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RECEIVED OPPT NCIC AR sa6 - /g T elomer Research Program 1200 South Hayes Street Arlington, Virginia 22202-5050 Tet (703)413-1100 ext 5364 Fac (703)4138111 Clan ant February 14, 2003 Mary F. Dominiak U.S. Environmental Protection Agency EPA East (Mail Code 7405M) 1200 Pennsylvania Avenue NW Washington, DC 20460 E-mail: dominiak.mary@epa.gov CD Dear Mary: The Telomer Research Program (TRP) and its member companies, Asahi Glass, Clariant, Daikin, and DuPont, are providing to U.S. EPA Docket# AR226 final reports on the acute hazard of 8-2 Telomer B Alcohol (3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecan-1-ol; CAS No. 678-39-7) when tested with standard freshwater aquatic test species. These reports present the results of the acute aquatic toxicity testing conducted by an independent laboratory at the request of the TRP. These studies were conducted according to existing OECD test guidelines and in accordance with the provisions of the appropriate Good Laboratory Practices (GLP) regulations. The physical-chemical characteristics of the test substance indicate that the test substance is a "difficult" substance, as indicated in OECD Monograph 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD 2000), and the experimental designs selected for the acute aquatic toxicity testing were reflective of this observation. Common practices for testing these types of materials, and for evaluating the results from tests of these types of materials, were used during the conduct of the studies and the preparation of the study reports. The TRP and its member companies believe that the data gathered from these studies are consistent with the hypothesis that the 8-2 Telomer B Alcohol is of low to medium concern in terms of its potential for causing acute effects on aquatic species. If there are questions concerning the technical aspects of the studies submitted, please contact me so that I may direct your questions to the appropriate TRP member company personnel. Sincerely, Katie D. Smythe TRP Administrator This document does not contain TSCA CBI. 000003 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- Alga, Growth Inhibition Test with Scenedesmus subspicatus, 72 h, Limit-Test with the Saturated Solution acc. to OECD-Guideline No. 201 for Testing of Chemicals (June 1984) Sponsor Telomer Research Project Asahi Glass atofina Clariant Daikin Industries Ltd Dupont Author Dirk Scheerbaum Test Facility Dr .u .Noack-Laboratorium FR ANGEWANDTE BIOLOGIE Kthe-Paulus-Str. 1 D-31157 Sarstedt Laboratory Project ID Project-No. 020109CK Study-No. SSOB4111 Study Completed On August 07, 2002 Page 1 of 39 Original 7 of 7 000004 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 2 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,1O-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Statement of GLP Compliance Title Guideline Test Item Testing Facility Deviations from GLP Principles 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoroAlga, Growth Inhibition Test with Scenedesmus subspicatus, 72 h, Limit-Test with the Saturated Solution OECD-Guideline No. 201 for Testing of Chemicals (1984) 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro(Batch number: P00/001) D r .U .N o a c k -L a b o r a t o r iu m f r a n g e w a n d t e B io l o g ie Kthe-Paulus-Str.1, D-31157 Sarstedt Phone. (+49) 050 66/706 70, Fax: (+49) 05066/706 789, E-mail: into@noack-lab.de None W e declare that this study was conducted and reported in compliance 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. August 07, 2002 August 07, 2002 August 07, 2002 (Dirk Scheerbaum, Study Director) (jd j% (Thomas Geffke, Scientist) (Dr. Udo Noack, Head of Testing Facility) 000005 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 3 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Statement of the Quality Assurance Unit Title Guideline Test Item Study Director 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoroAlga, Growth Inhibition Test with Scenedesmus subspicatus, 72 h, Limit-Test with the Saturated Solution OECD-Guideline No. 201 for Testing of Chemicals (1984) 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro(Batch number: P00/001) Dirk Scheerbaum The study was verified as follows: inspection study plan process based report dates 2002-03-12 2002-04-02 2002-04-07 2002-04-11 2002-04-12 2002-07-15 date of report 2002-03-12 2002-04-02 2002-04-08 2002-04-11 2002-04-12 2002-07-15 The reported results accurately and completely reflect the raw data of the study. Also methods, procedures, and observations are accurately and completely described in the report. The accordance of the study with its study plan and the principles of Good Laboratory Practice is guaranteed. August 07, 2002 C* 'it*' Gudrtr1Mhrmann-Kalabokidis 000006 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 4 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201_____________Study-No. SS084111 Personnel Involved Study Director: Dirk Scheerbaum (Biologist) Scientist: Thomas Geffke (Chemical Engineer) Responsible for concentration control analysis Technical Staff: Ute Kutzner Quality Assurance Unit: Gudrun Mhrmann-Kalabokidis (Biologist) Deputy: Susanne Becker (Biologist) Head of Testing Facility: Dr. Udo Noack (Biologist) 000007 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-heptadecafluoroAlga, Growth Inhibition Test with Scenedesmus subspicatus, 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201 Page 5 of 39 Project-No. 020109CK Study-No. SS084111 Table of Content Title Page Statement of GLP Compliance Statement of Quality Assurance Unit Personnel Involved Table of Content List of Tables List of Figures Page 1 2 3 4 5 6 6 1 2 3 4 4.1 4.2 4.3 5 6 7 8 9 10 10.1 10.2 10.2.1 10.2.2 10.2.3 10.2.4 10.2.5 10.3 10.4 10.5 11 12 13 Summary Characterisation Data of the Test Item Method Results Preliminary Test Definitive Test Test of the Reference Item Validity Criteria Conclusions Literature / References Graph Physicochemical Data GC - Analysis of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9, 10,10,10heptadecafluoroMethod Method Validation Linearity Quantification Limit and Detection Limit Precision (Repeatability) Peak Symmetry Accuracy Results Calibration Curves Chromatograms Raw Data Certificate of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- Analysis GLP-Certificate of Dr.U.Noack-Laboratorium 7 9 10 17 17 17 20 20 21 21 22 23 24 24 26 26 26 27 27 28 29 30 33 36 38 39 000008 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 6 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 List of Tables Tab. 1: Tab. 2: Tab. 3: Tab. 4: Tab. 5: Tab. 6: Tab. 7: Tab. 8: Tab. 9: Tab. 10: NOEC/LOEC and EC50-Values (0-72 h) of Biomass Inhibition and Rate-Related Inhibition Preliminary Test Results: Cell Density Values NOEC/LOEC and EC5o-Values (0-72 h) of BiomassInhibition and Rate-Related Inhibition Cell Densities Evaluation after 72 h EC5o-Values of Biomass Inhibition and Rate-Related Inhibitionof the Reference Item Water Parameters Repeatability of Injections of Test Item Mean Recovery Rate of Fortified Samples Concentrations of the Active Ingredient 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Page 8 17 18 18 19 20 23 27 28 29 List of Figures Fig. 1: Fig. 2: 30 Fig. 3: Fig. 4: Fig. 5: Fig. 6: Fig. 7: Cell Densities for Control and Limit Concentration (0-72 h) Calibration Curve of Standard - concentration range 0.01 - 0.06 mg/L Calibration Curve of Standard - concentration range 0.1 - 10 mg/L Calibration Curveof Standard - concentration range 0.1 - 0.6 mg/L Chromatogram of Standard Chromatogram of Test Item Chromatogram of Control 22 31 31 33 34 35 000009 Dr.U.noack-Laboratorium fr Angewandte Biologie Report Page 7 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 1 Summary The acute toxicity of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- (batch number P 00/001) to the unicellular freshwater green alga Scenedesmus subspicatus was determined according to the principles of OECD-Guideline 201, at D r .U . N o a c k - L a b o r a t o r iu m F r A n g e w a n d t e B io l o g ie in 31157 Sarstedt, Germany from April 01 to 04, 2002. The study was conducted under static conditions over a duration of 72 hours with an initial cell density of nominally 104 cells/mL and a saturated solution as limit concentration, containing 0.20 mg/L. For the preparation of the saturated solution a suspension with a nominal loading of 100 mg/L was shaken for 96 h and centrifuged. The clear supernatant was used for the test. Test vessels were coated with the test item for 14 days before the test started. Three replicates were tested for the limit concentration and 6 replicates for control. The test item was clearly dissolved throughout the test. Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities. W ater quality parameters of pHvalue, measured at 0 and 72 h, and room temperature, measured continuously, were determined to be within the acceptable limits. The concentration of the active ingredient 1-Decanol, 3,3,4,4,5,5,6,6, 7,7,8,8,9,9,10,10,10heptadecafluoro- were analysed at the limit concentration level at test start and test end. Details of the analytical results are given in Part 10. The initially measured concentration of 1Decanol, 3,3,4,4,5,5,6,6, 7,7,8,8,9,9,10,10,10-heptadecafluoro- was 0.20 mg/L. At the end of the test the concentration of the test item was below the detection limit of 0.009 mg/L. Sorption to the glass walls of the test vessels is unlikely as the walls were already pre-coated for 14 days before the test has started. The effects of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- based on Initially measured a.i. concentration are summarized in Table 1. OOOOIO Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 8 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Tab. 1: NOEC/LOEC and ECso-Values (0-72 h) of Biomass Inhibition and Rate-Related Inhibition NOEC/LOEC calculated using ANOVA (One Way Analysis of Variances) and Dunnett's test (a=0.05), based on initially measured concentration [m g/L]. EbCso 95 % confidence interval LOEC NOEC ErC5o 95 % confidence interval LOEC NOEC Inhibition of biomass growth [mg/L] > 0.20 Not applicable > 0.20 0.20 Rate-related inhibition [mg/L] > 0.20 Not applicable > 0.20 0.20 C o n c lu s io n In this study 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- was found not to inhibit the growth of the freshwater green alga Scenedesmus subspicatus at the. saturated solution level, containing 0.20 mg/L (initially measured concentration). O O O O ll dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 9 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 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 Active ingredient (EINECS) Purity C8-perfluoroalkylethanol 99.3 % (a/a), sum perfluoroalkylethanol 99.9 % (a/a) 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/cm3 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. 000012 Dr.U.Noack-Laboratorium fr angewandte Biologie Report Page 10 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 3 Method TEST GUIDELINE OECD-Guideline No. 201 for Testing of Chemicals (adopted 7June, 1984) TYPE AND PURPOSE OF THE STUDY Alga, growth inhibition test for determination of the effects on growth rate and biomass production with a saturated solution of the test item over a period of 72 h (Limit Test). TEST SYSTEM Test organism Reason for the selection of the test system Origin Date of receipt Method of cultivation Culture medium Scenedesmus subspicatus CHODAT SAG 86.81 According to the guideline Scenedesmus subspicatus CHODAT SAG 86.81 is suitable for this kind of study. Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universitt Gttingen, Nikolausberger W eg 18, D-37073 Gttingen 1997-04-07 Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 pE/m2 - s for 24 h per day. Nutrient medium Z according to LCittge et al. (1994). TEST ITEM Pretreatment of the test item 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- The test item was heated to 50 C before weighing for homogenisation. 000013 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 11 of 39 1-Oecanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Coating of the test vessels A coating phase of 14 days was carried out. Preparation 200 mg 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro-/L dilution water (total volume 2 L) were shaken in a rotating shaker with 20 rpm for 96 h at 20 C. 2 L -brown glass flasks were used. After phase separation 100 mL per test replicate was transferred from the clear liquid phase to 250 mL Erlenmeyer flasks and closed with plugs. Dilution water was used for the preparation of the coating solution. Coating period The readily filled and closed Erlenmeyer flasks were stored at nominally 23 2 C for 14 days. At the end of the coating period the flasks were emptied and used for the test. Preparation of the test medium (saturated solution) 200 mg 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- /L dilution water (total volume 2 L) were shaken in a rotating shaker with 20 rpm for 96 h at 20 C. 2 L -brown glass flasks were used. This suspension was centrifuged for 20 min at 3000 rpm. 100 mL per test replicate of the clear saturated solution was transferred to the coated Erlenmeyer flasks. The preparation method was chosen with regard to the test item properties and in accordance with the relevant guidance documents (OECD 2000, BAUA 2001). CONTROL Six replicates (without test item) were tested under the same test conditions as the test replicates. SELF FLUORESCENCE No self fluorescence was found at the saturated solution. REFERENCE ITEM Potassium dichromate p.a. (M e r c k ) CAS No. 7778 - 50 - 9 Purity 100.2 % Batch No. K 233 78864646 Test concentrations (nom.) 0.1 - 0.2 - 0.4 - 0.8 - 1 .6 - 3 .2 mg/L 000014 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 12 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Range of validity Experimental date 0.20 mg/L < EbC50 (72 h) < 0.75 mg/L 0.60 mg/L < ErC50 (72 h) < 1.03 mg/L November 27 to 30, 2001 TEST CONDITIONS Performance of the test Exposure duration Replicates Test container Test volume Dilution water Preculture Static procedure Experimental start date: April 01, 2002 Experimental completion date: April 04, 2002 Three replicates for limit concentration level, 6 per control Erlenmeyerflasks, 250 mL, with cotton wool plugs 100 mL According to the guideline: Comportant NH 4CI MgCI2 6 H2O CaCI2 2 H20 M gSO i 7 H20 KH2PO4 FeCI3 6 H2O Na2EDTA 2 H2O H3BO3 MnCI2 4 H2O ZnCI2 Na2MoO* 2 H2O C0 CI2 6 H2O CuCI2 2 H2O NaHCOa pH Concantration [mg/L] 15 12 18 15 1.6 0.08 0.1 0.185 0.415 3 x 10'3 7 x 10'3 1.5 x 10'3 1 x 10`5 50 8.2 t 0.2 A four day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with dilution water. For the start of the test the preculture was diluted with dilution water to receive an initial cell concentration of approximately 1 x 104 cells/mL in the replicates. All algae were from the same source and have not been used in a previous study. 000015 Dr.U.Noack-Laboratorium for Angewandte Biologie Report Page 13 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Application Temperature Agitation Light intensity Light regime At the test start fluorescence was measured after application of the test item. Nominally 23 2 C Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm. Nominally 60 - 120 pE/mz -s 24 h/d light KIND AND FREQUENCY OF MEASUREMENT Equipment Cell density was measured via Chlorophyll-a-fluorescence (excitation at 435 nm, emission at 685 nm). Each replicate was measured 6-fold. The cell density was measured at the beginning of the test and every 24 h. Filtrated dilution water was used as ground signal. The pH-value at the beginning of the test was measured out of one additional replicate of the limit concentration and control. At the end it was measured from a pool of all replicates. The room temperature was measured continuously by a hygrothermograph. Light intensities were measured prior to test start. Microscopic evaluation of the cells was determined at the start and end of the incubation. Alga cells were checked for any unusual cell shapes, color differences, differences in chloroplast morphology, flocculations, adherence of algae to test vessels or aggregation of alga cells. pH-Meter, pH 191 (W TW ) Hygro-thermographe (K l im a t h e r m ) Chlorophyll-impulsfluorometer (K l e in f e l d ) Shaker, SM 25 A (BOhler) Centrifuge: ZK 364, ZiRBUS Inverse microscope ( Z e is s IM 25) Quantum Sensor (S kye Instrum ents Lt d ) Rotating shaker 3040, GFL Centrifuge: ZK 364, ZiRBUS Fluorescent tubes, Osram L 36 W / 11-860, daylight (test) Fluorescent tubes, Lampi 18 W , 60 cm, warmwhlte (culture) 000016 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 14 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8.8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 DETERMINATION OF THE TEST ITEM The test concentration level and control was analytically verified by gas chromatography with mass spectrometric detection (m /z = 463 and 395) at test start and test end. At the end of the test replicates with alga were analysed. All details of the analytical method are given in part 10. COURSE OF THE STUDY Based on the results of a preliminary range finding test (NONGLP), the definitive test was performed with 1 limit concentration level with 3 replicates. A control with dilution water (without test item, six replicates) was tested under the same conditions as the test group. A four day-old preculture incubated at study conditions was used for the main study. Chlorophyll-fluorescence was determined at the beginning and after 24, 48 and 72 h. Other measurements were performed as specified above. CALCULATIONS AND STATISTICS Cell densities for each replicate were calculated from the fluorescence values 3-6 (see raw data). The fluorescence values were related to cell density values as follows: Calculation of cell density out of fluorescence values x = 63 Fq + 3625 x = cell density cells/mL 63 = factor cells/mL/rel.units/10 mL Fq = mean fluorescence value of all replicates per concentration rel.units/10 mL 3625 = cell density cells/mL Correlation dated April 12, 2001, r2 = 0.9963, n=8 The growth rate, the inhibition of biomass and the rate related inhibition after 72 h were calculated from the cell density values according to the formulas listed below. 000017 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 15 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Calculation of biomass integral N! - n0 n, + Ni -2 n0 n,,.,+ n,, - 2N 0 a = ------------- 1, + -------------------------- <t* - 1,) + ... + -------------------------- ( 1 ) 22 2 A = Area No = nominal number of cells/mL at time to Ni = measured number of cells/mL at time ti Nn = measured number of cells/mL at time t,, ti = time of first measurement after beginning of test t,, = time of n,h measurement after beginning of test Calculation of biomass inhibition lA = ((Ac - AT) / Ac) 100 % U = biomass-inhibition % (2) Ac = biomass-integral in the control after n d in cells/mL A t = biomass-integral in the test concentration after n d in cells/mL Calculation of growth rate p = (In (N,,) - In (N 0)) / (t,, - 10) M = growth rate cell density/d N,, = biomass after t,, d in cells/mL No = biomass at to In cells/mL to = time of beginning of test tn = time of n,h measurement after beginning of test (3) Calculation of rate related inhibition lMt = ((pc - pt) / pc) 1 0 0 % l,,, = rate related inhibition % (jc = growth rate of the control after n d Mi = growth rate of the test concentrations after n d (4) NOEC, LOEC and statistical analysis One way analysis of variance (ANOVA) , Du n nett's and W iiliam's test of growth rate and biomass integrals, respectively were carried out for the determination of statistically significant differences compared to control replicates. When running a one way analysis of variance a normality test and an equal variance test was done first. P-values for both normality and equal variance tests were 0.05. The cc-value for ANOVA and Dunnett's test (acceptable probability of incorrectly concluding that there is a difference) was a=0.05. 000018 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 16 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Software VALIDITY CRITERIA The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently if calculated manually based on the given data minor variations may occur from these figures. Calculations were carried out using software - SigmaPlot rel. 2000 (2000), Spss In c . - SigmaStat rel. 2.03 (1992-1997), S pss In c. - Excel 2000 (2000), Microsoft C orporation - TOXSTAT rel. 3.4 (1994), Western Ecosystems Technology, Inc., Cheyenne, W Y 82001 & David D. Gulley, University of Wyoming, WY, USA. The cell density had to increase at minimum 16-fold in the control replicates within 72 h. The temperature during the test had to be in the range of 21-25 C controlled at 2 C. The pH-value of the control replicates should not normally deviate by more than 1.0 units during the test. DEVIATIONS FROM THE GUIDELINE None DEVIATIONS FROM THE STUDY PLAN REPORTING ARCHIVING Purity was C8-perfluoroalkylethanol 99.3 % (ala). The pH-value at the beginning of the test was measured out of one additional replicate. LOD and LOQ were 0.009 and 0.027 mg/L, respectively. These deviations had no impact on the quality and integrity of the study. The final report is issued as paper copy (6 originals) and an electronic record (1 original). The following was retained in the archive of the test facility for the period as specified in the operative national GLP regulations: all raw data Study plan (Original 1 of 2) final report paper copy (Original 1 of 6) all records performed by the quality assurance programme including master schedules sample of test item Microfilms will be retained in a safe-deposit by Voiksbank Sarstedt, D-31157 Sarstedt. 000019 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 17 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedosmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 4 Results 4.1 Preliminary Test The results of the preliminary test conducted February 25 to 28, 2002 are presented below: Tab. 2: Preliminary Test Results: Cell Density Values (2 replicates each, control: 4 replicates) Dilution level Replicate No. 1:1 of the saturated solution 1:10 of the saturated solution Control 1 2 1 2 1 2 3 4 Cell density [cells/mL] 0 h 72 h 10965 10697 11705 11358 11626 11390 10587 11547 611103 471400 537078 473763 586848 598818 626065 593463 No self fluorescence was found at the saturated solution 4.2 Definitive Test Cell densities are listed in Table 4. Evaluations of biomass inhibition and rate-related inhibition are given in Table 5 (calculated out of cell density values). The growth inhibition effects of the test item are summarized in Table 3. Microscopic evaluation of the cells at the start and end of the incubation period revealed no morphological abnormalities. W ater quality parameters of pH-value, measured at 0 and 72 h, and room temperature, measured continuously, met the guideline requirements. 000020 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 18 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Tab. 3: NOEC/LOEC and ECso-Values (0-72 h) of Biomass Inhibition and Rate-Related Inhibition NOEC/LOEC calculated using ANOVA (One Way Analysis of Variances) and Dunnett's test (a=0.05), based on initially measured concentration [mg/L] . EbC5o 95 % confidence interval LOEC NOEC ErC5o 95 % confidence interval LOEC NOEC Inhibition of biomass growth [mg/L] > 0.20 Not applicable > 0.20 0.20 Rate-related inhibition [mg/L] > 0.20 Not applicable > 0.20 0.20 Tab. 4: Cell Densities Based on Initially Measured Concentrations Test item 0.20 mg/L Control Replicate No. 0 h 1 9075 2 9437 3 9453 mv 9322 1 9468 2 9342 3 9390 4 8602 5 9169 6 8854 mv 9138 mv = mean value Cell Density [cells/mL] 24 h 48 h 39000 200563 37724 39157 227968 196594 38627 208375 28242 31770 33219 32495 32904 165535 180624 177253 173190 189664 32873 182010 31917 178046 72 h 609055 609685 587478 602073 555505 598188 595038 631263 577398 637405 599133 000021 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 19 of 39 1-Oecanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Tab. 5: Evaluation after 72 h Statistically significant differences of biomass integrals and growth rates compared to control values are marked (+), not significant differences are marked (-). NOEC was calculated using ANOVA (One Way Analysis of Variances), Dunnett's and W illiam's test (a=0.05), based on initially measured concentrations. Test item Replicate Biomass Integral Inhibition of Biomass Growth Rate No. [%1 1 521403 - 7.13 1.40 0.20 mg/L 2 546942 - 12.38 1.39 3 505858 - 3.94 1.38 mv 524734 (+*) - 7.82 1.39 1 447860 1.36 2 488133 1.39 Control 3 484516 4 499812 1.38 1.43 5 488345 1.38 6 511451 1.43 mv 486685 1.39 mv = mean value negative Inhibitions = increase of growth * significant difference caused by increase of growth Rate-Related Inhibition [%] -0.56 0.35 1.28 (-) 0.36 000022 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 20 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 4.3 Test of the Reference Item The acute toxicity of potassium dichromate to the unicellular freshwater green alga Scenedesmus subspicatus was determined over a period of 72 h from November 27 to 30. 2001. Tab. 6: ECso-Values of Biomass Inhibition and Rate-Related Inhibition of the Reference Item based on nominal concentrations [mg/L], (0-72 h) EbCso 95 % confidence interval ErCso 95 % confidence interval Inhibition of biomass growth 0.49 0.45 - 0.54 Rate-related Inhibition 0.94 0.82-1.07 The EC50 - values of the reference item potassium dichromate after 72 h met the validity criteria according to EEC Directive 92/69/EEC Method C.3 Annex 2 (valid ranges are 0.20 0.75 mg/L and 0.60 - 1.03 mg/L potassium dichromate for inhibition of biomass growth and rate-related inhibition, respectively). 5 Validity Criteria The study met the validity criteria of the guideline: The cell growth increased 66-fold after 72 h in the control (required: 16-fold). The temperature during the test was in the range of 21-25 C. The difference from the mean was within 2 C: min.: 22.0 C, max.: 24.0 C mean value: 23.0 C. The pH-value in the control replicates increased not higher than 1.0 units: 8.13 to 8.54. 000023 Dr .U.Noack-Laboratorium fr Angewandte Biologie Report Page 21 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafIuoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 6 Conclusions In this study 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- was not found to inhibit the growth of the freshwater green alga Scenedesmus subspicatus tested with a saturated solution, containing 0.20 mg/L (initially measured concentration). 7 Literature / References (1) BAUA, Bundesanstalt fr Arbeitsschutz und Arbeitsmedizin (2001): Leitfaden fr Anmeldungen neuer Stoffe nach dem Chemikaliengesetz. 3. berarbeitete Auflage. (2) DuPont, Determination of physico-chemical parameters of Perfluorooctylethanol, CAS No. 67839-7, Unpublished results, Update Feb. 15,2002 (3) EEC Directive 92/69/EEC Method C.3, EEC L 383A/179 (December, 1992) (4) Lttge, U, Schnepf, E., LAuchli, A., Nagata, T. (editors, 1994): Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), T hiemeVerlag. (5) OECD - guideline no. 201 for testing of chemicals: Alga, growth inhibition test. Adopted June 7, 1984 (6) OECD Series on Testing and Assessment, No 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures ENV/JM/Mono (2000)6. 000024 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 22 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 8 Graph 0 24 48 Time h Fig. 1: Cell Densities for Control and Limit Concentration (0-72 h) 72 000025 Dr.u .Noack-laboratorium for Angewandte Biologie Report Page 23 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 9 Physicochemical Data Tab. 7: W ater Parameters Based on initially measured concentrations Control 0.20 mg/L pH-value Start: 0 h End;72 h 8.13 8.17 8.54 8.20 Temperature: min.: 22.0 C max.: 24.0 *C mean value: 23.0 C The mean light intensity was 67.2 pE/m2 s (minimal 61.5, maximal 74.2 pE/m2 s). 000026 Dr .U.Noack-Laboratorium fr Angewandte Biologie Report Page 24 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 10 GC - Analysis of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9, 10,10,10-heptadecafluoro- 10.1 Method METHOD OF D E T E R M IN A T IO N Equipment Reagents Analyses of various concentrations of 1-Decanol. 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecaflUorowere carried out via gas chromatography on a Fused Silica CPSIL 8 capillary column using external standards. Detection was performed with a mass selective detector (MS). Gas chromatograph Autosampler Detector Software : CP-3800, V a r ia n : CTC Combi PAL, CTC A n a l y t ic s : MS, Saturn 2000, V a r ia n : Saturn GC/MS Workstation 5.51, V a r ia n ferf.-butyl methyl ether 99.8 %, Batch 423370/1 31101, Fluka CONDITIONS OF ANALYSIS Column Injector Injector temperature Injection volume Temperature programme Chrompack CP-SIL 8 (Varian), 30 m, i.d.: 0.25 mm, LOW BLEED / MS, Film thickness: 0.25 pm Splitless (30 psi pressure puls) for 1 min 250 X 1 pL Heating rate [C/min] -- 10 30 Temperature [X ] 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 oooc Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 25 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 STANDARD PREPARATION OF STANDARDS PREPARATION OF SAMPLES Test item was used as standard. Two stock solutions of the standard of 200 mg/L were prepared in ferf.-butyl methyl ether, diluted (9 concentrations each) and used for calibration. For calibration range see part 10.2.1. 40 mL of the sample were extracted with 4.0 mL ferf.-butyl methyl ether. After phase separation the extract was injected directly into the GC/MS. METHOD VALIDATION Peak symmetry Repeatability Accuracy 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 limits 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 os/ (2 f) T Wo 05 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. 5 samples with 40 mL water were spiked with an amount of 6 pg 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro(80 pL of 100 mg/L) to give a final concentration of 200 pg/L. Afterwards samples were extracted as described above. 000028 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 26 of 39 1-Decanoi, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 10.2 Method Validation The method was validated and tested with satisfactory results with respect to linearity precision (repeatability) peak symmetry accuracy determination limit and detection limit. 10.2.1 Linearity The analytical system gave linear response in the range of 0.1 - 1 0 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 2 - 7 . For determination and detection limits see part 10.3. 10.2.2 Quantification Limit and Detection Limit The limit of quantification (LOQ) and limit of detection (LOD) were calculated using the blind value method described in DIN 32645 "Nachweis- und Bestimmungsgrenze". The LOD was determined to be 0.009 mg/L and therefore the LOQ was 0.027 mg/L (threefold the LOD). 000029 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 27 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8.9,9,10,10,1O-heptadecafluoro- Alga, Growth inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 10.2.3 Precision (Repeatability) The results of the repeatability tests are provided in the table below. Tab. 8: Repeatability of Injections of Test Item Peak area tMV's] Serial No 1 2 3 4 5 Mean SD CV [%] 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,B,9,9,10,10,10- heptadecafluoro- (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 10.2.4 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. 000030 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 28 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,1O-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201_____________Study-No. S S 084111 10.2.5 Accuracy Tab. 9: Mean Recovery Rate of Fortified Samples Fortified concentration of test item 0.1986 [mg/L] Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Mean SD (N-1) CV% LOQ [mg/L] LOD [mg/L] Linearity correlation coefficient ( r2 ) 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9.10,10,10 -heptadecafluoro- calc. cone. [mg/L] RR [%] 0.22 111 0.24 121 0.23 116 0.21 106 0.21 106 0.21 106 0.22 111 0.21 106 0.21 106 0.21 106 110 5.3 0.027 4.8 0.009 0.9906 calc. cone. RR LOQ LOD SD (N-1) CV calculated concentration, accumulation 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 00031 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 29 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,1O-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 10.3 Results Tab. 10: Concentrations of the Active Ingredient 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10, 1 0 ,1 0-h ep ta d e c a f luo ro - Sampling Date Start of Analysis 2002-04-01 Test Start 2002-04-05 2002-04-04 Test End 2002-04-05 Test Item Replicate No. 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10- heptadecafluoro- Calc. Cone. [mg/L] Calc. Cone. [mg/L] Saturated solution 1 2 0.17 0.18 0.22 0.22 < LOD < LOD < LOD < LOD Control LOQ [mg/L] LOD [mg/L] linearity ( r2 ) < LOD < LOD < LOD < LOD 0.027 | 0.009 || 0.9996 Mean11 [mg/L] 0.20 calc. cone. LOQ LOD 1) = calculated concentration, accumulation factor taken Into account = Limit of quantitation = Limit of detection = concentrations from the test end were not taken into account for mean value, because they were < LOD 000032 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 30 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scanedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 10.4 Calibration Curves The calibration curves shown were established for different concentration ranges and at different times Calibration Curve of Fluowet (2002-04-04) Fig. 2: Calibration Curve of Standard - concentration range 0.01 - 0.06 mg/L (dated 2002-04-04) 000033 Dr .U.Noack-Laboratorium fr angewandte Biologie Report Page 31 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8.8,9,9,10,10,1O-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201_____________Study-No. S S 0 8 4 1 11 Calibration Curve of Fluowet (2002-04-10) 500000 450000 400000 ^ 350000 300000 " 250000 2 200000 < 150000 100000 50000 0 024 6 8 Conc. [mgfL] 10 Fig. 3: Calibration Curve of Standard - concentration range 0.1 -1 0 mg/L (dated 2002-04-10) Calibration Curve of Fluowet (2002-06-19) Fig. 4: Calibration Curve of Standard concentration range 0.1 - 0.6 mg/L (dated 2002-06-19) 000034 ________________ Dr.U.Noack-Laboratorium fr Angewandte Biologie_______________ Report Page 32 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 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. 000035 ________________ Dr.UjNoack-Laboratorium fr Angewandte Biologie_______________ Report Page 33 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 10.5 Chromatograms T g M C o rrp oinJ Raporth i1 fm m S 4 1 1 0 .1 M .S M 9 SampleID: Instrument D: MeasurementType: AcquisitionDate: CalculationOats: SampleType: Inj. SempleNotes: Divisor: 8411 0.1 I Area 10.04.02 13:21 11.04.0206:08 Calibration 1.000000 Operator. laetCallbration: CalibrationType: DateFile: Method Multiplier: 11.04.0208:08 External Standeid c\... 8411 0.1 ij.sm cl... \8411_repZmth 1.000000 PCaormampoeutnerdNumber CRPeTAeSOkNfNfusaemmtbeer Status AQrusaenlone HAFimetigohutnt(RF) CalibrationEquation BaselineCoda 4,918+/- 0,200 68+395+483 >10 >-700 UnSar, Include.Nona A41.c9tu11almin. A0,00676min. 6F7lu6o-3wa-t7 42326883 44124,941 9y7-1 +4,41256+4*+0,0 BB Statue Pass Pats Pass S9 T>nt 2 Tlr*a(mln)r 3,485-15,315 Scans 2 K M 82B b a ia t o ; El A lio Pnparatkn: N on* P a nnila: 1 Fig. 5: Chromatogram of Standard 0.1 mg/L (dated 2002-04-10) 000036 Dr.u .Noack-Laboratorium fr Angewandte Biologie Report Page 34 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201_____________Study-No. SSQ84111 Target Compound Report 1er 1 from S 8 08 4t11 E L M .S M 8 SampiaIO Instrument : Meaeutimen! Type; AcquisitionDate: CalculationData; SampleType: Inj. SampleNotes; Divisor: SS084111 EGLI Aree 05.04.0223:07 0*04.0200:39 Analysis 1,000000 Operator LastCrifcration: CalibrationType: DataPile: Method: Multiplier: 05.04.02 1 3:46 External Standard c\...sao84111 agl i c\... \8411_rap2.m!h PCaormampoeutnerdNumber RReTteOnftfisoentTime PCeAaSkNNuammbeer SQAtruaeataurtetons AHmeioghutnt FCiatlibrationEquation BaselineCode SnorifeaHon 4,925 0,200 6>9-+130954463 >>-700,0000 Linear, Ignore, None Actual 50,0,18526m8i0n.min. 0Ft7u8o*w30e-t7 M 0 00,000 y58-04-2,0285e+4x+630.0130 Status Pass Fall Paas Fan S*9 Dnt 2 'nm(min)t 3,464-15,317 Scans 2IO-19W b n b a k n : E l Auto Preparation: N o r* C ha nn el: 1 Fig. 6: Chromatogram of Test Item Saturated solution at the test end (dated 2002-04-05) 000037 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 35 of 39 1-Oecanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition T est with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. S S 084111 T g rt Compound Im port f e r i i from SSOS4111_2 E Ktr L2.SMS SampiaID: InstrumentID: Measurement Typs: AcqultkmData; CaicJationData: SampleTyps: Inj. SamplsKotes: Divisor: SS084111 2EKtr1 Area 05.04.0222:45 08.04.0208:39 Anaiys 1,000000 Operator LastCalibration: CalibrationType: DataFila: Method: Multiplier: 06.04.02 13:46 External Standard c\...ao64l11_2e kt e\...\8411_rep2.mth 1.000000 Paramater CReotmenptoiuonndTNimusmber RPCeTAaSOkNfNfsuaemmtbeer Status QArueaan tons HAmeigohutnt FCBiaatlsiabtriantisoCnoEdqeuation Specification 4,925 +/- 0,200 69*395*463 >10 > 0,000 >700 linear, Ignora, Nona Actual 41,926min. 0,00148 mia 6FM7k6n-w39e*t7 000,000 465 y- +2,0285e+4x*630,0136 States Pm Fail PFaasis Staus and E ra s : M : Missino p e * . Fig. 7: Chromatogram of Control at test end (dated 2002-04-05) 000038 Dr.U.Noack-Laboratorium fr Angewandte Biologie Report Page 36 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 11 Raw Data Fluorescence a t te s t beginnin g control cuvetteno. F1 t 91 2 SB 3 92 4 76 5 85 6 60 F2 90 67 85 73 85 82 F3 92 87 86 76 87 83 F4 94 92 90 63 90 82 conc.: Saturataci solution(0.21 mg/L) cuvetteno. F1 F2 F3 F4 1 89 63 87 87 2 89 90 92 91 3 89 88 90 94 F5 94 94 97 77 68 82 F5 86 96 93 SB: Ku Prf-Nr. Projekt-Nr. 020t 09CK SSOB4111 Ffl mvF3-F6 coir, fluor. VC[%1 overall mv 91 92,75 92.75 0.0182 87,5 90 90,75 90,75 0,0329 91 91.5 91.5 0,0423 78 79 79 >,0343 87 68 88 0,0161 85 83 83 0,0170 F6 mvF3-F6 coir, fluor. 86 66,5 86.5 90 92,25 92,25 93 92.5 92,5 VCM 0,0067 0,0285 0,0187 overall mv 90,41666667 Fluorescence after 24 h Prf-Nr. 020109CK SB: Ku Projekt-Nr. SS084111 control cuvetteno. F1 F2 F3 F4 F5 F6 mvF3-F6 coir, fluor. 1 387 375 368 361 389 405 390,75 390,75 2 459 442 457 441 436 453 446,75 446,75 3 481 471 475 474 468 462 469,75 469,75 4 460 470 467 448 462 456 458,25 458,25 5 451 471 461 464 460 474 464,75 464,75 6 474 472 465 455 465 472 464,25 464,25 VC%1 0,0260 0,0221 0,0128 0,0179 0,0138 0,0151 overall mv 449,0833333 conc.: Saturated olutlon(0.2t mg/L.) cuvettano. FI F2 F3 F4 F5 F8 mvF3-F6 con. fluor. 1 571 582 566 563 564 553 561,5 561,5 2 532 528 537 547 544 537 541,25 541.25 3 590 575 561 561 564 570 564 564 vera 0,0103 0,0093 0,0075 overall mv 555,5833333 000039 Dr .U.Noack-Laboratorium for Angewandte Biologie Report Page 37 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,1O-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 Fluorescence after 48 h SB: Ku control cuvetteno. F1 F2 F3 F4 F5 F6 mvF3-F6 1 2668 2598 2602 2592 2546 2540 2570 2 2782 2834 2822 2826 2804 2786 2809,5 3 2672 2788 2768 2768 2756 2732 2756 4 2676 2720 2732 2684 2686 2664 2691,5 5 2942 2996 2986 2940 2924 2962 2953 6 2856 2824 2836 2812 2854 2824 2831,5 cone., Saturated eolutlon {0.21 rog/L) cuvetteno. F1 F2 F3 F4 F5 F6 mvF3-FB 1 3180 3086 3182 3116 3102 3104 3126 2 3478 3520 3592 3510 3600 3542 3561 3 3008 3110 3062 3046 3068 3076 3063 Prf-Nr. Projekt-Nr. 020109CK SS084111 corr. fluor. 2570 2809,5 2756 2691,5 2953 2631,5 VC|%1 0,0123 0,0065 0,0062 0,0107 0,0091 0,0063 overall mv 2768,583333 corr. fluor. 3126 3561 3063 VC1%1 0.0121 0,0120 0,0041 overall mv 3250 Fluorescence after 72 h control cuvetteno. F1 F2 1 9140 9040 2 9770 9720 3 9690 9760 4 9800 10030 5 9060 9120 6 9730 10120 F3 8820 9610 9450 10170 9250 10140 F4 8900 9430 9510 10090 9170 10080 cone.: Saturatedeolutlon(0.21 mfl/L) cuvetteno. F1 F2 F3 F4 1 9810 10080 9800 9650 2 9670 9850 9730 9560 3 9220 9420 9430 9310 F5 8630 9410 9330 9940 9060 10170 F5 9570 9460 9250 SB: Ku F6 mvF3-F6 8690 8760 9300 9437,5 9260 9387,5 9650 9962,5 8950 9107,5 9850 10060 F6 mvF3-F6 9420 9610 9730 9620 9080 9267,5 Prtf-Nr. Projekt-Nr. 020109CK SS084111 corr. fluor. 8760 9437,5 9387.5 9962,5 9107,5 10060 vero 0,0140 0,0136 0,0121 0,0230 0.0144 0,0144 overall mv 9452,5 corr. fluor. 9610 9620 9267,5 VCf%l 0,0165 0.0139 0,0157 overall mv 9499,166667 000040 Dr.U.Noack-Laboratorium for Angewandte Biologie Report Page 38 of 39 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 12 Certificate of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- Analysis CCQUhGam/ QGumatbidHu,aMWnaifiCt HGMctimM bif 044504 BurskirclMA Fm : +49 1679 / 2674 C la ria n t i aI cnc osr dpi n eg ct ot i EoN 1n0204-c3.1eB r t i f i c a t e DP aagtee:: 11 3/. 111 . 2 0 0 1 Qur consignment Material Material-no. nat-.iNo. I-Decanoi 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10, 1U-heptadecafluoro : 104105 POO/C'Jl it; z'iff bat.?.*., of wni r '..i? .-orisiardent is a part, the fol l o w i n g values ire c;T.ermi n e u . rt-.ey cont ji.r *..i<? .nqreed p roduct s p e c i f i c a t i o n Inspect L c - . a r i v v i i ;--met r.edipee ifcat ion C 3 -?e: i ..u o - s 1ky 1e cn.cr.al >:api.i:'i;y GC Sun Periluoroalfcyiethanol Capi 1 iar y C-C Sum Periluorai kylbutanol Cap Lila ry 3C 3_m ?etflucrs1kylathene Capillary GC Water content Karl ?i scher DIN 517T7 S~M echy_pyrrclidone content Capi. I lary GC > - 97,5 > - 98,0 <-1,0 <-C, 5 <~0, 2 <-0,2 A O Result 99.3 * [a/a ] 99,9%[a/a] < 0,1%[a/ai < 0,I t [a/ a ] < 0, l \ 7he a c c / e particulars d o not release the customer from Che obligation carry cut an inspect ion c: ^oods received. to Ms. sa-cri'.r-irtnor - Wc rks inspector 000041 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- Page 39 of 39 Alga, Growth Inhibition Test with Scenedesmus subspicatus, Project-No. 020109CK 72 h, Limit-Test, with the Saturated Solution acc. to OECD 201____________ Study-No. SSQ84111 13 GLP-Certificate of Dr.U.Noack-Laboratorium n Niedarachafachas Gute Laborpraxts/Good Laboratory Practice GLP-Beschelnlgung/Statement of GLP Compliance (gama/Bccorellrtg to $ 19 b Ab.1 ChenrlkaHaogesatz) Eine GLP-Inspektlon zur berwachung dar Einhaltung der GLP-Grundstze gemtD Chemikaliengesetz bzw. Richtlinie B8/320/EG wurde durchgefhrt In: Assasscnsnt of conformity with GLP according to Chemlkallengetetz and Directive 8B/320/EEC at: X Prfeinrichtung / Teat facility Prfstandort / Teat site O r. U. NOACK LABORATO RIUM fr angewandte Biologie Kthe-Paulus-Str. 1 D -31157 Sarstedt D r. U. NOACK LABO RATO RY of applied biology Kthe-Paulus-Str. 1 D -31157 Sarstedt (UnvarwacheatoaraBezeichnungundAdreeeWUnequtvocal nameandaddraaa) Prfungen nach Kategorien/Areas of Expertise (gemiSfeccorSIngChanWWV-OLPNr. S.lioeco guManos) 1 - Prfungen zur Bestimmung der physikalisch-chemischen Eigenschaften und Gehaltsbestfmmungen 3 - Prfungen zur Bestimmung der erbgutverndemden Eigenschaften (ln vitro und In vivo) 4 - Umwelttoxikologische Prfungen zu Auswirkungen auf aquatlsche und terrestrische Organismen 5 - Prfungen zum Verhalten im Boden, Im W asser und in der Luft. Prfungen zur Bioakkumulation und zur Metabolisierung B - Prfungen zur Bestimmung von Rckstnden 7 - Prfungen zur Bestimmung der Auswirkungen auf Mesokosmen und natrliche kosystem e Datum der Inspektion / D ate of Inspection (Tag.MonaLJahr / day.month.ysar) 25. - 27. Juni 2 0 0 1 1June 2 5 * - 2 7 *. 2001 29. A ugust und 21. S eptem ber 2001 / A ugust 2 9 * and Septem ber 2 1 *', 2001 Dia genannte Prfeinrichtung/Der gewannte PdHatandad befindet sich im nationalen GLPberwachungaverfahren und wird regelmJMUg auf Einhaltung dar GLP-Grundsitoe Obarwcht. Auf der Grundlagedes Inspektionsberichtes wirdhiermit besttigt, dass in dieser PrOfeinrichtungMeeem Prafstenden die oben genannten Prflingen unter Einhaltung der GLP-GrundsBtze durchgefOhrt werden knnen. Theabove mentionedtat facUtyl teat >Hs teincludedln the nattonai GLP Comptianoe Programme and hl inspectedona regularbasis. Baaed on the inspectionreport Itcm be confirmed, that this test fectlty t tset ska is able to conduct the aforementionedstudies incompRaneewiththe Principles of GLP. 11. Mine2002 ImAuftrge y Dr. Braedt 000042