Document vVZZ9j8ZX3GOE1ZXJr7EJ26yE

Ar a 6 _ i o Dr.U.Noack-Laboratorium fr Angewandte Biologie 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge acc. to OECD Guideline No. 209 for Testing of Chemicals adopted April 4, 1984 Sponsor Telomer Research Project A sahi Glass Atofina Clariant Daikin Industries Ltd Dupont Author Martina Noack Test Facility Dr .U .N o ack-Laborato rium FR ANGEWANDTE BIOLOGIE Kthe-Paulus-Str. 1 D -31157 Sarstedt Laboratory Project ID Project-No. 020109CK Study-No. BBR84111 Study-No. of the Study Plan BBR8411- Page 1 of 30 Study Completed September 26, 2002 Original 7 of 7 000083 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 2 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________Project-No. 020109CK Statement of GLP Compliance Title G u id e lin e Test Item Test Facility 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoroRespiration Inhibition Test with Activated Sludge OECD 209 for Testing of Chemicals (adopted April 04, 1984) 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro(Batch number: POO/001) Dr .U .N o ack-Laborato rium fr an g ew and te Biologie Kthe-Paulus-Str.1, D-31157 Sarstedt Phone: (+49) 050 66 / 706 70, Fax: (+49) 050 66 / 706 789 E-mail: info@ noack-lab.de Deviations from GLP principles None W e declare that this study was conducted and reported in com pliance with the actual OECD principles of Good Laboratory Practice and the national GLP regulations as specified in the law in force, except deviations mentioned above. 26.09.2002 (Martina Noack, S tikiy Director) 26.09.2002 (Thomas Geffke, Scientist) 26.09.2002 ^^1 (Dr. Udo Noack, Head of Test Facility) 000084 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 3 o f 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. B B R 84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No. 020109CK Statement of the Quality Assurance Unit Title G u id e lin e Test Item Study Director 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoroRespiration Inhibition Test with Activated Sludge OECD 209 for Testing of Chemicals (adopted April 04, 1984) 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro(Batch number: POO/001) Martina Noack The study was verified as follows: inspection study plan process based report date of inspection 2002-02-26 2002-03-12 2002-04-03 2002-04-08 2002-04-12 2002-09-06 2002-09-26 date of report 2002-02-26 2002-03-12 2002-04-03 2002-04-08 2002-04-12 2002-09-06 2002-09-26 The reported results accurately and completely reflect the raw data of the study. Also methods, procedures, and observations are accurately and com pletely described in the report. The accordance of the study with its study plan and the principles of Good Laboratory Practice is guaranteed. 26.09.2002 ............ Guoran Mohrmann-Kalabokidis 0000S5 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 4 of 30 1-Oecanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________ Project-No. 020109CK Personnel Involved Study Director: Martina Noack (Biologist) Deputy: Silke Fiebig (Engineer, Biotechnologist) Scientist T. Geffke (Chemical engineer) Responsible for test item analysis Address see above Technical Staff: Jan Schwerdtfeger Quality Assurance Unit: Deputy: Gudrun Mohrmann-Kalabokidis (Biologist) Susanne Becker (Biologist) Director of the Test Facility: Dr. Udo Noack (Biologist) 000086 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 5 o f 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No. 020109CK Table of Contents Title Page Statement of GLP Compliance Statem ent of Quality Assurance Unit Personnel Involved Table of Contents List of Tables and Figures 1 Summary 2 Characterisation data of the test item 3 Method 4 Results 4.1 Solubility of the test item 4.2 Volatisation of the test item 4.3 Respiration rates and inhibition of the respiration 4.4 EC-values 4.5 Biotic and abiotic factors 4.6 Reference test 5 Validity criteria 6 Literature / References 7 Raw data 8 Analytical method 8.1 Method 8.2 Method Validation 8.2.1 Linearity 8.2.2 Quantification Limit and Detection Limit 8.2.3 Precision (Repeatability) 8.2.4 Peak Symmetry 8.2.5 Accuracy 8.3 Results 8.4 Calibration Curves 8.5 Chrom atogram s 9 C ertificate of test item analysis 10 G LP -C ertificate o f D r .U .N o a c k -L a b o r a to r iu m Page 1 2 3 4 5 5 7 8 9 14 14 14 14 15 16 16 16 16 17 18 18 20 20 20 21 21 22 23 24 27 29 30 000087 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 6 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________Project-No. 020109CK List of Tables and Figures Tables: Table 1: EC-values of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoroTable 2: Respiration rates and inhibition of respiration of test and reference item concentrations Table 3: EC-values of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10 heptadecafluoroTable 4: Biotic and abiotic factors Table 5: Raw data and respiration rates of test and reference item concentrations Table 6: Repeatability of Injections of Test Item Table 7: Mean Recovery Rate of Fortified Samples Table 8: C oncentrations of the Active Ingredient 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- in the air trap 7 15 15 16 17 21 22 23 Figures: Figure 1: Calibration Curve of Standard - concentration range 0.01 Figure 2: Calibration Curve of Standard - concentration range 0.1 Figure 3: Calibration Curve of Standard - concentration range 0.1 Figure 4: Chrom atogram of Standard Figure 5: Chrom atogram of Test item - 0.06 mg/L 24 - 10 mg/L 25 - 0.6 mg/L 25 27 28 ooooss Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 7 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. B B R 84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No. 020109CK 1 Summary A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 and GLP principles was performed for 3 h with the test item 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafiuoro- batch-no. P00/001, on 2002-04-03 at the test facility D r .U .N o ac k-Labo r ato r iu m fr an g e w an d te B io lo g ie . Test system was activated sludge of the municipal treatm ent plant of D -31157 Sarstedt. The test method was static. Nominal concentrations of the test item were as follows: 100 - 180 - 320 - 580 - 1000 mg/L. The respiration rates of control, reference and test item were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determ ined in comparison to the control respiration rates. The inhibition ranged from 2 to 9 % (Table 2). The EC2o-, EC50- and ECao- values could not be calculated in the tested concentration range (Table 1). In order to check the activity of the test system and the test conditions a reference test is carried out once every 7 days with copper-(ll)-sulphate-pentahydrate as reference item and the reference toxicity is determ ined. The EC50- value for the reference item was 104 mg/L. Validity criteria of the test guideline were fulfilled. Table 1: EC-values o f 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- E C 20 EC-values [mg/L] > 1000 > 1000 > 1000 Confidence interval P = 95 [%] [mg/L] - - - 0un O ID m 0COO The test item 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- is not toxic in concentrations < 1000 mg/L to activated sludge of a municipal sewage treatm ent plant. 000089 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 8 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No. 020109CK 2 Characterisation data of the test item TEST ITEM 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Batch Number P00/001 CAS RN 678-39-7 Chemical characterization 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecane-1-ol (EINECS) Purity C8-perfluoroalkylethanol: 99.3 % (ala), sum perfluoroalkylethanol 99.9 % (ala) Appearance Yellowish solid wax W ater solubility 134 pg/L at 12 degree C, Std 29 pg/L, n=8 (DuPont, 2002) 137 pg/L at 25 degree C, Std 53 pg/L, n=17 (DuPont, 2002) Stability in water Stable at test conditions Density Ca. 1.7 g/cm 3 Expiry date Recommended storage Storage at testing facility Retention Identification parameter at testing facility Unlimited if stored under the recommended conditions. Room temperature (20 C), protected from moisture. Room temperature, protected from moisture and light. At least 1 g has been retained. Name, batch number and colour. The test item and the information concerning the test item were provided by the sponsor. 000090 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 9 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________ Project-No. 020109CK 3 Method GUIDELINE OECD Guideline 209 TYPE AND PURPOSE OF THE STUDY Respiration Inhibition Test with activated sludge to determine the EC2o-, EC50- and ECao-values. TEST SYSTEM Reason for the selection of study system Origin P re-treatm ent Nutrient solution Dry sludge concentration Activated sludge Non adapted activated sludge from the sewage plant at Sarstedt is well suited as it comprises m ostly household sewage and hardly industrial waste water. Municipal sewage treatm ent plant of D-31157 Sarstedt The sludge was washed with autoclaved tap water and diluted corresponding to a dry weight of 4.0 g/L 1 0 % . Synthetic waste water according to OECD 209 3.69 g/L TEST ITEM Stock solution Test concentrations Pretreatment of the test item 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- None, directly weighing 100 - 180 - 320 - 580 - 1000 mg/L The test item will be heated to 50 C, weighed directly into the Erlenmeyer flasks, filled with 250 mL autoclaved tap water and stirred for 24 h. CONTROL 2 control samples without test item were included in every test series at the beginning and at the end of the test. 000091 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 10 o f 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________Project-No. 020109CK REFERENCE ITEM CAS No. Batch-No. Purity Expiry date Test concentrations Range of validity (EC50) Test date Copper (II) sulphate pentahydrate p.a. Merck 7758-99-8 A265990 99.9 % 2005-11-30 58 - 100 - 180 mg/L 54 - 146 mg/L 2002-04-03 METHOD Test duration Test date R e p lic a te s Test vessels Volume of the study medium Application Test temperature Ventilation Static 3h 2002-04-03 One per concentration and two per control. The highest concentration was tested in duplicates for analysis of airflow-outlet. 500 mL Erlenmeyer flasks, DIN 12440 500 mL 200 mL inoculum, 16 mL synthetic waste water 250 + 34 mL autoclaved water Test item concentrations (directly weighing) 18 - 22 C Permanent TYPE AND FREQUENCY OF MEASUREMENT AND OBSERVATIONS The pH value of the activated sludge was m easured prior to testing and the tem perature in the measuring cell once during the measurement. The oxygen depletion of the control, reference and test item was measured after 3 h incubation by means of oxim eter and recorded for at least 3 minutes on a flat bed recorder. The room temperature during the test was recorded continuously by a hygrothermograph. 000092 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 11 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________ Project-No. 020109CK Equipment pH-meter, C o r n in g pH 240 Magnetic stirrer, Ikamag Reo or Ret Oximeter, W tw Oxi 530 Flat bed recorder, L inseis Membrane pump, Behr Tube pump, B iosens Hygrothermograph, Lufft Adjustm ent of the recorder Voltage-. Speed (y-direction): 500 mV 1 cm/min Calibration of the recorder The recorder had been calibrated to the relation of 5 mm (x-direction) and 0.1 mg/L oxygen reduction. DETERM INATION OF THE TEST ITEM IN AIR STREAM TEST DESCRIPTION Possible losses of the test item via air outlet due to the aeration were analytically verified by gas chromatography with mass spectrometric detection (m/z = 69, 395 and 463) from highest test concentration. For details see part 8. A range-finding test (NON-GLP-state) was carried out with three concentrations: 10 - 100 - 1000 mg/L. A fresh sample of activated sludge taken from the sewage plant Sarstedt, washed twice with autoclaved tap water and diluted corresponding to a dry weight of 4.0 g/L 10 %, was used for the test procedure. 5 concentrations within the range 100 - 1000 mg/L in a series with a dilution factor of 1.8 were tested. 2 controls without test item were included. Once every 7 days a reference test is carried out. The reference item was tested in three concentrations within the concentration range 58 - 180 mg/L with a dilution factor of 1.8. The respiration rates of all samples were determ ined subsequent to an incubation period of 3 h. Evaluation was carried out as described below. ooooa Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 12 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guidellne No. 209____________________________________ Project-No. 020109CK EVALUATION Determined are the distances recorded by a flat bed recorder during 3 min m easurement for each test vessel. 5 mm on the recorder are equivalent to a decrease of 0.1 mg 0 2/L. That leads to the factor f = 0.02 mg 0 2/L m m . The estimation of the respiration rate (R) according to the raw data was perform ed by Form ula (1). R = md f t R : respiration rate m g 0 2/L-h md : measured distance mm/3min f : 0.02 mgCh/L-mm t : 20 [3min/h] The calculation of respiration rates (R) and inhibition of respiration rates was performed by standard method: Inhibition [%] = 2 Rs I 00 /?C| + R-C1 (2) Rs = Respiration rate of test concentration in mg O2/L h Rei = Respiration rate of control 1 Rc2 = Respiration rate of control 2 For the controls the deviations of the respiration rates were calculated in %. STATISTICAL ANALYSIS The EC-values of the reference item were calculated with probit analysis using software S ig m aPlot (W indows) rel. 6.00, 2000, SPSS Corpo ration. Probit values were taken from W eber (1986). Confidence intervals were calculated according to a standard procedure (B reitig & TOm pling 1982). 000094 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 13 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________Project-No. 020109CK VALIDITY CRITERIA The results are valid if: The two control respiration rates differ not more than 15 %. The EC50 of the reference item is in the range of 54 - 146 mg/L. DEVIATIONS FROM THE TEST GUIDELINES In contrast to the guideline, it was decided to use copper (II) sulphate pentahydrate instead of the prescribed 3,5dichlorophenol. The hence of Dichlorophenol is unsuitable for routine lab use. DEVIATIONS FROM THE STUDY PLAN None REPORTING The final report is issued as paper copy (6 originals) and an electronic record (1 original). ARCHIVING The following was retained in the archive of the test fa cility for the period as specified in the operative national GLP regulations: all raw data s tu d y plan (Original 1 of 2) final report paper copy (Original 1 of 6) all records performed by the quality assurance program m e including master schedules sample of test item M icrofilm s will be retained in a safe-deposit by Volksbank Sarstedt, D-31157 Sarstedt. 000095 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 14 o f 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________Project-No. 020109CK 4 Results 4.1 Solubility of the test item Due to the low water solubility of the test item (0.13 mg/L) most of the test item was not dissolved during the test. 4.2 Volatisation of the test item Test item concentration [mg/L] Amount of test item in test solution [mg] Solid phase extraction cartridge* 1000 500 C8 1000 500 C 18 * Specification of cartridge type see part 8.1 Test item adsorbed [mg] 5.02 1.65 Test item adsorbed [%] 1.0 0.3 Aeration time (incubation): 3 h Aeration stream: > 180 L/h Only 1 % of the test item was stripped from the system, therefore losses by volatisation were negligible. 4.3 Respiration rates and inhibition of the respiration From the oxygen depletion the respiration rates were calculated in mg 0 2/L h (Table 2). The inhibition of the respiration rates from the test and reference item concentrations in % was calculated in com parison with the respiration rates from the control. The results are listed in Table 2. The raw data are listed in Table 5. 000096 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 15 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________Project-No. 020109CK Table 2: R e sp ira tio n rates and in h ib itio n o f re s p ira tio n o f te s t and reference item concentrations test item control 1 control 2 reference item concentration [mg/L] 1000 (1sl) 1000 (2nd) 580 320 180 100 - - 180 100 58 respiration rate [m g 0 2/L-h] 23.6 25.6 24.0 25.6 24.4 25.2 27.2 24.8 7.6 12.4 19.6 inhibition [%] 9 2 8 2 6 3 - - 71 52 25 4.4 EC-values The EC20-, EC50- and EC8o- values could not be calculated in the tested concentration range, the EC-values were > 1000 mg/L (Table 3). Table 3: EC-values o f 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10 h e p ta d e ca flu o ro - EC2o EC50 EC-values [mg/L] > 1000 > 1000 > 1000 Confidence interval P = 95 [%] [mg/L] - - - Oco O ID 000097 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 16 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________ Project-No. 020109CK 4.5 Biotic and abiotic factors The pH-value of the activated sludge, the dry sludge concentration and the test tem perature are documented in Table 4. Table 4: Biotic and abiotic factors D ifference of the control respiration rates [%] pH-value of the activated sludge Dry sludge concentration [g/L] Test temperature [C] 9.0 7.05 3.69 20.5 4.6 Reference test In the reference test the EC50 -value was determ ined by probit analysis. The EC50 -value of the reference item is: EC50 = 104 mg/L The EC50-value of the reference item copper(ll)-sulphate-pentahydrate is in the confidence range of 54 - 146 mg/L. 5 Validity criteria Validity criteria were fulfilled: The two control respiration rates differ not more than 15 %. . The EC50 (3 hours) of copper(ll)sulphate pentahydrate is in the accepted range of 54 - 146 mg/L. 6 Literature / References (1) OECD-Guideline No. 209 for Testing of Chemicals, adopted April 04, 1984 (2) W e b e r , E. (1986): Grundri der biologischen Statistik, 9. Auflage. (3) B r e it ig , G., T m plin g , W. (editors, 1982): Methoden der W asseruntersuchungen Bd.II. VEB G ustav F ischer V er lag , Jena. (4) DuPont, D eterm ination of physico-chem ical param eters of Perfluorooctylethanol, CAS No. 678-39-7, Unpublished results, Update Feb. 15, 2002 000098 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 17 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No. 020109CK 7 Raw data Table 5: Raw data and re s p ira tio n rates o f te s t and reference item c o n c e n tra tio n s concentration [mg/L] oxygenconcentration* [m g 0 2/L] measured distance** [m m /3m in] test item control 1 control 2 reference item 1000 (1st) 1000 (2nd) 580 320 180 100 - 180 100 58 7.58 4.96 2.00 2.38 1.68 1.89 5.88 2.67 7.15 6.69 5.56 59 64 60 64 61 63 68 62 19 31 49 * determined at the start of the 3 minute measurement ** 5 mm are related to 0.1 mgC>2/L (see Formula (1) on page 11) respiration rate [m g 0 2/L h ] 23.6 25.6 24.0 25.6 24.4 25.2 27.2 24.8 7.6 12.4 19.6 000099 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 18 o f 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No. 020109CK 8 Analytical method 8.1 Method METHOD OF DETERMINATION Equipment Analyses of the airflow-outlet from the highest test concentration of the active ingredient 1-Decanol,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10, 10-heptadecafluoro- was carried out via gas chromatography on a Fused Silica CP-SIL 8 capillary column using external standards. Detection was performed with a mass selective detector (MS). Gas chromatograph: A u to sam ple r Detector Software CP-3800, Varian CTC Combi PAL, CTC A nalytics MS, Saturn 2000, Varian Saturn GC/MS W orkstation 5.51, Varian Solid phase extraction cartridges Reagents HF Bond Elut C8, Varian HF Bond Elut C18, Varian ferf.-butyl methyl ether 99.8 %, Batch 423370/1 31101, Fluka CONDITIONS OF ANALYSIS Column Injector Injector temperature Injection volume Temperature programme Chrom pack CP-SIL 8 (Va r ia n ), 30 m, i.d .: 0.25 mm, LOW BLEED / MS, Film thickness: 0.25 pm Splitless (30 psi pressure puls) for 1 min 250 C 1 pL Heating rate [C/min] 10 30 T e m p eratu re [C] 40 90 220 Holding time [min] 0.0 1.0 5.0 Carrier gas Detector Ionisation mode Run time Retention time Helium 1 mL / min column flow MS Electron ionisation (El) 15.33 min approx. 4.9 min OOOIOO Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 19 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No. 020109CK STANDARD PREPARATION OF STANDARDS PREPARATION OF SAMPLES METHOD VALIDATION Peak symmetry Repeatability Accuracy Test item was used as standard. Two stock solutions of the standard of 200 mg/L were prepared in ferf.-butyl methyl ether, diluted (6 concentrations each) and used for calibration. For calibration range see part 8.2.1. The airflow-outlet from the test replicate was filtered through Varian HF Bond Elut C8 and Varian HF Bond Elut C18, respectively. The solid phase extraction cartridge was eluated with 5.0 mL ferf.-butyl methyl ether. The extract was injected directly into the GC/MS. Quantification of the test item was calculated by peak area of the masses 69 + 395 + 463 (m/z) based on the external standards. The calibration procedure and the principle for the determination of detection and quantification lim its were adapted from DIN 32645. The method was validated as described below: Peak symmetries were calculated from chromatograms of analytical standards and test item according to the following equation: T = W 0.o5 / (2-f) T W o.os f = Peak symmetry = Peak width at 5 % of peak height [mm] = first sector of peak width ( divided by the perpendicular line from the peak maximum ) [mm] 5 sub-samples of the highest and lowest concentration prepared from a single homogenous sample were analysed. Mean values, standard deviations and variations of coefficients were calculated. Three C8 SPE-cartridges were spiked with an am ount of 5000 pg 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro(250 pL of 20000 mg/L). After spiking and removing of excess of solvent, air was sucked through the cartridges with approx. 150 L/h for 3 h. Afterw ards cartridges were eluted each with 5.0 mL tert.butyl methyl ether. The extracts were then each diluted by factor 100 with ferf.-butyl methyl ether and injected into the GC/MS. O O O lO l Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 20 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________Project-No. 020109CK 8.2 Method Validation The method was validated and tested with satisfactory results with respect to linearity precision (repeatability) peak symmetry accuracy determination lim it and detection limit. 8.2.1 Linearity The analytical system gave linear response in the range of 0.1 - 10 mg/L of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro-. The calibration curve and chromatograms obtained for standard and test item are given in figures 1 - 3. For determ ination and detection lim its see part 8.3. 8.2.2 Quantification Limit and Detection Limit The lim it of quantification (LOQ) and limit of detection (LOD) were calculated using the blind value method described in DIN 32645 "Nachwels- und Bestim m ungsgrenze" . The LOD was determ ined to be 0.009 mg/L and therefore the LOQ was 0.027 mg/L (threefold the LOD). 000102 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoroRespiration Inhibition Test with Activated Sludge acc. to OECD-G uideline No. 209 Page 21 of 30 Study-No. B B R 84111 Project-No. 020109CK 8.2.3 Precision (Repeatability) The results of the repeatability tests are provided in the table below. Table 6: R e p e ata b ility o f In je ctio n s o f Test Item Peak area [pV*s] Serial No 1 2 3 4 5 Mean SD CV [%] 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9.10,10,10heptadecafluoro- (m/z = 69+395+463) 0.1 mg/L 0.6 mg/L 2098 2098 1978 1958 2341 2094.6 152.5 12141 13563 13291 13597 13500 13218.4 613.9 7.3 4.6 8.2.4 Peak Symmetry The peak symmetry of the test item 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8 ,9,9,10,10,10heptadecafluoro- was 1.8. 000103 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 22 o f 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________ Project-No. 020109CK 8.2.5 Accuracy Table 7: Mean R ecovery Rate o f F o rtifie d Sam ples Fortified amount of test item 5000 [pig] Replicate 1 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10 ,10-heptadecafluoro- calc, amount RR [p g ] [%] 5760 115 Replicate 2 5846 117 Replicate 3 5762 115 Mean SD ( N - 1 ) CV % Linearity correlation coefficient ( r2 ) 0.9851 116 1.2 1.0 calc, amount RR LOQ LOD SD (N-1 ) CV = calculated amount, dilution factor taken Into account = Recovery rate related to the nominal concentration of the test item = Limit of quantification = Limit of detection = standard deviation = coefficient of variation 000104 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 23 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________ Project-No. 020109CK 8.3 Results Table 8: C o n ce n tra tio n s o f the A ctive In g re d ie n t 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10, 10,10-heptadecafiuoro- in the a ir tra p Sampling Date Start of Analysis Test Item Replicate Nominal No. [mg/L] 2002-04-03 2002-04-09 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,1 0-heptadecafluoro- Calc. Amount in the air trap [mg] Mean of sum of two elution steps* 1st Elution step* 2nd Elution step* [mg] 1000 1 (C8) 2 (C 18) 5.00 0.2811 1.66 1.59 0.0036 0.040 0.020 0.020 5.02 1.65 LOQ [mg/L] LOD [mg/L] linearity ( r2 ) 0.027 0.009 0.9996 calc. cone. LOQ LOD 1) * = calculated amount, accumulation factor taken into account = Limit of quantification = Limit of detection = Injection error, not included in calculations = Eluent: ferf.-butyl methyl ether 000105 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 24 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No. 020109CK 8.4 Calibration Curves The calibration curves shown were established for different concentration ranges and at different tim e s Calibration Curve of Fluowet (2002-04-04) 2500 2000 - r--, *>3. 1500 no 1000 < _ "0 < -' ' c ,.q \ o\o \ \ 500 - 00 S I y = 3 2 2 6 3 x -79,967 R2= 0,9655 ------1-------------------------------- 0,01 0,02 0,03 0,04 0,05 0,06 Cone. [mg/L] Figure 1: C a lib ra tio n Curve o f S tandard - c o n ce n tra tio n range 0.01 - 0.06 m g/L (dated 2002-04-04) 000106 Dr.U.Noack-Laboratorium fur Angewandte Biologie Report Page 25 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to OECD-Guideline No. 209____________________________________ Project-No. 020109CK Calibration Curve of Fluowet (2002-04-10) 500000 450000 400000 350000 300000 250000 200000 150000 100000 50000 0^ 5 2 y = 44250x - 2011,7 R2 = 0,9996 III 4 6 81 Cone. [mg/L] Figure 2: C a lib ra tio n Curve o f S tandard - co n c e n tra tio n range 0.1 - 10 m g/L (dated 2002-04-10) Calibration Curve of Fluowet (2002-06-19) Figure 3: C a lib ra tio n Curve o f S tandard - c o n ce n tra tio n range 0.1 - 0.6 mg/L (dated 2002-06-19) 000107 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 26 o f 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No, 020109CK The first calibration curve was established 2002-04-04 but the concentration range (0.01 - 0.06 mg/L) was not broad enough to be used for all tests running. Therefore the second curve (2002-04-10) which covers a much wider range (0.1 - 10 mg/L) was used for the calculations in this test. The concentration calculations using the two different curves do not differ more than 10 % from each other. The calibration curve from 2002-06-19 with a concentration range of 0.1 - 0.6 mg/L shows that the GC/MS system has a good stability over a long period of time. 000108 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 27 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BBR84111 acc. to O ECD-Guideline No. 209____________________________________ Project-No. 020109CK 8.5 Chromatograms Target Compound Report fcr #1 from 8411 0.1 I_1.SMS Sample ID: Instrument ID: Measurement Type: Acquisition Date: Calculation Date: Sample Type: Inj. Sample Notes: Divisor: 8411 0.1 I Area 10.04.02 13:21 1 1.04.02 08:08 Calibration 1,000000 Operator: Last Calibration: Calibration Type: Data File: Method: 11.04.02 08:08 External Standard c:\... 8411 0.1 i j . s m c:\... \8411_rep2.mth Multiplier: 1,000000 Parameter Compound Number Retention Time RT Offset Peak Name CAS Number Status Guan Ions Area Height Amount RF) Fit Calibration Equation Baseline Code Specification 4,918 * /- 0,200 69+395+463 >=10 >=700 Linear, Include, None Actual 1 4,911 min. Ao,00676 min. Fluowet 678-39-7 4368 2283 44124,941 971 y = +4,4125e+4x+0,0 BB Status Pass Pass Pass Figure 4: C hrom atogram o f S tandard 0.1 mg/L (dated 2002-04-10) 000109 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 28 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. B B R 84111 acc. to OECD-Guideline No. 209____________________________________ Project-No, 020109CK Print Date: 10 Apr 2002 17:55:48 Target Compound Report for #1 from BBR84111 100QRep1_1 SMS Sample ID: Instrument ID: Measurement Type: Acquisition Date. Calculation Date. Sample Type: Inf. Sample Notes: Divisor: BBR84111 1000Rep1 Area 10.04 02 11:54 10.04.02 17:50 Analysis 1:100 1,000000 _____ Parameter Compound Number Retention Tim e RT Offset Peak Name CAS Number Status Quan Ions Area Height Amount Fit Calibration Equation Baseline Code SDecification 4,925 +/- 0,200 69+395+463 >=10 >= 0,000 >=700 Linear, Force, None O 'Operator: Last Calibration Calibration Type: Data File: Method: 09.04.02 16:09 External Standard c:\... bbr84111 1000re \3411_rep2.mfrt Multiplier:_____________ 1,000000 Actual 1 4,916 min. 0 ,00916 min. Fluowet 678-39-7 XD 440506 244324 440506,156 Counts 996 BB Status Pass Error Pass Pass Pass status and hrrors D Can't calibrate. Default to raw peak size. F ig u re 5. C hrom atogram o f Test item 1000 mg/L (dated 2002-04-10) OOOllO Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 29 of 30 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge Study-No. BB R 84111 acc. to O ECD-Guideline No. 209____________________________________Project-No. 020109CK 9 Certificate of test item analysis G ira n t GmbH. Werk Gendorf CQG / Q u iliu c s m a n a g c fiie n t D-84504 B u rg lu rc fte n Fax: +-*-49 3679 / :6 7 6 Clariant :. n 3 p e c t i o n c e r t i f i c a t e a tccrdlr. to ENl020 -1-3.18 Date: 13.11.2001 Page: 1 / 1 i - r ,,onargnnent N!- r r i a 1 L- D e c a n o i , 3 , 3 , 4 , 4 , 5 , 5 , 6 , 6 , 7 , 7 , 3 , 8 , 9 , 9 , 1 0 , 1 0 , 10-r:ep tade <:a tluofo 104 70S wp,l :- -,c -,-TiLIancer.t ia a part, tae c 1 low.oq value: ::orr / caused produce, spcification tod Spec ].vcat lori >= 97,5 99, 3*[a ! a ] 3 _rt Pcr 11u :rca 1 y let hacci >= 98,0 99,9% [a/a] Sc.-t, Per z 1 uora -cyioutanol C P ^r:l_:r,:.;y:echenS Su sene: DIN 5l ' > ' n inc:re -cnt- " A o ro <-l, 0 <=0, 5 <-0,2 < 0 ,1'i (a /a ] < 0,1% [a/a ' < 0,1i < 0, 1% COCO lease the customer iron the obligation O O O lll Dr.U.Noack-Laboratorium fr Angewandte Biologie Report 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Respiration Inhibition Test with Activated Sludge acc. to OECD-G uideline No. 209 ___________________________ Page 30 of 30 Study-No. BBR84111 Project-No. 020109CK 10 GLP-Certificate of Dr.U.Noack-Laboratorium n Niederschsisches UmweltministBrium Gute Laborpraxis/Good Laboratory Practice GLP-Bescheinigung/Statement of GLP Compliance (gemll/according to 19 b Abs.1 Chemikaliengesetz) Eine GLP-lnspektion zur berwachung der Einhaltung der GLP-Grundstze gem Chemikaliengesetz bzw. Richtlinie 88/320/EG wurde durchgefhrt in: Assessment of conformity with GLP according to Chemikaliengesetz and Directive 88/320/EE C at: X Prfeinrichtung / Test facillty Prfstandort /T e s t site Dr. U. NOACK LABORATORIUM fr angewandte Biologie Kthe-Paulus-Str. 1 D -31 157 Sarstedt Dr. U. NOACK LABORATORY of applied biology Kthe-Paulus-Str. 1 D -31157 Sarstedt (Unverwechselbare Bezeichnung und Adresse/Unequivocal name and address) Prfungen nach Kategorien/Areas of Expertise (gem/accordmg ChemVwV-GLP Nr. 5.3/OECD guldance) 1 - Prfungen zur Bestimmung der physikalisch-chemischen Eigenschaften und Gehaltsbestimmungen 3 - Prfungen zur Bestimmung der erbgutverndemden Eigenschaften (in vitro und in vivo) 4 - Umwelttoxikologische Prfungen zu Auswirkungen auf aquatische und terrestrische Organismen 5 - Prfungen zum Verhalten im Boden, im W asser und in der Luft, Prfungen zur Bioakkumulation und zur Metabolisierung 6 - Prfungen zur Bestimmung von Rckstnden 7 - Prfungen zur Bestimmung der Auswirkungen auf Mesokosmen und natrliche kosysteme Datum der Inspektion / Date of Inspection (Tag.Monat.Jahr / day.month.year) 25. - 27. Juni 2001 / June 25,h - 2 7 \ 2001 29. August und 21. September 2001 / A ugust 29"' and Septem ber 21" , 2001 Die genannte F befindet sich im nationalen GLPberwachungsverfahren und wird regelmig auf Einhaltung der GLP-Grundstze berwacht. Auf der Grundlage des Inspektionsberichtes wird hiermit besttigt, dass in dieser Prfeinrichtung/diesem Rrfstandort die oben genannten Prfungen unter Einhaltung der GLP-Grundstze durchgefhrt werden knnen. Niederschsisches Umweltministerium Referat 33 Archivstrae 2 30169 Hannover The above mentioned test facility / tost site is included in the national GLP Compliance Programme and is Inspected on a regular basis. Based on the inspection report it can be confirmed, that this test facility l test site is able to conduct the aforementioned studies in compliance with the Principles of GLP. 11. Mrz 2002 Im Auftrge Dr. Braedt 000112