently, at least one further test was necessary. 3.1.3 Growth inhibition The results after 72 and 96 hours are presented in tables 2, 3,4 and 5. The following results are based on nominal concentrations. Inhibitions of the growth rate at 0.01, 0.1, 1, 10 and 100 mg/L were respectively 0%, 0%, 0%, 0% and 4%, relative to the control, at T72 hours. Inhibitions of the growth rate at 0.01, 0.1, 1, 10 and 100 mg/L were respectively 0%, 2%, 1%, 1% and 1%, relative to the control, at T96 hours. Inhibitions of the biomass at 0.01, 0.1, 1, 10 and 100 mg/L were respectively 0%, 1%, 10%, 8% and 31%, relative to the control, at T72 hours. Inhibitions of the biomass at 0.01, 0.1, 1, 10 and 100 mg/L were respectively 0%, 4%, 5%, 4% and 17%, relative to the control, at T96 hours. Acccording to the results obtained in this test, the 72-hour and 96-hour EC50s were > 100 mg/L. After 72 and 96 hours, the growth rate was equivalent to that of the control at 100 mg/L (highest concentration tested) while there was evidence of effect on biomass at this concentration. A further test was therefore performed (limit test not applicable). 3.2 FIRST TEST 3.2.1 Water quality The test solution was a translucent colorless solution at all test item concentrations. The minimum and maximum parameters measured during the first test were: . pH: 6.99 and 9.37 between TO and T72 hours; 6.44 and 9.63 between TO and T96 hours, . temperature: 21.9C and 24.6C, . lighting: 6600 lux and 7580 lux. The temperature and pH data are presented in table 6. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 21 of 53 3.2.2 Chemical analysis No chemical analysis was performed since this first test was not retained for determination of all toxicity parameters. 3.2.3 Growth inhibition The results after 72 and 96 hours are presented in tables 7, 8, 9 and 10. The following results are based on nominal concentrations. According to the results obtained in this test, the 72-hour and 96-hour EC50s were > 400 mg/L. After 72 hours, the growth rate was significantly lower than that of the control at 400 mg/L. NOEC at 72 hours was 200 mg/L using the two replicates giving valid results at this concentration. After 96 hours, the growth rate was significantly lower than that of the control at and above a concentration of 12.5 mg/L. The NOEC at 96 hours was 6.25 mg/L. However, after 96 hours, inhibition of the growth rate at 400 mg/L was only 14%. After 72 hours, the biomass was not significantly lower than that of the control at 400 mg/L. Therefore, the NOEC at 72 hours was 400 mg/L. After 96 hours, the biomass was significantly lower than that of the control at and above 200 mg/L. The NOEC at 96 hours was 100 mg/L. The large differences between the growth rate NOECs at 72 and 96 hours and between the NOECs at 96 hours for growth rate and biomass, were considered unusual, and it was considered possible that the low slope of the concentration-effect curve may have prevented determination of precise NOECs. The results were not considered conclusive and, consequently, a second test was performed to determine whether the results of the first test were reproducible, using the same range of concentrations. 3.3 SECOND TEST 3.3.1 Water quality The test solution was a translucent colorless solution at all test item concentrations. The minimum and maximum parameters measured during the second test were: . pH: 6.66 and 10.24 between TO and T72 hours; 6.61 and 10.74 between TO and T96 hours, . temperature: 22.6C and 23.8C, . lighting: 6710 lux and 7620 lux. The temperature and pH data are presented in table 11. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 22 of 53 3.3.2 Growth inhibition and chemical analysis The results after 72 and 96 hours are presented in tables 12, 13, 14 and 15. The following results are based on nominal concentrations. The second test confirmed that EC50 values after 72 and 96 hours were > 400 mg/L. After 72 hours, the growth rate was significantly lower than that of the control at 400 mg/L. The NOEC at 72 hours, i.e. 200 mg/L, was the same as that obtained in the first test. After 96 hours, the growth rate was significantly lower than that of the control at and above 25.0 mg/L. The NOEC at 96 hours, i.e. 12.5 mg/L, was only twice that obtained in the first test (6.25 mg/L), confirming a significant reduction of the growth rate at a much lower concentration after 96 hours compared to 72 hours. However, inhibition of growth rate at 400 mg/L was once again low after 96 hours (15%). After 72 hours, the biomass was significantly lower than that of the control at 400 mg/L. The NOEC at 72 hours corresponded to 200 mg/L while it was 400 mg/L in the first test. After 96 hours, the biomass was significantly lower than that of the control at and above 25.0 mg/L. At the maximum concentration tested (400 mg/L), the biomass after 96 hours was 43% lower than that of the control. The NOEC at 96 hours, i.e. 12.5 mg/L, was therefore eight times lower than in the first test but equivalent to the NOEC at 96 hours based on growth rate obtained in this second test. Then, the second test generally confirmed that the low slope of the concentration-effect curve made it difficult to determine precisely NOECs after 72 and 96 hours using statistical analysis, as observed in the first test. However, the second test was considered to provide more reliable data because there was not the large difference in the growth rate and biomass NOEC values that had been observed in the first test. Therefore, the second test was considered as the reference test for determination of all toxicity parameters and, consequently, all samples taken during this test were analyzed to measure actual concentrations of AMMONIUM PERFLUOROOCTANOATE (APFO) in test solutions. Results of chemical analysis are presented in table A, appendix 3. Measured concentrations in the solutions of the second test with and without algae were within 20% of the corresponding nominal values at the beginning (TO hour) and the end (T96 hours) of the test. Hence, the test item was stable in solution throughout the test and was not significantly bioaccumulated or adsorbed at the surface of the cells. Furthermore, the study results could be based on nominal concentrations. All EC50s (24, 48, 72, and 96-hour ErC50s and 72 and 96-hour EbC50s) were not calculated since the inhibition percentage of the growth rate or biomass at the highest concentration (400 mg/L) was systematically lower than 50% at the corresponding time. Consequently, the ErC50 at each of the measured growth intervals was as follows: ____Time (h)_____ErC50 (mg/L) (1)______95% confidence limits (mg/L)______ 24 > 400 48 > 400 72 > 400 _(1_) e_xp_re_sse9d6as_th_e_AP_F_O_(s_ol_ids>) c4on0te0nt_o_f th_e_tes_t _ite_m __________________________ Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 23 of 53 The 72-hour EbC50 and 96-hour EbC50 were as follows: Time(h)____EbC50 (mg/L)(l)______95% confidence limits (mg/L) 72 > 400 96 > 400 (1) expressed as the APFO (solids) content of the test item The NOEC at 72 hours was calculated as 200 mg/L (p = and biomass data. The NOEC at 96 hours was calculated as 12.5 mg/L (p = and biomass data. 0.05) based on the specific growth rate 0.05) based on the specific growth rate 4. CONCLUSION Under our experimental conditions, the 72-hour ErC50 and the 72-hour EbC50 of AMMONIUM PERFLUOROOCTANOATE (APFO) in a static test system were > 400 mg/L for Pseudokirchneriella subcapitata. The NOEC at 72 hours was 200 mg/L based on the specific growth rate and biomass data. The NOEC at 96 hours was 12.5 mg/L based on the specific growth rate and biomass data. These results are based on the APFO (solids) content of the test item. 5. REFERENCES Bliss, C.I.: The determination of dosage-mortality curves from small number. Quart. J. Pharm. 1L 192-216 (1938). Fieller: A fundamental formula in the statistics of biological assay and some applications. Quarterly Journal of Pharmacy and Pharmacology, 117-123 (1944). Weber, E: Grunden der biologischen Statistic Gustav Fisher Verlag, Stuttgart, 1972. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 24 of 53 Table 1: Range-finding test - temperature and pH Nominal concentration (mg/L) 0 100 pH at T 0 hour 7.80 7.76 pH at T 72 hours 9.67 6.98 Temperature measured between 0 and 2 hours: 23.6C Temperature at 24 hours (measured in the same test vessel): 23.9C Temperature at 48 hours (measured in the same test vessel): 23.8C Temperature at 72 hours (measured in the same test vessel): 23.9C Temperature at 96 hours (measured in the same test vessel): 24.3C pH at T 96 hours 10.35 7.93 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 25 of 53 Table 2: Range-finding test - T72 hours - number of cells and calculation of the specific growth rate at each concentration Nominal concentration (mg/L) 0 0.01 0.1 1 10 100 Rep. 1 2 3 4 5# 6# 1 2 1 2 1 2 1 2 1 2 3# Total No. of cells at 72 hours (104/^lL) 133.0 154.0 157.0 150.0 LnNo. of cells 14.10 14.25 14.27 14.22 MeanLn No. of cells Specific growth rate % Inhibition of growth rate 14.21 0.07 163.0 179.0 168.0 148.0 135.0 159.0 170.0 131.0 121.0 122.0 14.30 14.40 14.33 14.21 14.12 14.28 14.35 14.09 14.01 14.01 14.35 14.27 14.20 14.22 14.01 0.07 0.07 0.07 0.07 0.07 -2.84 -1.24 0.23 -0.14 3.97 Ln number of cells at TO (10 000 cells) = 9.21 Rep.: replicate # Values obtained in two control replicates (Nos. 5 and 6) and one replicate at 100 mg/L (No. 3) were not retained for data interpretation since the value(s) obtained after 72 and/or 96 hours was/were at least 3 times lower than the mean of other replicate values. Variation coefficient of the control cell density at 72 hours: 7.22% Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 26 of 53 Table 3: Range-finding test - T96 hours - number of cells and calculation of the specific growth rate at each concentration Nominal concentration (mg/L) 0 0.01 0.1 1 10 100 Rep. 1 2 3 4 5# 6# 1 2 1 2 1 2 1 2 1 2 3# Total No. of cells at 96 hours (lO^mL) 338.0 396.0 336.0 388.0 - LnNo. of cells 15.03 15.19 15.03 15.17 MeanLn No. of cells Specific growth rate % Inhibition of growth rate 15.11 0.06 - 364.0 370.0 334.0 318.0 338.0 360.0 368.0 338.0 346.0 324.0 - 15.11 15.12 15.02 14.97 15.03 15.10 15.12 15.03 15.06 14.99 15.12 15.00 15.06 15.08 15.02 0.06 0.06 0.06 0.06 0.06 -0.16 1.85 0.70 0.51 1.39 Ln number of cells at TO (10 000 cells) = 9.21 Rep.: replicate # Values obtained in two control replicates (Nos. 5 and 6) and one replicate at 100 mg/L (No. 3) were not retained for data interpretation since the value(s) obtained after 72 and/or 96 hours was/were at least 3 times lower than the mean of other replicate values. Variation coefficient of the control cell density at 96 hours: 8.76% Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 27 of 53 Table 4: Range-fmding test - Number calculation of the area under 72 hours. Area = cell x mL"' x hour of cells (104 cells the growth curve x ml/') at each observation time and (biomass) at each concentration after Nominal concentration Rep. (mg/L) 0 1 2 3 4 5# 6# 0.01 1 2 0.1 1 2 1 1 2 10 1 2 100 1 2 3# Total number of cells at 24 hours 6.30 5.70 4.60 5.30 at 48 hours 33.50 45.70 29.00 42.00 at 72 hours 133.00 154.00 157.00 150.00 5.60 4.50 3.80 5.90 4.50 3.50 4.20 5.40 1.90 1.50 44.70 38.00 34.30 30.50 28.00 28.00 30.30 26.00 20.80 18.60 163.00 179.00 168.00 148.00 135.00 159.00 170.00 131.00 121.00 122.00 Oto24 hours 63.60 56.40 43.20 51.60 Biomass 24 to 48 hours 48 to 72 hours Oto72 hours Average (0-72 hours) 453.60 592.80 379.20 543.60 1974.00 2372.40 2208.00 2280.00 2491.20 3021.60 2630.40 2875.20 2754.60 Inhibition (%) 55.20 42.00 33.60 58.80 42.00 30.00 38.40 52.80 10.80 6.00 579.60 486.00 433.20 412.80 366.00 354.00 390.00 352.80 248.40 217.20 2468.40 2580.00 2403.60 2118.00 1932.00 2220.00 2379.60 1860.00 1677.60 1663.20 3103.20 3108.00 2870.40 2589.60 2340.00 2604.00 2808.00 2265.60 1936.80 1886.40 3105.60 2730.00 2472.00 2536.80 1911.60 -12.74 0.89 10.26 7.91 30.60 Rep.: replicate # Values obtained in two control replicates (Nos. 5 and 6) and one replicate at 100 mg/L (No. 3) were not retained for data interpretation since the value(s) obtained after 72 and/or 96 hours was/were at least 3 times lower than the mean of other replicate values. Variation coefficient of the control cell density at 72 hours: 7.22% Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME - 28 of 53 Table 5: Range-finding test - Number calculation of the area under 96 hours. Area = cell x mL"1 x hour of cells (104 cells the growth curve x mL'') at each observation time and (biomass) at each concentration after Nominal concentration Rep. (mg/L) 0 1 2 3 4 5S 6# 0.01 1 2 0.1 1 2 1 1 2 10 1 2 100 1 2 3# Total number of cells at 24 hours 6.30 5.70 4.60 5.30 - - 5.60 4.50 3.80 5.90 4.50 3.50 4.20 5.40 1.90 1.50 - at 48 hours 33.50 45.70 29.00 42.00 - - 44.70 38.00 34.30 30.50 28.00 28.00 30.30 26.00 20.80 18.60 - at 72 hours 133.00 154.00 157.00 150.00 - - 163.00 179.00 168.00 148.00 135.00 159.00 170.00 131.00 121.00 122.00 - at 96 hours 338.0 396.0 336.0. 388.0 - - 364,0 370.0 334.0 318.0 338.0 360.0 368.0 338.0 346.0 324.0 - Biomass Oto24 hours 24 to 48 hours 48 to 72 hours 72 to 96 hours Oto96 hours Average (0-96 hours) 63.60 56.40 43.20 51.60 453.60 592.80 379.20 543.60 1974.00 2372.40 2208.00 2280.00 5628.00 6576.00 5892.00 6432.00 8119.20 9597.60 8522.40 9307.20 8886.60 55.20 42.00 33.60 58.80 42.00 30.00 38.40 52.80 10.80 6.00 579.60 486.00 433.20 412.80 366.00 354.00 390.00 352.80 248.40 217.20 2468.40 2580.00 2403.60 2118.00 1932.00 2220.00 2379.60 1860.00 1677.60 1663.20 6300.00 6564.00 6000.00 5568.00 5652.00 6204.00 6432.00 5604.00 5580.00 5328.00 9403.20 9672.00 8870.40 8157.60 7992.00 8808.00 9240.00 7869.60 7516.80 7214.40 9537.60 8514.00 8400.00 8554.80 7365.60 Rep.: replicate 9 Values obtained in two control replicates (Nos. 5 and 6) and one replicate at 100 mg/L (No. 3) were not retained for data interpretation since the value(s) obtained after 72 and/or 96 hours was/were at least 3 times lower than the mean of other replicate values. Variation coefficient of the control cell density at 96 hours: 8.76% (%) -7.33 4.19 5.48 3.73 17.12 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 29 of 53 Table 6: First test - temperature and pH Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 pH at T 0 hour 7.70 7.77 7.66 7.63 7.57 7.60 7.59 7.67 pH at T 72 hours 9.37 9.22 8.89 7.82 7.63 7.05 6.99 7.05 Temperature measured between 0 and 2 hours: 21.9C Temperature at 24 hours (measured in the same test vessel): 24.2C Temperature at 48 hours (measured in the same test vessel): 24.2C Temperature at 72 hours (measured in the same test vessel): 24.3C Temperature at 96 hours (measured in the same test vessel): 24.6C pH at T 96 hours 9.63 9.27 9.10 8.94 8.37 7.64 6.73 6.44 Company Sanitized. Does not contain TSCA CB1 CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 30 of 53 Table 7; First test - T72 hours - number of cells and calculation of the specific growth rate at each concentration Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 Rep. 1 2 3 4 5 6 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2# 3 1 2 3 Total No. of cells at 72 hours (104/mL) LnNo. of cells MeanLn No. of cells Specific growth rate % Inhibition of growth rate 189.0 235.0 205.0 230.0 242.0 197.0 223.0 220.0 256.0 233.0 208.0 255.0 241.0 204.0 227.0 240.0 289.0 216.0 239.0 234.0 217.0 222.0 14.45 14.67 14.53 14.65 14.70 14.49 14.62 14.60 14.76 14.66 14.55 14.75 14.69 14.53 14.63 14.69 14.88 14.59 14.69 14.67 14.59 14.61 14.58 14.66 14.65 14.62 14.72 14.65 14.58 0.07 0.08 0.08 0.08 0.08 0.08 0.07 -1.42 -1.32 -0.69 -2.51 -1.21 0.04 208.0 183.0 139.0 164.0 14.55 14.42 14.14 14.31 14.29 0.07 5.42* Ln number of cells at TO (10 000 cells) = 9.21 Rep.: replicate # Values obtained in this replicate (No. 2) at 200 mg/L were not retained for data interpretation since the value(s) obtained after 72 and/or 96 hours was/were at least 3 times lower than the mean of other replicate values. * Significant inhibition (relative to the control) Variation coefficient of the control cell density at 72 hours: 10.2% Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 31 of 53 Table 8: First test - T96 hours - number of cells and calculation of the specific growth rate at each concentration Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 Rep. 1 2 3 4 5 6 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2# 3 1 2 3 Total No. of cells at 96 hours (lO^mL) 379.0 470.0 432.0 418.0 466.0 436.0 408.0 422.0 378.0 396.0 322.0 388.0 404.0 308.0 340.0 364.0 288.0 348.0 334.0 324.0 366.0 270.0 LnNo. of cells MeanLn No. of cells Specific growth rate % Inhibition of growth rate 15.15 15.36 15.28 15.25 15.35 15.29 15.22 15.26 15.14 15.19 14.98 15.17 15.21 14.94 15.04 15.11 14.87 15.06 15.02 14.99 15.11 14.81 15.28 15.21 15.12 15.06 15.01 15.04 14.82 0.06 0.06 0.06 0.06 0.06 0.06 0.06 1.19 2.70* 3.56* 4.37* 3.92* 7.64* 274.0 183.0 185.0 180.0 14.82 14.42 14.43 14.40 14.42 0.05 14.20* Ln number of cells at TO (10 000 cells) = 9.21 Rep.: replicate # Values obtained in this replicate (No. 2) at 200 mg/L were not retained for data interpretation since the value(s) obtained after 72 and/or 96 hours was/were at least 3 times lower than the mean of other replicate values. * Significant inhibition (relative to the control) Variation coefficient of the control cell density at 72 hours: 7.73% Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 32 of 53 Table 9: First test - Number of cells (104 cells x mL'') at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 72 hours. Area = cell x mL'1 x hour Total number of cells Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 Rep. 1 2 3 4 5 6 2 3 2 3 2 3 1 2 3 2 3 1 2 3 1 2 3 at 24 hours 2.10 3.90 5.90 4.90 4.90 3.60 3.60 4.30 4.80 3.90 4.70 4.10 3.80 4.00 4.10 4.40 2.50 3.50 3.70 4.30 3.30 3.40 3.70 2.70 2.10 2.40 at 48 hours 26.00 34.30 29.80 34.70 39,70 45.70 39.30 33.70 40.70 38.70 43.70 33.30 50.50 38.70 34.30 38.00 35.30 38.00 42.30 37.70 40.00 41.70 at 72 hours 189.00 235.00 205.00 230.00 242.00 197.00 223.00 220.00 256.00 233.00 208.00 255.00 241.00 204.00 227.00 240.00 289.00 216.00 239.00 234.00 217.00 222.00 38.00 46.30 35.70 33.30 208.00 183.00 139.00 164.00 Biomass Oto24 hours 24 to 48 hours 48 to 72 hours Oto72 hours Average (0-72 hours) 13.20 34.80 58.80 46.80 46.80 31.20 31.20 39.60 45.60 34.80 44.40 37.20 33.60 36.00 37.20 40.80 18.00 30.00 32.40 39.60 27.60 28.80 313.20 434.40 404.40 451.20 511.20 567.60 490.80 432.00 522.00 487.20 556.80 424.80 627.60 488.40 436.80 484.80 429.60 474.00 528.00 480.00 495.60 517.20 2556.00 3207.60 2793.60 3152.40 3356.40 2888.40 3123.60 3020.40 3536.40 3236.40 2996,40 3435.60 3474.00 2888.40 3111.60 3312.00 3867.60 3024.00 3351.60 3236.40 3060.00 3140.40 2882.40 3676.80 3256.80 3650.40 3914.40 3487.20 3645.60 3492.00 4104.00 3758.40 3597.60 3897.60 4135.20 3412.80 3585.60 3837.60 4315.20 3528.00 3912.00 3756.00 3583.20 3686.40 3478.00 3747.20 3751.20 3711.20 3893.60 3750.40 3561.60 32.40 20.40 13.20 16.80 476.40 564.00 429.60 404.40 2928.00 2727.60 2072.40 2343.60 3436.80 3312.00 2515.20 2764.80 2864.00 Inhibition (%) -7.74 -7.86 -6.71 -11.95 -7.83 -2.40 17.65 Rep.: replicate # Values obtained in this replicate (No. 2) at 200 mg/L were not retained for data interpretation since the value(s) obtained after 72 and/or 96 hours was/were at least 3 times lower than the mean of other replicate values. Significant inhibition (relative to the control) Variation coefficient of the control cell density at 72 hours: 10.2% Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 33 of 53 Table 10: First test - Number of cells (104 cells x mL'1) at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 96 hours. Area = cell x mL"1 x hour Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 Rep. 1 2 3 4 5 6 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 2# 3 1 2 3 Total number of cells at 24 hours 2.10 3.90 5.90 4.90 4.90 3.60 3.60 4.30 4,80 3.90 4.70 4.10 3.80 4.00 4.10 4.40 2.50 3.50 3.70 4.30 3.30 3.40 at 48 hours 26.00 34.30 29.80 34.70 39.70 45.70 39.30 33.70 40.70 38.70 43.70 33.30 50.50 38.70 34.30 38.00 35.30 38.00 42.30 37.70 40.00 41.70 at 72 hours at 96 hours 189.00 235.00 205.00 230.00 242.00 197.00 223.00 220.00 256.00 233.00 208.00 255.00 241.00 204.00 227,00 240.00 289.00 216,00 239.00 234.00 217.00 222.00 379.00 470.00 432.00 418.00 466.00 436.00 408.00 422.00 378.00 396.00 322.00 388.00 404.00 308.00 340.00 364.00 288.00 348.00 334.00 324.00 366.00 270.00 3.70 2.70 2.10 2.40 38.00 46.30 35.70 33.30 208.00 183.00 139.00 164.00 274.00 183.00 185.00 180.00 Biomass Inhibition (%) Oto24 hours 24 to 48 hours 48 to 72 hours 72 to 96 hours Oto 96 hours Average (0-96 hours) 13.20 34.80 58.80 46.80 46.80 31.20 31.20 39.60 45.60 34.80 44.40 37.20 33.60 36.00 37.20 40.80 18.00 30.00 32.40 39.60 27.60 28.80 313.20 434.40 404.40 451.20 511.20 567.60 490.80 432.00 522.00 487.20 556.80 424.80 627.60 488.40 436.80 484.80 429.60 474.00 528.00 480.00 495.60 517.20 2556.00 3207.60 2793.60 3152.40 3356.40 2888.40 3123.60 3020.40 3536.40 3236.40 2996.40 3435.60 3474.00 2888.40 3111.60 3312.00 3867.60 3024.00 3351.60 3236.40 3060.00 3140.40 6792.00 8436.00 7620.00 7752.00 8472.00 7572.00 7548.00 768000 7584.00 7524.00 6336.00 7692.00 7716.00 6120.00 6780.00 7224.00 6900.00 6744.00 6852.00 6672.00 6972.00 5880.00 9674.40 12112.80 10876.80 11402.40 12386.40 11059.20 11193.60 11172.00 11688.00 11282.40 9933.60 11589.60 11851.20 9532.80 10365.60 11061.60 11215.20 10272.00 10764.00 10428.00 10555.20 9566.40 11252.00 11351.20 10935.20 10583.20 10849.60 10582.40 9381.60 -0.88 2.82 5.94 3.58 5.95 16.62* 32.40 20.40 13.20 16.80 476.40 564.00 429.60 404.40 2928.00 2727.60 2072.40 2343.60 5760.00 4368.00 3864.00 4104.00 9196.80 7680.00 6379.20 6868.80 6976.00 38.00* Rep.: replicate # Values obtained in this replicate (No. 2) at 200 mg/L were not retained for data interpretation since the value(s) obtained after 72 and/or 96 hours was/were at least 3 times lower than the mean of other replicate values. * Significant inhibition (relative to the control) Variation coefficient of the control cell density at 96 hours: 7.73% Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 34 of 53 Table 11: Second test - temperature and pH Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 pH at T 0 hour 7,30 7.47 7.45 7.44 7.44 7.42 7.41 7.40 PH at T 72 hours 10.24 9.90 9.66 9.52 9.38 8.58 6.73 6.66 Temperature measured between 0 and 2 hours: 22.6C Temperature at 24 hours (measured in the same test vessel): 23.6C Temperature at 48 hours (measured in the same test vessel): 23.3C Temperature at 72 hours (measured in the same test vessel): 23.4C Temperature at 96 hours (measured in the same test vessel): 23.8C pH at T 96 hours 10.74 10.03 10.11 9.94 9.87 9.50 7.13 6.61 Company Sanitized. Does not contain TSCA CBI CIT/StudyNo. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 3 5 of 53 Table 12: Second test - T72 hours - number of cells and calculation of the specific growth rate at each concentration Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 Rep. 1 2 3 4 5 6 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total No. of cells at 72 hours (lO^mL) 115.0 114.0 154.0 127.0 129.0 133.0 121.0 129.0 141.0 130.0 179.0 126.0 145.0 134.0 148.0 133.0 131.0 135.0 129.0 138.0 143.0 117.0 120.0 123.0 104.0 113.0 103.0 LnNo. of cells MeanLn No. of cells Specific growth rate % Inhibition of growth rate 13.95 13.95 14.25 14.05 14.07 14.10 14.01 14.07 14.16 14.08 14.40 14.05 14.19 14.11 14.21 14.10 14.09 14.12 14.07 14.14 14.17 13.97 14.00 14.02 13.85 13.94 13.84 14.06 14.08 14.17 14.17 14.10 14.13 14.00 13.88 0.07 0.07 0.07 0.07 0,07 0.07 0.07 0.06 -0.33 -2.30 -2.17 -0.79 -1.33 1.34 3.78* Ln number of cells at TO (10 000 cells) =9.21 Rep.: replicate * Significant inhibition (relative to the control) Variation coefficient of the control cell density at 72 hours: 11.3% Company Sanitized. Does not contain TSCA CBI CIT/StudyNo. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 36 of 53 Table 13: Second test - T96 hours - number of cells and calculation of the specific growth rate at each concentration Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 Rep. 1 2 3 4 5 6 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total No. of cells at 96 hours (lO^mL) 476.0 420.0 474.0 510.0 442.0 518.0 394.0 460.0 520.0 368.0 460.0 446.0 328.0 298.0 370.0 316.0 348.0 350.0 316.0 332.0 292.0 208.0 248.0 238.0 184.0 200.0 186.0 LnNo. of cells Mean Ln No. of cells Specific growth rate % Inhibition of growth rate 15.38 15.25 15.37 15.44 15.30 15.46 15.19 15.34 15.46 15.12 15.34 15.31 15.00 14.91 15.12 14.97 15.06 15.07 14.97 15.02 14.89 14.55 14.72 14.68 14.42 14.51 14.44 15.37 15.33 15.26 15.01 15.03 14.96 14.65 14.46 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.05 0.59 1.80 5.78* 5.44* 6.68* 11.63* 14.79* Ln number of cells at TO (10 000 cells) = 9.21 Rep.: replicate * Significant inhibition (relative to the control) Variation coefficient of the control cell density at 96 hours: 8.00% Company Sanitized. Does not contain TSCA CBI CIT/StudyNo. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 37 of 53 Table 14: Second test - Number of cells (104 cells x mL"1) at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 72 hours. Area = cell x mL'1 x hour Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 Rep. 1 2 3 4 5 6 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Total number of cells at 24 hours 6.50 8.60 6.40 6.30 8.10 5.60 6.70 7.20 6.60 6.30 6.50 6.80 5.90 6.80 6.40 5.50 7.00 6.60 5.50 5.20 5.60 5.70 7.10 5.90 4.60 4.50 4.00 at 48 hours 31.00 42.30 33.70 37.00 39.00 37.00 33.30 39.00 36.70 32.00 38.00 30.50 31.50 32.00 31.00 30.50 40.30 39.30 33.30 30.80 28.00 21.00 39.70 31.00 27.50 27.00 28.30 at 72 hours 115.00 114.00 154.00 127.00 129.00 133.00 121.00 129.00 141.00 130.00 179.00 126.00 145.00 134.00 148.00 133.00 131.00 135.00 129.00 138.00 143.00 117.00 120.00 123.00 104.00 113.00 103.00 Oto24 hours 66.00 91.20 64.80 63.60 85.20 55.20 68.40 74.40 67.20 63.60 66.00 69.60 58.80 69.60 64.80 54.00 72.00 67.20 54.00 50.40 55.20 56.40 73.20 58.80 43.20 42.00 36.00 Biomass 24 to 48 hours 48 to 72 hours Oto72 hours Average (0-72 hours) 426.00 586.80 457.20 495.60 541.20 487.20 456.00 530.40 495.60 435.60 510.00 423.60 424.80 441.60 424.80 408.00 543.60 526.80 441.60 408.00 379.20 296.40 537.60 418.80 361.20 354.00 363.60 1728.00 1851.60 2228.40 1944.00 1992.00 2016.00 1827.60 1992.00 2108.40 1920.00 2580.00 1854.00 2094.00 1968.00 2124.00 1938.00 2031.60 2067.60 1923.60 2001.60 2028.00 1632.00 1892.40 1824.00 1554.00 1656.00 1551.60 2220.00 2529.60 2750.40 2503.20 2618.40 2558.40 2352.00 2596.80 2671.20 2419.20 3156.00 2347.20 2577.60 2479.20 2613.60 2400.00 2647.20 2661.60 2419.20 2460.00 2462.40 1984.80 2503.20 2301.60 1958.40 2052.00 1951.20 2530.00 2540.00 2640.80 2556.80 2569.60 2447.20 2263.20 1987.20 Inhibition (%) -0.40 -4.38 -1.06 -1.57 3.27 10.55 21.45* Rep.: replicate Significant inhibition (relative to the control) Variation coefficient of the control tell density at 72 hours: 11.3% Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME - 38 of 53 Table 15: Second test - Number of cells calculation of the area under the 96 hours. Area = cell x mL x hour (104 cells x mL"1) at each observation time and growth curve (biomass) at each concentration after Nominal concentration (mg/L) 0 6.25 12.5 25.0 50.0 100 200 400 Rep. 1 2 3 4 5 6 1 2 3 1 2 3 2 3 1 2 3 2 3 1 2 3 1 2 3 Total number of cells at 24 hours at 48 hours at 72 hours at 96 hours 6.50 8.60 6.40 6.30 8.10 5.60 6.70 7.20 6.60 6.30 6.50 6.80 5.90 6.80 6.40 5.50 7.00 6.60 5.50 5.20 5.60 5.70 7.10 5.90 4.60 4.50 4.00 31.00 42.30 33.70 37.00 39.00 37.00 33.30 39,00 36.70 32.00 38.00 30.50 31.50 32.00 31.00 30.50 40.30 39.30 33.30 30.80 28.00 21.00 39.70 31.00 27.50 27.00 28.30 115.00 114.00 154.00 127.00 129.00 133.00 121.00 129.00 141.00 130.00 179.00 126.00 145.00 134.00 148.00 133.00 131.00 135.00 129.00 138.00 143.00 117.00 120.00 123.00 104.00 113.00 103.00 476.00 420.00 474.00 510.00 442.00 518.00 394.00 460.00 520.00 368.00 460.00 446.00 328.00 298.00 370.00 316.00 348.00 350.00 316.00 332.00 292.00 208.00 248.00 238.00 184.00 200.00 186.00 Oto24 hours 66.00 91.20 64.80 63.60 85.20 55.20 68.40 74.40 67.20 63.60 66.00 69.60 58.80 69.60 64.80 54.00 72.00 67.20 54.00 50.40 55.20 56.40 73.20 58.80 43.20 42.00 36.00 Biomass Inhibition (%) 24 to 48 hours 48 to 72 hours 72to96 hours Oto96 hours Average (0-96 hours) 426.00 586.80 457.20 495.60 541.20 487.20 456.00 530.40 495.60 435.60 510.00 423.60 424.80 441.60 424.80 408.00 543.60 526.80 441.60 408.00 379.20 296.40 537.60 418.80 361.20 354.00 363.60 1728.00 1851.60 2228.40 1944.00 1992.00 2016.00 1827.60 1992.00 2108.40 1920.00 2580.00 1854.00 2094.00 1968.00 2124.00 1938.00 2031.60 2067.60 1923.60 2001.60 2028.00 1632.00 1892.40 1824.00 1554.00 1656.00 1551.60 7068.00 6384.00 7512.00 7620.00 6828.00 7788.00 6156.00 7044.00 7908.00 5952.00 7644.00 6840.00 5652.00 5160.00 6192.00 5364.00 5724.00 5796.00 5316.00 5616.00 5196.00 3876.00 4392.00 4308.00 3432.00 3732.00 3444.00 9288.00 8913.60 10262.40 10123.20 9446.40 10346.40 8508.00 9640.80 10579.20 8371.20 10800.00 9187.20 8229.60 7639.20 8805.60 7764.00 8371.20 8457.60 7735.20 8076.00 7658.40 5860.80 6895.20 6609,60 5390.40 5784.00 5395.20 9730.00 9576.00 9452.80 8224.80 8197.60 7823.20 6455.20 5523.20 1.58 2.85 15.47* 15.75* 19.60* 33.66* 43.24* Rep.: replicate * Significant inhibition (relative to the control) Variation coefficient of the control cell density at 96 hours: 8.00% Company Sanitized. Does not contain TSCA CBI CIT/StudyNo. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 39 of 53 APPENDICES Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 40 of 53 1. Analytical certificate and composition of the test item Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONTUM PERFLUOROOCTANOATE (APFO)/ APME 41 of 53 Certificate of Compliance/Analysis' This is to certifythat the material shippedon this order is m conformance wna 3M specifications dated April 25,2001 with the following analysis: 3M Invoice 3M Product ID: 3M Protect Codt: DefcriptioD: Lot Nan 10004 OrderQty: 40 LBS Customer 3M/PBRFMATLSLAB MIKESIERAKOWSKI BLDG236-2A-01 ST PAUL, MN 55144 ProdnctTMt pH 2 % Aqueous Solution Solid* APHA Color boa Based on 100% Solids CurtoBtrP.OJftr; SAMPLE/SIERAKOWS =\ LotQty: 40LBS 1DRUM@40LBS Ship Date: Jan 24,2002 DatcOfMrg;Mir30,2001 SpeeUle*lioiu Loww Upper Unft Umft Tireet Limtt Result/ Range Tect Method 5.0 6.5 6.0 30.2.C % ppm 19.5 - 20.5 19.6 14.20.C 50 15 96.1.C 5.0 02 58.47.C ITU . % is/cm - % 3 3 47.5.C 0.08 0.02 71.5.I.C O.oO 0.04 71.5.1.C 1.4 0.1 300.114 96.5 99.7 300.114 Shelf Life: The shelf life of this nuteril w two yew from die date of manufacture if left in fee origin*!, unopened conteiner under recomaendedftonge conditions. . TTiesc are introductory (pecificationsbued upon limited diti md ire fubjectto chmge, Important Node* to rurcluwr m* blalalwmilinanlI*niiunddaalnInaaguJoar*rwl winnnmleitaa..ipru<orlmpil(Kt: sta^onlyoC8Jiai(hJblorwta(udii3u*niityo>lt)eproduaprevdtDbe Btos*fUtUwtl.^inueow.AUreM(nrT*(Bnn*d8rndtKorttMnbnenihleBlUthtt*MftcluuriiaU6wM^lKoi^y(<.olhfo<awperprofdo(^ldilunca^rh.rt^(flhlrliif^<r eMr(iMau(sua<qivuiau^<i<i>iiunugnoieu3lrtaArt^uainadotloabr3ta^ii^^ MfMrftt. Tti* (ongoing nay nolMdianoailaiOBct by *n*ereinnt tio id by w officw ofultw.' 3M Specialty Materials 3M Cntor BIdg. 223.6S-04 St Paul, MN 55144-1000 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 42 of 53 3M Corporate Toxicology and Regulatory Services 3M Center, Building 0220.02-E-02 St. Paul, MN 55144-1000 651 733 1773 Fax May 2,2003 _-wj|W| Dr. Jacques L'Haridon err BP563 27005 Evreux, France Dear Dr. L'Haridon: This is to confirm that the sample of ammonium to CIT, BP 563, 27005 Evreux, France (3M^ would be expected to contain apprqximate^H material (mole percentages)flfys adfsolution of ammonium perfluorooctanoate in' water^An example analysis on a batch of solid ammonium perfluorooctanoate (3M Product Codef^^B------jgave the following isomer distribution, determined using "F-NMR techniques: Sincerely, ^L^^.gS^^' John L. Butenho^ Ph-D., CIH, DABT Staff Scientist JLB/nn Company Sanitized. Does not contain TSCA CBI CIT/StudyNo: 23685 EAA/AMMONTUM PERFLUOROOCTANOATE (APFO)/ APME - 43 of 53 2. Substances required for the preparation ofLC reconstituted water Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 44 of 53 LC RECONSTITUTED WATER (as described in the French guideline T 90-304) Final Concentrations Solution No. 1: - calcium nitrate (Ca (NO^tH?) Solution No. 2: ............................................................ - potassium nitrate (KNO^) Solution No. 3: .......................................................................... - magnesium sulphate (MgSO^JH^O) ......................................................... Solution No. 4: - monohydrogen potassium phosphate (KyHPO^)........................................... Trace element solutions: Solution No. 5: - copper sulphate (CuSt^JH^O) ................................................................. - ammonium heptamolybdate [(NFL^N^O^H^O] ................................. - zinc sulphate (ZnS04.7H;>0) ...................................................................... - cobalt chloride (CoCl2.6H;>0) .................................................................... - manganese nitrate (Mn(N03)2.4H20) ........................................................ - citric acid (CeHgO^.H^O) ............................................................................ - boric acid ^3803) ................................................................................... Solution No. 6: - iron citrate (ffl) (C^tLFeC^) - iron sulphate (H) (FeSt^JH?..)..................................................................................................................................... - iron chloride (m) (FeC^ti^O) .................................................................. 40 mg/L 100 mg/L 30 mg/L 40 mg/L 15 ug/L 30 ug/L 30 ug/L 30 ug/L 30 ug/L 30 ug/L 30 ug/L 0.616 mg/L 0.3125 mg/L 0.3125 mg/L All solutions are made up with injectable grade deionized water (conductivity < Prepared solutions are kept for no more than 1 week after autoclaving. Final pH = 7.5 0.3; hardness = approximately 34 mg/L as CaCOs. 10 uScm-1). Company Sanitized. Does not contain TSCA CBI CIT/StudyNo. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 45 of 53 3. Chemical analysis of test solutions Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 46 of 53 CHEMICAL ANALYSIS Principle An aliquot of each sample was diluted and analyzed by Ion Chromatography with Electrochemical Detection (Conductimetry). The concentrations of AMMONIUM PERFLUOROOCTANOATE (APFO) were determined from a calibration curve of peak area against concentration of AMMONIUM PERFLUOROOCTANOATE (APFO) in standard solutions. Sample preparation All samples were mixed and those with algae were centrifiiged (4000 rpm, 15 min, +4C) to discard algae for analysis. If necessary, samples were then diluted with Milli-Q water to achieve concentrations in the range 1-100 mg/L of AMMONIUM PERFLUOROOCTANOATE (APFO). Chromatographic conditions Pump Mobile phase : GP 50 Gradient Pump (Dionex) : phase A: aqueous sodium hydroxide solution 50 mM* phase B: Milli-Q water phase C: acetonitrile * Aqueous sodium hydroxide solution 50 mM: 2.6 mL of sodium hydroxide solution at 46/48% (Fisher Chemicals, Ref: S/4930/05) was added to 1 L of Milli-Q Water. Time (min) 0 5 12 13 25 phase A (%) 10 40 40 10 10 phase B (%) 85 0 0 85 85 phase C (%) 5 60 60 5 5 curve 6 6 - Flow rate Precolumn Column Temperature Detector Neutralization : 1 mL/min : lonpac ATC-3, 4 mm (Dionex) : lonpac AS 16 (Dionex) length = 250 mm, inner diameter = 4 mm :30C : ED 50 Electrochemical Detector (Dionex) Conductimetry after eluent neutralization : AMMS ffl (Dionex) Company Sanitized. Does not contain TSCA CBI CIT/StudyNo. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 47 of 53 Injector : AS50 (Dionex), at 10C Injected volume : 25 uL Data acquisition software : Multichrom 2 (Fisons Instruments) Retention time Analysis time : APFO, approx. 9 min : 30 min Calibration curve Peak areas were determined for standard solutions ranging from 1 to 100 mg/L of AMMONIUM PERFLUOROOCTANOATE (APFO) (six levels). A calibration curve was obtained by plotting test item peak areas against concentrations. The regression analysis of the calibration data gave an equation of the following form: where Y=aX+b Y = test item peak area (nVs) X = concentration of test item (mg/L) a = slope value b = intercept Samples of AMMONIUM PERFLUOROOCTANOATE (APFO) were analyzed by Ion Chromatography with Electrochemical Detection (Conductimetry). One dilution was prepared for each sample and one injection (of 25-uL aliquots) was performed for each final dilution. The peak area was determined for each sample. The concentration of AMMONIUM PERFLUOROOCTANOATE (APFO) in each sample was calculated using the equation obtained from the calibration data. All the results are expressed as mg/L of APFO. Company Sanitized. Does not contain TSCA CBI CIT/Study N6;23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME - 48 of 53 Table A: Concentration of AMMONIUM PERFLUOROOCTANOATE (APFO) in the test solutions of the second test (mg/L) Nominal 6.25 12.5 25.0 50.0 100 200 400 0 hour without algae 5.73 10.8 22.4 45.8 96.7 179 364 (92) (86) (90) (92) (97) (90) (91) Measured (1) 96 hours with algae without algae 6.03 12.7 24.3 47.3 95.9 180 362 (96) (102) (97) (95) (96) (90) (91) 5.53 10.6 21.4 45.9 95.0 183 383 (88) (85) (86) (92) (95) (92) (96) (I): numbers in brackets represent percentages of the nominal concentration Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 49 of 53 VALIDATION OF THE ANALYTICAL METHOD The validation of the analytical method was performed according to CIT Standard Operating Procedures. The specificity, limit of quantification, linearity, repeatability of injections, accuracy and precision (Coefficient of Variation: CV %) of the analytical method were determined. Specificity The specificity of the analytical method was demonstrated as follows: . analysis of a standard solution of AMMONIUM PERFLUOROOCTANOATE (APFO) in Milli-Q water, . analysis of Milli-Q water or test water (LC, M4 or dechlorinated: desionised water) without dilution, No relevant interference between the test item peak and Milli-Q water or test water was observed on chromatograms. Limit of quantification The limit of quantification of the analytical method was established as 1 mg/L for a standard solution of APFO. This limit corresponds to a limit of quantification of 1 mg/L for the test item in aqueous phase. Linearity Linearity was checked by analysis of three different sets of six standard solutions containing 1, 5, 10, 20, 50 and 100 mg/L of AMMONIUM PERFLUOROOCTANOATE (APFO) in Milli-Q water. Satisfactory linearity was demonstrated in the range 1 to 100 mg/L since the coefficients of determination obtained were higher than 0.999. Repeatability of injections Replicate analysis (n = 10) of a solution containing 50 results since the coefficients of variation values obtained . 1% based on peak height, . 1% based on peak area. Based on these results, the repeatability of injections taking into account peak area. mg/L of the test were as follows: of the analytical item gave satisfactory method was validated Comp'any Sanitized. Does not contain TSCA CBI CIT/StudyNo. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 50 of 53 Accuracy and precision Six analyses of solutions containing 2.03 and 50.7 mg/L of the test item in Milli-Q water were carried out. The accuracy and the precision (CV%) obtained were as follows: Concentration (mg/L) Dilution CV Accuracy Nominal 2.03 50.7 Mean measured*_____factor_____(%)_______(%) 2.02 1 6 99 53.7 1 2 106 *: mean values of six determinations Conclusion The analytical method was validated and considered to be suitable for the analysis of the samples of the study. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 51 of 53 4. Comparison of areas under the growth curves Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 52 of 53 COMPARISON OF AREAS UNDER THE GROWTH CURVES (Biomass) The areas between the growth curves and the horizontal line N = N9 were calculated according to the formula: Ni - No Ni + N2 - 2No 2 2 A= ---------------- Xtl+ -------------------------- where, A = area X (t2 - tl) + + ... N()= number ofcells/mL at time tp (beginning of the test), Ni = measured number ofcells/mL at time t) Nn= measured number ofcells/mL at time t,, t, = time in hours of first measurement after beginning of test tn = time in hours ofn* measurement after beginning of test n = number of measurements taken after the beginning of the Nn + Nn-i - 2No ------------------------------ 2 test. X(tn-tn.l) The percentage inhibition of the cell growth at each test item concentration (1^) was calculated according to the formula: Ac-A. IA= ___Ac__- xlOO where, A(; = area between the control growth curve and the horizontal line N = No A( = area between the growth curve at the concentration t and the horizontal line N = Ng IA values are plotted on semilogarithmic paper or on semilogarithmic probit paper against the corresponding concentrations. If plotted on probit paper, the points are fitted by a straight line, either by eye or by a computed regression. The EC50 is estimated from the regression line by reading off the concentration that is equivalent to a 50% inhibition (IA = 50%). To denote this value unambiguously in relation to this method of calculation, it is proposed to use the symbol E^o is quoted with the appropriate exposure period, e.g. EbCso(0-96h). Company Sanitized. Does not contain TSCA CBI CIT/StudyNo. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 53 of 53 COMPARISON OF GROWTH RATES The average specific growth rate (u) for exponentially growing cultures was calculated as: Ln N,, - Ln No H= tn-to where to is the time at the beginning of the test and t^ the time in hours ofv* measurement after beginning of test. The percentage inhibition of specific growth rate at each test item concentration (It) was calculated according to the formula: "c-Hi I^= ______xlOO He where, He= mean control specific growth rate Ui = mean specific growth rate for the test concentration t The percentage reduction of average specific growth rate at each test item concentration compared with the control value is plotted against the logarithm of the concentration. The EC50 may be red from the resulting graph. To denote unambiguously the EC50 derived by this method it is proposed to use the symbol ErCso. The times of measurement must be indicated, e.g. if the value relates to times 0 and 96 hours, the symbol becomes-E,C5o(0-96h). Note: specific growth rate is a logarithmic term and small changes in growth rate may lead to great changes in biomass. E^C and E^C values are therefore not numerically comparable. CALCULATION OF THE NOEC The No Observed Effect Concentration was determined by first checking for normality (i.e. Chi-square test or Shapiro-Wilks test) as well as variance homogeneity (i.e. Bartlett test) and then by analysis of variance (i.e. Bonferroni T-test), using the individual replicate values of the areas under the growth curves or the specific growth rates. Company Sanitized. Does not contain TSCA CBI