Document ykXXMD0y7y6nOnVJ2QYmnwvXD

AR226-2815 SPONSOR APME Avenue E. van Niewenhuyse 4 BOX 3 B-l 160 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 LUaridon EXPERIMENTAL COMPLETIONDATE 7 March 2003 DATE OFISSUE J<G <eo(^ TEST FACILITY CIT BP 563 - 27005 Evreux - France LABORATORYSTUDY NUMBER 23685 EAA IFM Recherche S.N.C. au Capital de 846.092 7 0 8 0 6 0 46 2 R.C.S. Ev.au* VAT: FRI 9 788 060 465 Agrment Crdit Impt Recherche Page 1 of 53 Safety & Health Research Laboratories B.P. 563 27005 Evreux Cedex Tl. : +33 {0)2 32 29 26 26 Fax : +33 (0)2 32 67 87 05 E-mail : QT@cHox.com Web site : www.citox.com France Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 2 o f 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 o f 53 STATEMENT OF TTTF STTIDV 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. . Dcret N 98-1312 du 31 dcembre 1998 concernant les Bonnes Pratiques de Laboratoire, (Journal Officiel du 1erjanvier 1999) Ministre de l'Economie, des Finances et de l'Industrie. . Commission Directive 1999/11/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 harmonisation o f 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 o f International Trade and Industry, Good Laboratory Practice Standards, Basic Industries Bureau, KanHogyo No. 39, March 31,1984. . Japanese Ministry o f 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: Doctor of Ecotoxicology Study Director JfivliAU. R. Groult Date: Doctor of Chemistry Scientific Management OTHER SCIENTIST INVOLVED IN THIS STUDY For Pharmacy: X. Monciaux Doctor of Pharmacy Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 4 o f 53 STATEMENT OF QUALITY ASSURANCE UNIT Type of inspections Study plan Study Report Inspections 24 July 2002 14 October 2002 30 July 2003 Dates Reported to Study Director (*) 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 C1T Management. The inspections were performed in compliance with CIT 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: J C Jan. #oC>^ Ing. Biol. Head of Quality Assurance Unit (*) 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 5 o f 53 PAGE RESERVED FO R 40 CFR 158.34 (c) (1) CERTIFICATION (To be completed by the Sponsor) Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 6 o f 53 CONTENTS STATEMENT OF CONFIDENTIALITY OR NO CONFIDENTIALITY CLAIMS STATEMENT OF THE STUDY DIRECTOR AND CIT SCIENTIFIC MANAGEMENT OTHER SCIENTIST INVOLVED IN THIS STUDY STATEMENT OF QUALITY ASSURANCE UNIT PAGE RESERVED FOR 40 CFR 158.34 (c) (1) CERTIFICATION SUMMARY 1. INTRODUCTION 2. 2.1 2.1.1 2.1.2 2.2 2.2.1 2.2.2 2.2.3 2.3 2.3.1 2.3.2 2.4 2.5 2.5.1 2.5.2 2.6 2.6.1 2.6.2 2.7 2.8 MATERIALS AND METHODS TEST ITEM Identification Preparation of the test solutions TEST SYSTEM Algae Environmental conditions during culture Environmental conditions during the test TREATMENT Study design Time schedule OBSERVATIONS CHEMICAL ANALYSIS Range-finding test First and second tests DATA EVALUATION Determination o f the ErC50 and the EbC50 Determination o f the No Observed Effect Concentration (NOEC) ARCHIVING STUDY PLAN ADHERENCE - 2 3 3 4 5 9 13 13 13 13 14 15 15 15 16 17 17 17 17 18 18 18 18 18 19 19 19 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 7 o f 53 3. RESULTS 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. 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 - 20 20 20 20 20 20 20 21 21 21 21 22 23 5. REFERENCES Table 1: Range-finding test - temperature and pH Table 2: Range-finding test - T72 hours - number o f cells and calculation o f the specific growth rate at each concentration Table 3: Range-finding test - T96 hours - number of cells and calculation of the specific growth rate at each concentration Table 4: 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'1x hour Table 5: Range-finding test - Number of cells (104 cells x m L'1) at each observation time and calculation o f the area under the growth curve (biomass) at each concentration after 96 hours. Area = cell x mL'1x hour Table 6: First test - temperature and pH Table 7: First test - T72 hours - number o f cells and calculation of the specific growth rate at each concentration Table 8: First test - T96 hours - number o f cells and calculation o f the specific growth rate at each concentration Table 9: First test - Number o f cells (104 cells x mL'1) at each observation time and calculation o f the area under the growth curve (biomass) at each concentration after 72 hours. Area = cell x mL' 1x hour Table 10: First test - Number o f cells (104 cells x m L'1) at each observation time and calculation o f the area under die growth curve (biomass) at each concentration after 96 hours. Area = cell x mL'1x hour - Table 11: Second test - temperature and pH 23 24 25 26 27 28 29 30 31 32 33 34 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 8 o f 53 Table 12: Second test - T72 hours - number of cells and calculation o f the specific growth rate at each concentration Table 13: Second test - T96 hours - number of cells and calculation o f the specific growth rate at each concentration Table 14: Second test - Number of cells (104 cells x mL'1) at each observation time and calculation o f the area under the growth curve (biomass) at each concentration after 72 hours. Area = cell x mL'1x hour Table 15: Second test - Number of cells (104 cells x m L 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'1x hour 35 36 37 38 APPENDICES 1. Analytical certificate and composition o f the test item 2. Substances required for the preparation o f LC reconstituted water 3. Chemical analysis of test solutions 4. Comparison o f 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 o f 53 SUMMARY At the request o f 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 ,31st July 1992, . OECD Guideline No. 2 0 1 ,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 o f the Environmental Agency, No. 1039 o f the Ministry of Health and Welfare and No. 1014 o f Ministry o f 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 o f 50% in comparison with the control. This criterion is evaluated as: . Die ErC50 - the concentration of test item resulting in 50% reduction o f the specific growth rate with respect to the control. Specific growth rate can be defined as the rate of increase in the natural log o f 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 CaC0 3 . The study included three tests. Ranee-finding test Three replicate solutions o f algae with an initial dilution o f 1 x 104 cells/mL were exposed to a nominal concentration o f 100 mg/L expressed as the APFO (solids) content o f the test item, while four other groups o f two replicate flasks were exposed to nominal concentrations o f 0.01, 0.1, 1 and 10 mg/L expressed as the APFO (solids) content o f the test item. A further group of six replicate algal solutions without test item was used as the control. The number of replicates o f 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 o f 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 o f 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 o f 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 o f cell growth were recorded at 24, 48, 72 and 96 hours. Second test A second test was undertaken since the results o f the range-finding test were not in accordance with those o f the first test. This second test was considered as the reference test for determination o f all toxicity parameters. Seven concentrations o f 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 o f 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 o f the second test, except for the control, at the beginning (TO hour) and the end (T96 hours) o f the test. Results Ranee-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 o f 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 CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 11 o f 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 o f 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 o f the concentration-effect curve may have prevented determination o f 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 o f 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 o f AMMONIUM PERFLUOROOCTANOATE (APFO) in test solutions. Measured concentrations in the solutions o f the second test with and without algae were within 20% o f the corresponding nominal values at the beginning (TO hour) and the end (T96 hours) o f the test. Hence, the test item was stable in solution throughout the test and was not significantly bioaccumulated or adsorbed at the surface o f 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 o f the test item EbC50 >400 >400 NOEC 200 12.5 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONUM PERFLUOROOCTANOATE (APFO)/ APME 12 o f 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 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. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 13 o f 53 1. INTRODUCTION The. objective o f this study was to assess the acute toxicity o f AMMONIUM PERFLUOROOCTANOATE (APFO), to Pseudokirchneriella subcapitata, in a 96-hour static test based on: . Directive 92/69/EEC C.3, 31st July 1992, . OECD Guideline No. 201, 7th June 1984, . US EPA/OPPTS 850.5400 Guidelines, April 1996, . Requirements o f the Japanese Government under the revised Chemical Substance Law according to the Notification No. 700 of the Environmental Agency, No. 1039 o f the Ministry of Health and Welfare and No. 1014 of Ministry o f International Trade and Industry, 9th December 1986. The criterion measured is the EC50 (Median Effective Concentration), a statistically derived concentration o f the test item in water which can be expected to cause a reduction of algal growth o f 50% in comparison with the control. This criterion is evaluated as: . The ErC50 - the concentration of test item resulting in 50% reduction o f the specific growth rate with respect to the control. . The EbC50 - the concentration of test item resulting in 50% reduction o f 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 plmi^MM QhD PERFLUOROOCTANOATE (APFO) - lab eiin g ^ H m H | Both namesconcgponcHp Be same test item. . batch n u m b e rp B H 0 l . description at receipt: colorless liquid , . description on the analytical certificate: . containers: three drums . date o f receipt: 28 Januaiy 2002 . storage conditions: at room temperature . purity] Data relating to the characterisation of the test item are documented in an analytical certificate and in the composition o f the test item (presented in appendix 1) provided by the Sponsor. The test concentrations were expressed as concentrations ofAPFO , based on the stated APFO content of the test iteml Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 14 o f 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 o f preparation o f the stock solutions are reported in the following table: Range-finding test Quantity of test item (as received) (m g) 510.2 Quantity of APFO (solids) (mg) 100 First test 2041 400 Second test 2041 400 (1) expressed as the APFO (solids) content of the test item Concentration of the test item (mg/L) (1) Duration of the agitation (minutes) 100 10 400 15 400 15 The agitation was continued until the stock solutions were used to prepare the test solutions. Test solutions were prepared by further dilution o f the stock solution with LC reconstituted water to provide a geometric series o f concentrations (expressed as the APFO (solids) content o f 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 Erlenmeyer 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 o f 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 o f pH before addition o f the algae. For the test solution at 100 mg/L in the range-finding test and all test solutions o f the first and second tests (except for the controls), a further group o f 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 o f 53 2.2 TEST SYSTEM 2.2.1 Algae Species: Pseudokirchneriella 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: CIT. Origin: Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Far Sawrey, Ambleside, Cumbria, LA22 OLP, U K 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 o f 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 o f our laboratory. Culture period: algae are cultivated for 7 days before harvesting for use in a new culture. Temperature: between 21C and 25C in water. Algae are maintained at 2C during the acclimation period. Temperature is checked daily. Illumination: 24 hours per day o f constant illumination maintained at approximately 2000 lux throughout the culture period. For lighting details see 2.2.3. Aeration: the cultures are constantly aerated with air filtered using a 0.22 pm porosity filter to maximize the CO2 availability (and thereby the cell growth) while maintaining the sterility. Preculture loading: new cultures are loaded at a concentration o f 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 o f 53 2.2.3 Environmental conditions during the test Test water: reconstituted-LC (see 2.2.2). Temperature: controlled daily in a flask containing test water but no algae, run alongside the test. Between 21C and 25C (regulated to be as close as possible to 23C at the beginning of the test). Illumination: continuous. A set o f 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 die height o f the cultures in their conical flasks (corresponding to an irradiance o f 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. Duration of test: 96 hours. Transfer: the number of cells in the week-old culture was counted and the quantity o f algal pre-culture to be added to the fresh test medium calculated to give a test solution concentration o f 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. Culture homogeneity: the solutions were agitated throughout the test. pH: 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 o f the tests and after 72 and 96 hours. Hardness: water hardness was measured once in LC reconstituted water before the start of the test. Forced aeration: 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 o f 53 2.3 TREA TM EN T 2.3.1 Study design The study included three tests. The duration of each test was 96 hours. Ranee-finding test Nominal concentration ' (mg/L) (1) Control Treated Treated Treated Treated Treated 0 0.01 0.1 1 10 100 (1) expressed as the APFO (solids) content of the test item Number of replicates with algae without algae 60 20 20 20 20 31 The number o f 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 Nominal concentration Number of replicates _____ (mg/L) (1)___________with algae_________ without algae Group 1 (control) Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 0 6.25 12.5 25.0 50.0 100 200 400 6 3 3 3 3 3 3 3 0 1 1 1 1 1 1 1 (1) expressed as the APFO (solids) content of the test item. 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 lO^cells/mL. For the remaining observation times (T24, T48, T72 and T96 hours), cell numbers were determined using a Malassez cell counter. An observation o f less than 1 x 104 cells/mL is below the sensitivity o f 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 o f 53 2.5 CHEM ICAL 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 o f adsorption (at the surface o f 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, 172 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 o f 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) o f 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 o f solution x time in hours and therefore represents an area (cells.mL`l.h). 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 o f biomass at 72 or 96 hours to 50% that o f 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 o f 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 o f 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 o f 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 o f the in vivo phase o f the study: . Study plan and possible amendments, . raw data, . correspondence, . final report and possible amendments. On completion o f this period, the archived study materials will be returned to the Sponsor, or may be archived at CIT for a farther period (at additional cost). . The total duration of archiving (depending on regulations) will be the responsibility o f 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 PLA N 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 o f 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 o f 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 fbrther 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 o f 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 o f 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 o f 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 o f the control at 100 mg/L (highest concentration tested) while there was evidence o f 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 o f 53 3.2.2 Chemical analysis No chemical analysis was performed since this first test was not retained for determination o f 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 o f 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 o f the concentration-effect curve may have prevented determination o f precise NOECs. The results were not considered conclusive and, consequently, a second test was performed to determine whether the results o f 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 o f 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 o f 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 o f 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 o f growth rate at 400 mg/L was once again low after 96 hours (15%). After 72 hours, the biomass was significantly lower than that o f 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 o f 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 o f 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 o f 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) o f the test. Hence, the test item was stable in solution throughout the test and was not significantly bioaccumulated or adsorbed at the surface o f 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 o f 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) 24 >400 48 > 400 72 > 400 96 > 400 (1) expressed as the APFO (solids) content of the test item 95% confidence limits (mg/L) Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 23 o f 53 The 72-hour EbC50 and 96-hour EbC50 were as follows: Time (h) EbC50 (mg/L) (1) 72 > 400 96 > 400 (1) expressed as the APFO (solids) content of the test item 95% confidence limits (mg/L) The NOEC at 72 hours was calculated as 200 mg/L (p = 0.05) based on the specific growth rate and biomass data. The NOEC at 96 hours was calculated as 12.5 mg/L (p = 0.05) based on the specific growth rate and biomass data. 4. CONCLUSION Under our experimental conditions, the 72-hour ErC50 and the 72-hour EbC50 o f 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 o f dosage-mortality curves from small number. Quart. J. Pharm. U , 192-216(1938). Fieller: A fundamental formula in the statistics o f biological assay and some applications. Quarterly Journal of Pharmacy and Pharmacology, 117-123 (1944). Weber, E: Grnden der biologischen Statistik, Gustav Fisher Verlag, Stuttgart, 1972. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 24 o f 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 o f 53 Table 2: Range-finding test - T72 hours - number o f cells and calculation o f the specific growth rate at each concentration Nominal concentration (mg/L) Rep. Total No. o f cells at 72 hours LnNo. of cells % Inhibition Mean Ln Specific o f growth No. o f cells growth rate rate (104/mL) 0 1 133.0 14.10 14.21 0.07 2 154.0 14.25 3 157.0 14.27 4 150.0 14.22 5# - 6# - 0.01 1 163.0 14.30 14.35 0.07 -2.84 2 179.0 14.40 0.1 1 168.0 14.33 14.27 0.07 -1.24 2 148.0 14.21 1 1 135.0 14.12 14.20 0.07 0.23 2 159.0 14.28 10 1 170.0 14.35 14.22 0.07 -0.14 2 131.0 14.09 100 1 121.0 14.01 14.01 0.07 3.97 2 122.0 14.01 3# - Ln number o f 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 o f 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 o f 53 Table 3: Range-finding test - T96 hours - number of cells and calculation o f the specific growth rate at each concentration Total No. Nominal concentration (mg/L) Rep. % Inhibition of cells at LnNo. Mean Ln Specific o f growth 96 hours o f cells No. of cells growth rate rate (104/mL) 0 1 338.0 15.03 15.11 0.06 2 396.0 15.19 3 336.0 15.03 4 388.0 15.17 . 5# - 6# - 0.01 1 364.0 15.11 15.12 0.06 -0.16 2 370.0 15.12 0.1 1 334.0 15.02 15.00 0.06 1.85 2 318.0 14.97 1 1 338.0 15.03 15.06 0.06 0.70 2 360.0 15.10 10 1 368.0 15.12 15.08 0.06 0.51 2 338.0 15.03 100 1 346.0 15.06 15.02 0.06 1.39 2 324.0 14.99 3# - Ln number o f 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 o f 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 o f 53 Table 4: Range-finding test - Number o f 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 m L'1x hour Total number of cells Nominal concentration Rep. (mg/L) 01 2 3 4 5# 6# 0.01 1 2 0.1 1 2 11 2 10 1 2 100 1 2 3# 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 - Biomass Oto 24 24 to 48 48 to 72 Oto 72 Average hours hours hours hours (0-72 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 2491.20 3021.60 2630.40 2875.20 2754.60 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 3103.20 2580.00 3108.00 2403.60 2870.40 2118.00 2589.60 1932.00 2340.00 2220.00 2604.00 2379.60 2808.00 1860.00 2265.60 1677.60 1936.80 1663.20 1886.40 3105.60 2730.00 2472.00 2536.80 1911.60 Inhibition (%) -12.74 0.89 10.26 7.91 30.60 R ep.: 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 o f other replicate values. Variation coefficient o f 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 o f 53 Table 5: Range-finding test - Number o f cells (104 cells x mL'1) at each observation time and calculation o f the area under the growth curve (biomass) at each concentration after 96 hours. Area = cell x m L'1x hour Total numb' of cells Nominal concentration Rep. (mg/L) 01 2 3 4 5# 6# 0.01 1 2 0.1 1 2 11 2 10 1 2 100 1 2 3# 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 0 to 2 4 24 to 48 48 to 72 72 to 96 0 to 9 6 Average hours hours hours houts hours (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 Inhibition (%) -7.33 4.19 5.48 3.73 17.12 Rep. : replicate U Values obtained in two control replicates (Nos. 5 and 6) and one replicate at 100 rag/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 o f other replicate values. Variation coefficient o f 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 29 o f 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 CBI 9 CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 30 o f 53 Table 7: First test - T72 hours - number of cells and calculation of the specific growth rate at each concentration Total No. Nominal concentration (mg/L) Rep. o f cells at 72 hours LnNo. o f cells % Inhibition Mean Ln Specific of growth No. of cells growth rate rate (104/mL) 0 1 189.0 14.45 14.58 0.07 2 235.0 14.67 3 205.0 14.53 4 230.0 14.65 5 242.0 14.70 6 197.0 14.49 6.25 1 223.0 14.62 14.66 0.08 -1.42 2 220.0 14.60 3 256.0 14.76 12.5 1 233.0 14.66 14.65 0.08 -1.32 2 208.0 14.55 3 255.0 14.75 25.0 1 241.0 14.69 14.62 0.08 -0.69 2 204.0 14.53 3 227.0 14.63 50.0 1 240.0 14.69 14.72 0.08 -2.51 2 289.0 14.88 3 216.0 14.59 100 1 239.0 14.69 14.65 0.08 -1.21 2 234.0 14.67 3 217.0 14.59 200 1 222.0 14.61 14.58 0.07 0.04 2# - 3 208.0 14.55 400 1 183.0 14.42 14.29 0.07 5.42* 2 139.0 14.14 3 164.0 14.31 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 o f 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 o f 53 Table 8: First test - T96 hours - number o f cells and calculation o f the specific growth rate at each concentration Total No. Nominal concentration (mg/L) Rep. o f cells at LnNo. MeanLn Specific % Inhibition 96 hours o f cells No. of cells growth rate o f growth rate (104/mL) 0 1 379.0 15.15 15.28 0.06 2 470.0 15.36 3 432.0 15.28 4 418.0 15.25 5 466.0 15.35 6 436.0 15.29 6.25 1 408.0 15.22 15.21 0.06 1.19 2 422.0 15.26 3 378.0 15.14 12.5 1 396.0 15.19 15.12 0.06 2.70* 2 322.0 14.98 3 388.0 15.17 25.0 1 404.0 15.21 15.06 0.06 3.56* 2 308.0 14.94 3 340.0 15.04 50.0 1 364.0 15.11 15.01 0.06 4.37* 2 288.0 14.87 3 348.0 15.06 100 1 334.0 15.02 15.04 0.06 3.92* 2 324.0 14.99 3 366.0 15.11 200 1 270.0 14.81 14.82 0.06 7.64* 2# - 3 274.0 14.82 400 1 183.0 14.42 14.42 0.05 14.20* 2 185.0 14.43 3 180.0 14.40 Ln number o f 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 o f 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 o f 53 Table 9: First test - Number o f cells (104 cells x mL'1) at each observation time and calculation o f the area under the growth curve (biomass) at each concentration after 72 hours. Area = cell x mL'1x hour Nominal concentration Rep (mg/L) 01 2 3 4 5 6 6.25 1 2 3 12.5 1 2 3 25.0 1 2 3 50.0 1 2 3 100 1 2 3 200 1 2 3 400 1 2 3 Total number o f cells Biomass 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 at 72 0 to 24 24 to 48 48 to 72 0 to 7 2 Average houis houis hours hours hours (0-72 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 - 38.00 46.30 35.70 33.30 189.00 235.00 205.00 230.00 242.00 197.00 223.00 220.00 256.00 233.00 208.00 255.00 241.00 204.00 227.00 240.00 289.00 216.00 239.00 234.00 217.00 222.00 - 208.00 183.00 139.00 164.00 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 32.40 20.40 13.20 16.80 313.20 434.40 404.40 451.20 511.20 567.60 490.80 432.00 522.00 487.20 556.80 424.80 627.60 488.40 436.80 484.80 429.60 474.00 528.00 480.00 495.60 517.20 2556.00 2882.40 3207.60 3676.80 2793.60 3256.80 3152.40 3650.40 3356.40 3914.40 2888.40 3487.20 3123.60 3645.60 3020.40 3492.00 3536.40 4104.00 3236.40 3758.40 2996.40 3597.60 3435.60 3897.60 3474.00 4135.20 2888.40 3412.80 3111.60 3585.60 3312.00 3837.60 3867.60 4315.20 3024.00 3528.00 3351.60 3912.00 3236.40 3756.00 3060.00 3583.20 3140.40 3686.40 476.40 564.00 429.60 404.40 2928.00 2727.60 2072.40 2343.60 3436.80 3312.00 2515.20 2764.80 3478.00 3747.20 3751.20 3711.20 3893.60 3750.40 3561.60 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 va!ue(s) obtained after 72 and/or 96 hours was/werc at least 3 times lower than the mean o f other replicate values. * Significant inhibition (relative to die control) Variation coefficient o f 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 o f 53 Table 10: First test - Number o f cells (104 cells x mL'1) at each observation time and calculation o f the area under the growth curve (biomass) at each concentration after 96 hours. Area = cell x mL'1x hour Nominal concentration Rep. (mg/L) 01 2 3 4 5 6 6.25 1 2 3 12.5 1 2 3 25.0 1 2 3 50.0 1 2 3 100 1 2 3 200 1 2# 3 400 i 2 3 Total number o f 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 at 72 at 96 hours hours 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 41.70 189.00 235.00 205.00 230.00 242.00 197.00 223.00 220.00 256.00 233.00 208.00 255.00 241.00 204.00 227.00 240.00 289.00 216.00 239.00 234.00 217.00 222.00 379.00 470.00 432.00 418.00 466.00 436.00 408.00 422.00 378.00 396.00 322.00 388.00 404.00 308.00 340.00 364.00 288.00 348.00 334.00 324.00 366.00 270.00 3.70 38.00 208.00 274.00 2.70 46.30 183.00 183.00 2.10 35.70 139.00 185.00 2.40 33.30 164.00 180.00 0 to 2 4 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 32.40 20.40 13.20 16.80 Biomass 24 to 48 48 to 72 72 to 96 hours hours hours 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 5880.00 476.40 564.00 429.60 404.40 2928.00 2727.60 2072.40 2343.60 5760.00 4368.00 3864.00 4104.00 Inhibition (%) 0 to 96 Average hours (0-96 hours) 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* 9196.80 7680.00 6379.20 6868.80 6976.00 38.00* Rep.: replicate U 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 o f other replicate values. * Significant inhibition (relative to the control) Variation coefficient o f 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 o f 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/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 35 o f 53 Table 12: Second test - T72 hours - number of cells and calculation o f the specific growth rate at each concentration Total No. Nominal concentration (mg/L) Rep. o f cells at 72 hours % Inhibition LnNo. Mean Ln Specific o f growth of cells No. of cells growth rate rate (104/mL) 0 1 115.0 13.95 14.06 0.07 2 114.0 13.95 3 154.0 14.25 4 127.0 14.05 5 129.0 14.07 6 133.0 14.10 6.25 1 121.0 14.01 14.08 0.07 -0.33 2 129.0 14.07 3 141.0 14.16 12.5 1 130.0 14.08 14.17 0.07 -2.30 2 179.0 14.40 3 126.0 14.05 25.0 1 145.0 14.19 14.17 0.07 -2.17 2 134.0 14.11 3 148.0 14.21 50.0 1 133.0 14.10 14.10 0.07 -0.79 2 131.0 14.09 3 135.0 14.12 100 1 129.0 14.07 14.13 0.07 -1.33 2 138.0 14.14 3 143.0 14.17 200 1 117.0 13.97 14.00 0.07 1.34 2 120.0 14.00 3 123.0 14.02 400 1 104.0 13.85 13.88 0.06 3.78* 2 113.0 13.94 3 103.0 13.84 Ln number o f cells at TO (10 000 cells) = 9.21 R ep .: 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/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 36 o f 53 Table 13: Second test - T96 hours - number of cells and calculation of the specific growth rate at each concentration Nominal concentration (mg/L) Rep. Total No. o f cells at 96 hours LnNo. of cells % Inhibition Mean Ln Specific o f growth No. of cells growth rate rate (104/mL) 0 1 476.0 15.38 15.37 0.06 2 420.0 15.25 3 474.0 15.37 4 510.0 15.44 5 442.0 15.30 6 518.0 15.46 6.25 1 394.0 15.19 15.33 0.06 0.59 2 460.0 15.34 3 520.0 15.46 12.5 1 368.0 15.12 15.26 0.06 1.80 2 460.0 15.34 3 446.0 15.31 25.0 1 328.0 15.00 15.01 0.06 5.78* 2 298.0 14.91 3 370.0 15.12 50.0 1 316.0 14.97 15.03 0.06 5.44* 2 348.0 15.06 3 350.0 15.07 100 1 316.0 14.97 14.96 0.06 6.68* 2 332.0 15.02 3 292.0 14.89 200 1 208.0 14.55 14.65 0.06 11.63* 2 248.0 14.72 3 238.0 14.68 400 1 184.0 14.42 14.46 0.05 14.79* 2 200.0 14.51 3 186.0 14.44 Ln number o f cells at TO (10 000 cells) = 9.21 R ep .: 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/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 37 o f 53 Table 14: Second test - Number of cells (104 cells x m L'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'1x 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 0 to 24 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 48 to 72 0 to 72 Average hours hours hours (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 2220.00 1851.60 2529.60 2228.40 2750.40 1944.00 2503.20 1992.00 2618.40 2016.00 2558.40 1827.60 2352.00 1992.00 2596.80 2108.40 2671.20 1920.00 2419.20 2580.00 3156.00 1854.00 2347.20 2094.00 2577.60 1968.00 2479.20 2124.00 2613.60 1938.00 2400.00 2031.60 2647.20 2067.60 2661.60 1923.60 2419.20 2001.60 2460.00 2028.00 2462.40 1632.00 1984.80 1892.40 2503.20 1824.00 2301.60 1554.00 1958.40 1656.00 2052.00 1551.60 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* R ep.: replicate * Significant inhibition (relative to the control) Variation coefficient o f the control cell density at 72 hours: 11.3% Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME ....... 38 o f 53 Table 15: Second test - Number of cells (104 cells x m l/1) at each observation time and calculation of the area under the growth curve (biomass) at each concentration after 96 hours. Area = cell x m l/1x hour Nominal concentration Rep. (mg/L) 0i 2 3 4 5 6 6.25 1 2 3 12.5 1 2 3 25.0 1 2 3 50.0 1 2 3 100 1 2 3 200 1 2 3 400 1 2 3 Total number of cells at 24 hours at 48 hours at 72 hours at 96 hours 6.50 31.00 115.00 476.00 8.60 42.30 114.00 420.00 6.40 33.70 154.00 474.00 6.30 37.00 127.00 510.00 8.10 39.00 129.00 442.00 5.60 37.00 133.00 518.00 6.70 33.30 121.00 394.00 7.20 39.00 129.00 460.00 6.60 36.70 141.00 520.00 6.30 32.00 130.00 368.00 6.50 38.00 179.00 460.00 6.80 30.50 126.00 446.00 5.90 31.50 145.00 328.00 6.80 32.00 134.00 298.00 6.40 31.00 148.00 370.00 5.50 30.50 133.00 316.00 7.00 40.30 131.00 348.00 6.60 39.30 135.00 350.00 5.50 33.30 129.00 316.00 5.20 30.80 138.00 332.00 5.60 28.00 143.00 292.00 5.70 21.00 117.00 208.00 7.10 39.70 120.00 248.00 5.90 31.00 123.00 238.00 4.60 27.50 104.00 184.00 4.50 27.00 113.00 200.00 4.00 28.30 103.00 186.00 Biomass Inhibition (%) 0 to 24 24 to 48 48 to 72 72 to 96 0 to 96 Average hours hours hours hours hours (0-96 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 36.00 426.00 586.80 457.20 495.60 541.20 487.20 456.00 530.40 495.60 435.60 510.00 423.60 424.80 441.60 424.80 408.00 543.60 526.80 441.60 408.00 379.20 296.40 537.60 418.80 361.20 354.00 363.60 1728.00 7068.00 1851.60 6384.00 2228.40 7512.00 1944.00 7620.00 1992.00 6828.00 2016.00 7788.00 1827.60 6156.00 1992.00 7044.00 2108.40 7908.00 1920.00 5952.00 2580.00 7644.00 1854.00 6840.00 2094.00 5652.00 1968.00 5160.00 2124.00 6192.00 1938.00 5364.00 2031.60 5724.00 2067.60 5796.00 1923.60 5316.00 2001.60 5616.00 2028.00 5196.00 1632.00 3876.00 1892.40 4392.00 1824.00 4308.00 1554.00 3432.00 1656.00 3732.00 1551.60 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/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 39 o f 53 APPENDICES # Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 40 o f 53 1. Analytical certificate and composition of the test item Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 41 o f 53 Certificate of Compliance/Analysis T his is to certify that the material shipped on this order is in conform ance with 3M sp ecification s dated April 2 5 ,2 0 0 1 with die follow in g analysis: 3M Invoice Nb 3M Product ID: 3M Product D escription: LotNbn 10004 O rder Q ty: 40 LBS Costom en 3M/ PERF MATLS LAB MIKE SIERAKOWSKI BLDG236-2A-01 ST PAUL, MN 55144 Product Test pH 2 % Aqueous Solution Solids APHA Color Iron Based on 100% Solids Customer P.O JIbn SAMPLE/SIERAKOWS Lot Qty: 40 LBS 1DRUM @ 40 LBS Ship Date:Jan 24,2002 Date O fM fg:M ir 30,2001 Specification! Lower Unit Lim it T ercet Upper L im it R esu lt/ Ranae T aff M ethod 5.0 - 6.5 6.0 30.2.C % 19.5 ppm 20.5 19.6 14.20.C 50 15 96.1.C 5.0 0 2 58.47.C 3 3 47.5.C 0.08 0.02 71.5.1.C 0.60 0.04 71.5,1,C 1.4 0.1 300.114 99.7 300.114 ShelfLife: The shelflife of this materisl is two yean from the date of manufacture if left in fee original, unopened . container under recommended storage conditions. These are introductory specifications based upon limited data tnd are subject to change. Important NoUaoto PurdUMR T h e feSewlns la made In tau ofae to m m ie s , e s p rits er biased: s o * only o M p a fa ish lb sio m dses sued quantity of fee product prousd to be defeca, sifiler s t a i not be table fer rnnr M uy, los* er d m m , dliect or eofuemieMial, arising n il ofdie use of or Sie n ab m y io use M p ro d u rt Befere usino, user SMH eternine Cie uiabty of dw produci fer Stotr Mended use and users assumes <9 risk and BabBty w tettoevw In eonneellen Swrandth. f n a ferapoinp n a y not be changed scapi by n agreement tipred by an officer of toller.* -3M Specialty Materials 3M Center B ldg. 223-68-04 S t Paul, M N 55144-1000 Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 42 o f 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 3M Dr. Jacques L'Haridon CIT BP 563 27005 Evreux, Fiance Dear Dr. L'Haridon; This is to confirm that the sample o f ammonium perfluoro to CIT, BP 563, 27005 Evreux, France would be expected to contain approx ________________________________________ material (mole percentages).gflBRQps a g ^ g so lu tio n o f ammonium perfluorooctanoate in water^ A n example analysis on a batch o f solid ammonium perfluorooctanoate (3M Product C o d e ^ P ^ ^ ^ P B H M ^ g a v e the following isomer distribution, determined using ,9F-NMR techniques; Sincerely, a. John L. ButenhofT Ph-D., CIH, DABT Staff Scientist JLB/rm Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME - 43 o f 53 2. Substances required for the preparation o f LC reconstituted water # Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 44 o f 53 LC RECONSTITUTED WATER (as described in the French guideline T 90-304) Final Concentrations Solution No. 1: - calcium nitrate (Ca (N 0 3)2.4H20 ) ............................................................... Solution No. 2 : - potassium nitrate (K N 03) ............................................................................. Solution No. 3: - magnesium sulphate (M gS04.7H20 ) ........................................................... Solution No. 4 : - monohydrogen potassium phosphate (K2H P04)............................................. Trace element solutions: Solution No. 5: - copper sulphate (CuS04.5H20 ) .................................................................... - ammonium heptamolybdate [(NH4)6Mo70 24.4H20 ] .................................. - zinc sulphate (ZnS04.7H20 ) ......................................................................... - cobalt chloride (CoCl2.6H20 ) ....................................................................... - manganese nitrate (M n(N03)2.4H20 ) .......................................................... - citric acid (C6H80 2.H20 ) ............................................................................... - boric acid (H3B 0 3) ......................................................................................... Solution No. 6: - iron citrate (HI) (C6H5F e07) ...................................................=..................... - iron sulphate (H) (FeS04.7H20 ) ................................................ .................. - iron chloride (HI) (FeCl3.6H20 ) .................................................................... 40 mg/L 100 mg/L 30 mg/L 40 mg/L 15 pg/L 30 pg/L 30 pg/L 30 pg/L 30 pg/L 30 pg/L 30 pg/L 0.616 mg/L 0.3125 mg/L 0.3125 mg/L All solutions are made up with injectable grade deionized water (conductivity < 10 pScnr1). Prepared solutions are kept for no more than 1 week after autoclaving. Final pH = 7.5 0.3; hardness = approximately 34 mg/L as CaCC>3 . Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 45 o f 53 3. Chemical analysis of test solutions Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 46 o f 53 CHEMICAL ANALYSIS Principle An aliquot o f each sample was diluted and analyzed by Ion Chromatography with Electrochemical Detection (Conductimetry). The concentrations o f AMMONIUM PERFLUOROOCTANOATE (APFO) were determined from a calibration curve of peak area against concentration o f AMMONIUM PERFLUOROOCTANOATE (APFO) in standard solutions. Sample preparation All samples were mixed and those with algae were centrifuged (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 : GP 50 Gradient Pump (Dionex) Mobile phase : 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 o f 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 : Ionpac ATC-3, 4 mm (Dionex) : Ionpac AS 16 (Dionex) length = 250 mm, inner diameter 4 mm : 30C : ED 50 Electrochemical Detector (Dionex) Conductimetry after eluent neutralization : AMMS DI (Dionex) Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 47 o f 53 Injector AS50 (Dionex), at 10C Injected volume 25 pL Data acquisition software : Multichrom 2 (Fisons Instruments) Retention time APFO, approx. 9 min Analysis time 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 o f the following form: Y = aX + b where Y = test item peak area (pVs) X = concentration of test item (mg/L) a = slope value b = intercept # Assay 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-pL 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 o f APFO. Company Sanitized. Does not contain TSCA CBI CIT/Study N. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME - 48 o f 53 Table A: Concentration o f 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 (92) 10.8 (86) 22.4 (90) 45.8 (92) 96.7 (97) 179 (90) 364 (91) Measured (1) 96 hours with algae without algae 6.03 (96) 12.7 (102) 24.3 (97) 47.3 (95) 95.9 (96) 180 (90) 362 (91) 5.53 (88) 10.6 (85) 21.4 (86) 45.9 (92) 95.0 (95) 183 (92) 383 (96) (1): 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 o f 53 VALIDATION OF THE ANALYTICAL METHOD The validation o f the analytical method was performed according to CIT Standard Operating Procedures. The specificity, limit o f quantification, linearity, repeatability o f injections, accuracy and precision (Coefficient o f Variation: CV %) o f the analytical method were determined. Specificity The specificity o f the analytical method was demonstrated as follows: . analysis o f 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 o f quantification o f 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 o f injections Replicate analysis (n = 10) o f a solution containing 50 mg/L of the test item gave satisfactory results since the coefficients of variation values obtained were as follows: . 1% based on peak height, . 1% based on peak area. Based on these results, the repeatability of injections o f the analytical method was validated taking into account peak area. . Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 50 o f 53 Accuracy and precision Six analyses of solutions containing 2.03 and 50.7 mg/L o f the test item in Milli-Q water were carried out. The accuracy and the precision (CV%) obtained were as follows: Concentration (mg/L) Nominal Mean measured* 2.03 50.7 2.02 53.7 *: mean values o f six determinations Dilution factor 1 1 CV (%) 6 2 Accuracy (%) 99 106 Conclusion The analytical method was validated and considered to be suitable for the analysis of the samples o f the study. Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 51 o f 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 o f 53 COMPARISON OF AREAS UNDER THE GROW TH CURVES (Biomass) The areas between the growth curves and the horizontal line N = N0 were calculated according to the formula: N] - No N] + N2 - 2No Nn+ Nn_i - 2No A = -------------- x t i + --------------------- x (t2 - ti) + ... + ------------------------- x ( tn-t,,.i) where, A = area N0= number o f cells/mL at time t0 (beginning of the test), N, = measured number o f cells/mL at time t, Nn= measured number o f cells/mL at time tn t, = time in hours of first measurement after beginning o f test tn = time in hours of nlh measurement after beginning o f test n = number o f measurements taken after the beginning of the test. The percentage inhibition of the cell growth at each test item concentration (IA) was calculated according to the formula: A c-A , IA= ________ _ x 100 Ac where, Ac= area between the control growth curve and the horizontal line N = N0 A, = area between the growth curve at the concentration t and the horizontal line N = N0 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 EbC50 is quoted with the appropriate exposure period, e.g. EbC50(0-96h). Company Sanitized. Does not contain TSCA CBI CIT/Study No. 23685 EAA/AMMONIUM PERFLUOROOCTANOATE (APFO)/ APME 53 o f 53 COMPARISON OF GROWTH RATES The average specific growth rate (p) for exponentially growing cultures was calculated as: Ln N n - Ln N0 F= V to where tg is the time at the beginning of the test and tn the time in hours o f n1*1measurement after beginning o f test. The percentage inhibition of specific growth rate at each test item concentration (I^t) was calculated according to the formula: Pc-Ft IMt= ---------------- x 100 Fc where, pc = mean control specific growth rate pt = 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 ErC50. The times o f measurement must be indicated, e.g. if the value relates to times 0 and 96 hours, the symbol becomesErC50(0-96h). Note: specific growth rate is a logarithmic term and small changes in growth rate may lead to great changes in biomass. EbC and ErC 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 o f the areas under the growth curves or the specific growth rates. Company Sanitized. Does not contain TSCA CBI