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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- Daphnia magna Reproduction Test (21 d) Limit Test according to OECD-Guideline 211 for Testing of Chemicals (adopted September 198) Sponsor Telomer Research Project Asahi Glass Clariant Daikin Industries Ltd Dupont Author Martina Noack Testing Facility D r .U .N o a c k -L ab o r a to r iu m FR ANGEWANDTE BIOLOGIE Kthe-Paulus-Str. 1 D-31157 Sarstedt Laboratory Project ID Project-No. 020719CK Study-No. DRE84111 Study-No. of the Study Plan: DRE8411- Page 1 of 50 Study Completed on 2003-10-17 DR.U.NOACK-LABORATORIUM FR ANGEWANDTE BIOLOGIE Report i 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Daphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 2 of 50 Project-No. 020719CK Study-No. DRE84111 Statement of GLP Compliance Title Guideline Test Item Testing Facility 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoroDaphnia magna Reproduction Test (semi-static, 21 d), Limit Test OECD No. 211 for Testing of Chemicals 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro(Batch number: P00/001) Dr .U.Noack-Laboratorium fr angew andte Biologie Kthe-Paulus-Str.1, D-31157 Sarstedt Phone: (+49) 050 66/706 70, Fax: (+49) 05066/706 789 E-mail: info@ noack-lab.de Deviations to GLP Regulations None We 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. October 17, 2003 (Martina Noack, Study Director) October 17, 2003 October 17, 2003 (Thomas Geffke, Scientist) of Testing Facility) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 3 of 50 Project-No. 020719CK Study-No. DRE84111 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-heptadecafluoroDaphnia magna Reproduction Test (semi-static, 21 d), Limit Test OECD No. 211 for Testing of Chemicals 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro(Batch number: P00/001) Martina Noack The study was verified as follows: Inspection study plan study based report dates 2003-06-23 2003-06-30 2003-07-02 2003-09-09 2003-09-10 2003-10-15 2003-10-16 2003-10-17 date of report 2003-06-23 2003-06-30 2003-07-02 2003-09-09 2003-09-10 2003-10-15 2003-10-17 2003-10-17 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. October 17, 2003 s&r: Gudruh Mhrmann-Kalabokidls DR.U.NOACK-LABORATORIUM FUR ANGEWANDTE BIOLOGIE Report 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 4 of 50 Project-No. 020719CK Study-No. DRE84111 Personnel Involved Study Director: Martina Noack (Biologist) Scientist: (Analytical part) Thomas Geffke (Chemical Engineer) Technical Staff: Anke Gehlert Gwendolln Kraetzlg Jens Lange Quality Assurance Unit: Deputy: Gudrun Mhrmann-Kalabokidis (Biologist) Susanne Becker (Biologist) Director of Testing Facility: Dr. Udo Noack (Biologist) 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- Daphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 5 of 50 Project-No. 020719CK Study-No. DRE84111 Table of Content Page Title Page 1 Statem ent of GLP Compliance 2 Statem ent of Quality Assurance Unit 3 Personnel Involved 4 Table of Content 5 List of Tables 6 List of Figures______________________________________________________ ______________ 6_ 1 Summary 7 2 Characterisation Data of the Test Item 11 3 Method 12 4 Results 19 4.1 Preliminary Tests 19 4.2 Results of the Definitive Test 20 4.2.1 Reproductive Output 20 4.2.2 Immobile Juveniles 22 4.2.3 Number of Aborted Eggs Producing no Juveniles 22 4.2.4 First Appearence of Juveniles 23 4.2.5 Immobilisation of the Parental 23 4.2.6 Growth (Total Length and Dry Weight) of the Parent Animals 23 4.2.7 Presence of Males 24 4.2.8 Occurrence of Ephippia (Winter Eggs) 24 4.2.9 Water Parameters 24 4.2.10 Acute Immobilisation Test of the Reference Item 24 4.2.11 Conclusions 24 5 Validity Criteria 25 6 Literature / References 25 7 Observations at Test and Control Groups 26 8 Water Parameters 29 9 GC - Analysis of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9, 10,10,10- heptadecafluoro- 30 9.1 Method 30 9.2 Method Validation 33 9.2.1 Linearity 33 9.2.2 Quantification Limit and Detection Limit 33 9.3 Results 34 9.4 Calibration Curves 41 9.5 Chromatograms 43 10 Certificate of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10- heptadecafluoro- Analysis 49 11 GLP-Certificate of Dr .U.Noack-Laboratorium 50 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 6 of 50 Project-No. 020719CK Study-No. DRE84111 List of Tables Page Table 1: Table 2: Table 3: Table 4: Table 5: Table 6: Table 7: Table 8: Table 9: Table 10: Table 11: Table 12: Table 13: Table 14: Table 15: Table 16: Table 17: Table 18: Table 19: Table 20: Summary of Test Item Related Effects in the Limit Concentrations Immobilisation Rates of the Preliminary Acute Immobilisation Test [%] Number of Juveniles in the Control, Solvent Control and Test Groups after 21 Days Number of Immobile Juveniles Number of Aborted Eggs First Appearance of Juveniles in the Individual Groups Total Length and Dry Weight of the Parent Animals Observations at the Control Group Observations at the Solvent Control Group Observations at the Limit Concentration pH-Values Dissolved Oxygen Concentration [mg/L] Total Hardness [mg/L] as CaC03 Temperature [C] Dilution of Samples of Stock Solution Concentrations of the Active Ingredient 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10, Concentrations of the Active Ingredient 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10, Concentrations of the Active Ingredient 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10, Geometric Mean Exposure Concentration of 1-Decanol, 3.3.4.4.5.5.6.6.7.7.8.8.9.9.10.10.10- heptadecafluoroTime Weighted Mean Exposure Concentration of 1-Decanol, 3.3.4.4.5.5.6.6.7.7.8.8.9.9.10.10.10- heptadecafluoro- 9 19 21 22 22 23 23 26 27 28 29 29 29 29 31 34 36 38 40 40 List of Figures Figure 1: Course of the Test Item Concentration during 3 Renewal Intervals Figure 2: Course of Total Number of Hatched Juveniles after 21 Days Figure 3: Calibration Curve of the Test Item Figure 4: Calibration Curve of the Test Item Figure 5: Chromatogram of Standard of Test Item Figure 6: Chromatogram of Sample at 0 h, day 14 Figure 7: Chromatogram of Sample at 48 h, day 16 Figure 8: Chromatogram of Control, day 16 Figure 9: Chromatogram of Solvent Control, day 16 Figure 10: Chromatogram of Stock Solution, day 16 Page 8 20 41 42 43 44 45 46 47 48 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 7 of 50 Project-No. 020719CK Study-No. DRE84111 1 Summary The Daphnia magna Reproduction Test (21 d) of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- (Batch No. P00/001) was conducted according to the OECD Guideline No. 211 (September 1998) from 2003-06-30 to 2003-07-21 at Dr.U.Noack-Laboratorium fr angew andte Biologie in Sarstedt, Germany. Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held were used for the limit concentration, control and solvent control. At test start they were 2 to 24 hours old. A semi static exposure method with renewal of the test media every 48 h was carried out. This exposure method had been chosen based on the findings of preliminary investigations. They had been carried out as a tiered approach as recommended in the OECD guidance document on aquatic toxicity testing of difficult substances. Open and closed systems, with and without headspace, had been tested under semi-static and flow-through conditions. Different flow-through test designs, which had been run in preliminary Investigations, failed to give reproducible test item concentrations. During these tests deviating test item concentrations were caused by the high volatility of the test item. Therefore a semi static method was found to be more suitable. The test design had been chosen in accordance with recommendations of the OECD guidance document on aquatic toxicity testing of difficult substances. The study was carried out with one limit concentration of the test item, chosen in relation to the water solubility level: nominal 0.13 mg test item/L. With regard to the low solubility and high volatility of the test item the study was carried out with a solvent. Methanol was used for the preparation of the stock solution and the test item concentration. The methanol concentration in the test replicates and the solvent control replicates was 0.1 mL/L. The aim of the Daphnia Reproduction Test over 21 days was to assess the effects on the reproduction capacity and other test item-related effects on parameters such as body weight, length, immobilisation of the parental, time of production of first brood, aborted eggs and immobile juveniles. The 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 determined during three 48 h lasting periods within the study. The sampling intervals were 0, 6, 12, 24 and 48 h. The measuring periods were between the study days 0 - 2, 8 - 10 and 14 - 16. The concentrations in the corresponding stock solutions were also analysed. As expected the analytical findings well demonstrated that due to the high volatility of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- the initially found concentrations dropped down following a second order polynomial regression curve with coefficients r2 > 0.985 (see figure 1 and table 19). Details of the analytical results are given in part 9. Using the analytically verified concentrations the geometric mean exposure concentration was calculated to be 45.3 pg 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro-/L and all effect levels are based on figure 1 (for details please refer to page 32 (equation 1) and page 40 (results)). As an alternative, the time weighted mean measured concentration was calculated to be 64.3 pg a.i./L (for details please refer to page 32 (equation 2,3) and page 40 (results)). 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 8 of 50 Project-No. 020719CK Study-No. DRE84111 1st Measuring Period 2nd Measuring Period Time [h] 3rd Measuring Period Figure 1: Course of the Test Item Concentration during 3 Renewal Intervals 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 9 of 50 Project-No. 020719CK Study-No. DRE84111 The average number of juveniles per parent in the untreated control group was 72.4 and in the solvent control group 60.5 after 21 days. At the limit concentration the reproductive output was statistically not significant reduced compared to the control and solvent control group (ANOVA, Bonferroni t-test, p < 0.05, Table 1 and 3). The coefficient of variation around the mean number of living offspring produced per parent in the control and solvent control group was < 25 % (Table 3). Immobile juveniles were observed at the solvent control and at the limit concentration (Table 1 and 4). Aborted eggs were observed at the solvent control and at the limit concentration (Table 1 and 5). No winter eggs occurred in control, solvent control or limit concentration. Immobile juveniles and aborted eggs occurred in the limit concentration and solvent control. The reproduction rate is not significantly reduced in the treatment group. A significant effect is not observable due to the effects in the solvent control group. There is no statistically significant difference at the endpoint between the solvent control and the tested limit concentration. The no observed effect concentration (NOEC) was determined to be the geometric mean exposure concentration 45.3 pg a.i./L. No mortality of the parentals (> 20 %) occurred at any of the tested treatments. At the limit concentration mortality of the parentals came to 10 %. No males were present neither in control nor in test groups. A summary of all test item related effects is given in table 1. Table 1: Summary of Test Item Related Effects in the Limit Concentrations Effects Number of Juveniles (Reproduction Rate) Coefficient of Variation (C V )1) Immobile Juveniles Aborted Eggs Immobilisation of the Parental2) Occurrence of Males, Winter Eggs Control no no no no no no Geometric Solvent Control Mean Exposure Concentration 45.3 pg a.i./L yes no no no yes yes yes yes no no no no no = no significant effect " = CV > 25 % 21 = > 20 % after 21 days yes = significant effect 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 10 of 50 Project-No. 020719CK Study-No. DRE84111 Statistical significance of the achieved data were controlled by One Way Analysis of Variance (ANOVA): Bonferroni t-test (number of juveniles). In order to prove the validity of the test system and test conditions at the testing facility, an acute immobilisation test according to OECD 202 (1999, Draft) was carried out with potassium dichromate as reference item once per month. The EC50 of the reference item at 1.49 mg/L after 24 hours was within the validity range of 1.0 to 2.5 mg/L according to DIN 38412 L 30 (for details see part 4.2.10). Water quality parameters as pH-value, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 11 of 50 Project-No. 020719CK Study-No. DRE84111 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 Water 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. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 12 of 50 Project-No. 020719CK Study-No. DRE84111 3 Method GUIDELINE TYPE AND PURPOSE OF THE TEST TEST SYSTEM Test organism Reason for the selection of the test organism Origin Culture Culture medium Feeding OECD Guideline 211 for Testing of Chemicals, adopted September 1998 Reproduction test on Daphnia magna STRAUS to determine the effects of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- on reproduction and immobilisation of daphnids at the water solubility level of 0.13 mg/L as limit concentration. 0.13 mg/L were chosen in relation to the water solubility level and according to the sponsor's request. Daphnia magna STRAUS (Clone 5) Daphnia magna STRAUS belongs, in accordance with the study guideline, to the suitable Daphnia types and are bred in the test facility. Institut fr Wasser-, Boden- und Lufthygiene des Bundes gesundheitsamtes, Corrensplatz 1, D-14195 Berlin In 2-3 L glass vessels with approximately 1.8 L culture medium, at 21 C (temperatures of 20 - 25 C are tolerated), in an incubator, 16 h illumination, illumination strength max. 20 pmol-m"2 -s'1. Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaC03/L. 5 - 7 x weekly ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106cells/mL. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 13 of 50 Project-No. 020719CK Study-No. DRE84111 TEST ITEM Pre-treatment Stock solution Dispersion treatment Limit concentration 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- The test item was heated to 50 - 70 C before weighing for homogenisation. A stock solution of 1.3 g test Item/L was prepared in methanol. The stock solution was not longer used than 21 d. Agitation 0.13 mg 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro-/L dissolved in 0.1 mL methanol/L (0.1 mL of the stock solution were mixed with dilution water to give a final volume of 1 L). CONTROL 10 replicates with dilution water without test Item tested under same conditions as the test group. SOLVENT CONTROL TEST METHOD Test duration Test vessels Volume of the test medium Dilution water Number of study organisms Number of replicates Age of the study organisms at test begin 10 replicates without test Item tested under same conditions as the test group. Methanol (0.1 mL/L; HPLC Gradient Grade, > 99.8 %, J.T. Baker) was used. Semi-static, renewal of test medium every 48 h 21 d Glass beakers (5 (ID) x 8 (H) cm), 100 mL, covered with parafilm 50 mL Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaC03/L. 10 animals, Individually held 10 per concentration, solvent control and control 2-24 h 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 14 of 50 Project-No. 020719CK Study-No. DRE84111 Feeding Test temperature Illumination Photoperiod Daily with algae Desmodesmus subspicatus and Chlorella vulgaris, 0.1 - 0.2 mg C / Daphnia and day. The food quantity was a Desmodesmus subspicatus (0.24 - 0.68 mL) and Chlorella vulgaris (0.24 - 0.93 mL) suspension per test vessel. It varied according to the growth of the daphnids, but was the same for all test groups on any particular day. On days between water exchange the algae were introduced by pricking a pipette tip through parafilm and covering with new parafilm. 18 - 22 C, 2 C constant Max. 15-20 pmol-m'2 s'1 16 / 8 hours light / dark cycle REFERENCE TEST A reference test was conducted as Static Acute Immobilisation Test (acc. to DIN 38412 211) with a test duration of 24 h once per month in order to prove the validity of the test system and test condition at the testing facility. Reference item Potassium dichromate p.a. (Merck) Purity 100 % Batch No. K 28974764 Storage stability 2006-02-28 Test concentrations 0 .5 8 -1 .0 -1 .8 -3 .2 -5 .8 mg/L Range of validity 1.0 mg/L < EC60 (24 hours) < 2.5 mg/L Test duration 24 h, 2003-07-08 to 2003-07-09 Test vessels Glass beakers (5 cm ID x 8 cm H), 50 mL volume will be used. Volume of the study medium 20 mL Dilution water Number of study organisms per concentration/control According to EEC 92/69 L383A C.2. Annex 20 animals, divided into 2 parallel samples, each with 10 animals Number of animals per test vessel 10 Number of parallel samples 2 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- Daphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 15 of 50 Project-No. 020719CK Study-No. DRE84111 TEST DESCRIPTION The reproduction study was performed with a nominal concentration of the test item of 0.13 mg/L at the water solubility level. 2 - 24 hours old daphnids were used for the test. They had been obtained by removing the mother animals twice within 22 h. 10 daphnids individually held were used for the limit concentration and for the solvent control and control groups. Feeding as specified above. All test vessels were placed in an acclimated incubation room (light / dark cycle 16 / 8 h) at 18 - 22 C, 2 C constant. Every 48 h the solutions were renewed at the limit concentration, solvent control and control. Observations, measurements and evaluation were carried out as described below. A reference test was performed once per month according to DIN 38412 211 in order to prove the validity of the test system and test conditions at the testing facility. TYPE AND FREQUENCY OF MEASUREMENTS AND OBSERVATIONS Measurements Observations Dissolved oxygen concentration, pH-values, total hardness and water temperature were measured once a week, in fresh and old media, in one replicate of the control, the solvent control and the test item concentration. The oxygen concentration should be > 3 mg/L at the beginning and during the test. The total hardness should be above 140 mg/L CaC03. The pH-value should be in the range of 6 - 9. Normally deviations of initial and final pH values should be < 1.5 units. The temperature in the incubator was recorded with a thermohygrograph during the test. Mortality of parent Daohnia Mortality of parent Daphnia was observed and recorded at least at every water renewal. Daphnia were considered to be dead, if they were unable to move after gentle agitation with a laboratory pin. Dead specimen were removed. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 16 of 50 Project-No. 020719CK Study-No. DRE84111 New born First appearance of juveniles was checked daily. The number of young Daphnia (mobile and immobile) were counted and recorded from the first day of production of young Daphnia until the end of the test at only every water renewal and not daily as recommended in the guideline to prevent volatility of the test item. After counting the young Daphnia were discarded. Condition of parent Daphnia Abnormalities (e.g. swimming behaviour, number of males and winter eggs) were observed and recorded at the observation days. Total length and body weight of parent Daphnia At the end of the test total length of each Daphnia and body weight of the alive Daphnia at the test item concentration, solvent control and control were determined. Measuring instruments Oxygen pH value Temperature Water hardness Fluorescence of algae : Oximeter, Oxi 530, (WTW) : pH-Meter inoLab pH Level 1 (WTW) : Hygro-Thermograph (Lufft) : Spectrophotometer, Cadas 100 LPG 158 (Dr . Lan g e) : Chlorophyll-Fluorometer (Kleinfeld) EVALUATION NOEC (No observed effect loading) LOEC (Lowest effect level) The NOEC for the reproduction was determined directly from the results. The LOEC could not be determined due to no significant effects at the tested limit concentration. The comparison of the control and the solvent control indicates a significant difference between the controls. The solvent control was used for statistical comparisons versus the limit concentration. There was no statistically significant difference at the endpoint between the solvent control and the tested limit concentration. The NOEC was reported as the calculated geometric mean exposure concentration. The tested concentration of the test item at which the test item is observed to have no significant effect when compared to the solvent control. The tested concentration of the test item at which the test item is observed to have a significant effect when compared to the solvent control. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 17 of 50 Project-No. 020719CK Study-No. DRE84111 Statistical significance Body length Biomass Reference test Software DETERMINATION OF THE TEST ITEM Sampling for analytical determination / sample preparation Reproduction rates of the treatment groups with mean values, standard deviation and the coefficient of variation were determined. The results were expressed as percentage reduction of reproduction relative to the solvent control and control. The coefficient of variation should have been < 25 %. Significant deviations concerning the reproduction rate were determined in comparison with the solvent control and control group using the One Way Analysis of Variance (ANOVA): Multiple Comparison Method using Bonferroni t-test (Table 1 and 3). The body length of all daphnids of treatment and control groups was measured (Table 7). The mean dry weights of all treatment and control groups were determined (Table 7). EC10- and ECS0-values with 95 % confidence intervals were calculated only for the reference item. Probit values were taken from W eber (1986). Confidence intervals were calculated according to a standard procedure (Br e it ig & T mpling 1982). EC-ioo-value was determined directly from the results obtained. The calculations were carried out using the software Sigma Stat of Jandel Scientific Version No. 2.0 (Windows) and Sigma Plot rel. 2000 (2000), Spss Co rporation. The test item concentration of the fresh and old media were analytically verified by gas chromatography with mass spectrometric detection (m/z = 463 and 395) three times within the study period after 0, 6, 12, 24 and 48 h. Test item concentrations were analysed in triplicates. The control, solvent control and stock solution were analysed after 0 and 48 h in one replicate. The overall recovery rates were < 80 % related to the nominal concentration. All results are based on the geometric mean exposure concentration (for details please refer to page 32 (equation 1) and page 40 (results)). As an alternative, the time weighted mean measured concentration was calculated (for details please refer to page 32 (equation 2,3) and page 40 (results)). At six water renewal intervals samples were prepared after 0, 6, 12, 24 and 48 h. For sampling after 6, 12, 24 and 48 h three additional test beakers without daphnids were prepared and incubated like the test beakers. Additionally, before the study starts 3 replicates were randomly selected by using a random generator (EXCEL) for sampling of old (48 h) test solutions out of the test chambers (with daphnid). After preparation these samples were stored in a freezer. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 18 of 50 Project-No. 020719CK Study-No. DRE84111 VALIDITY CRITERIA All samples were maintained as separate replicates. For details see part 9. Samples were directly given into ferf.-methyl-butyl ether (MTBE) and prepared for the analytical determination immediately. At three of the six sampling intervals samples were analysed immediately or stored (closed, in a freezer) until analyses. At the other three intervals the samples were stored in a freezer and only analysed, if necessary. For details see part 9. Mortality of the parent animal in the control group should not exceed 20 % at the end of the test. The mean number of live offspring produced per parent animal surviving at the end of the test should be > 60 in the control groups. The coefficient of variation around the mean number of living offspring produced per parent in the control group should be < 25 %. DEVIATIONS FROM THE TEST GUIDELINE None DEVIATIONS FROM THE STUDY PLAN None REPORTING The final report is issued as paper copy (5 originals) and an electronic record (1 original). ARCHIVING The following will be retained in the archive of the test facility for the period specified in the operative national GLP regulations: all raw data study plan (Original 1 of 2) final report (Original 1 of 5) all records performed by the quality assurance programme including master schedules samples of test and reference items Microfilms will be retained in a safe-deposit by Volksbank Sarstedt, D-31157 Sarstedt. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 19 of 50 Project-No. 020719CK Study-No. DRE84111 4 Results 4.1 Preliminary Tests A preliminary test according to OECD 202 (1999, Draft) was carried out with the saturated solution (Lab. ID: 020109CK, DAI84111). The percentage immobility was determined in all test item and control groups after 24 and 48 hours of exposure under semi-static conditions. The immobilisation rates are given in table 2. Table 2: Immobilisation Rates of the Preliminary Acute Immobilisation Test [%] (n = 20) Saturation concentration [mg/L] 0.16 Control 1. 0 0 IMMOBILISATION [%] 24 h Replicates 2. 3. 4. MV 1. 000 00 000 00 MV Mean value 48 h Replicates 2. 3. 4. 000 000 MV 0 0 Based on the results of this preliminary test an extensive number of more preliminary tests (NON-GLPState) were conducted with different test designs: under flow-through conditions semi-static test designs with and without pre-coating of the test vessels semi-static test designs with and without leaving a headspace in the test vessels (by using different test volumes or test vessels with different size). semi-static test designs with different covering material (parafilm and foil) semi-static test designs with feeding after 24 h by opening the parafilm or pricking through the parafilm with a pipette tip. A test design under flow-through conditions was refrained, due to a strong loss of the test item in the system. There were strong variations of test item concentrations measured at the inlets and the outlets of the test vessels. There were no significant differences measured if using pre-coated or not pre-coated test vessels with foil or parafilm covered, respectively. The preliminary studies showed that a headspace is necessary as in test vessels without headspace the oxygen concentrations were < than the recommended oxygen concentration for daphnids. Mortality occurred. For feasibility reasons it was not possible to prepare a saturated aqueous solution of the test item for the chronic test in analogy to the acute test. Therefore a solvent needed to be used for the preparation of test solutions. Methanol was found to be suitable and the concentration used was in compliance to the guideline. Based on the results of all preliminary tests the definitive test was conducted as described above. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 20 of 50 Project-No. 020719CK Study-No. DRE84111 4.2 Results of the Definitive Test 4.2.1 Reproductive Output The number of juveniles in all replicates of control, solvent control and test groups was recorded at every water renewal. Results concerning the number of juveniles of all test and control groups for every test day are shown in tables 8 -1 0 . The number of juveniles produced in 10 replicates of the control, solvent control and test groups between the 10thand 21st day were added to the total turnover (Table 3) and plotted versus the test item concentration (Figure 2). The mean number of offspring produced per parent animal surviving at the end of the test came to 72.4 juveniles in the control group, to 60.5 (alive and immobile) in the solvent control group and to 67.3 in the G.M.E.C. (Table 3). Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 Replicate 6 0 5 Replicate 7 Replicate 8 O Replicate 9 Replicate 10 05 O -0O5 oo control solvent control 45.3 geometric mean exposure concentration [pig a.i./L] Figure 2: Course of Total Number of Hatched Juveniles after 21 Days Number of counted juveniles per replicate (10 replicates per control, solvent control and limit concentration) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 21 of 50 Project-No. 020719CK Study-No. DRE84111 Table 3: Number of Juveniles in the Control, Solvent Control and Test Groups after 21 Days No. G.M.E.C. Number of Juveniles in Replicate No. Total of No. Parents alive [pg a.i./L] 1 2 3 4 5 6 7 8 9 10 x Mean Juveniles per Parent alive MV + SD 45.3 49 - 70 82 63 67 70 69 68 60 598 + Immobile juveniles 9 67.3+ 8.8 8 606 Solvent 56 61 57 62 60 72 60 40 65 51 584 Control + 10 60.5 6.2 Immobile 21 605 juveniles Comp arison CV versus Control Solvent Control [%] 13.1 no no 10.2 yes - Control 70 81 78 75 74 78 69 64 69 66 724 10 72.4 5.6 7.79 ~ yes G.M.E.C. = Geometric mean exposureconcentration No. = Number MV SD = Mean value Standard deviation CV = Coefficient of variation yes = Statistically significant,ANOVA, Bonferroni t-test (p < 0.5) no = Statistically not significant, ANOVA, Bonferroni t-test (p < 0.5) = Not applicable The coefficient of variation around the mean number of living offspring produced per parent in the control group came out to 7.79 % and in the solvent control group to 10.2 % (Table 3). The validity criteria that the coefficient of variation in the control should be < 25 % is fulfilled. The mean number of juveniles of the surviving daphnids of the treatment groups was compared by One Way Analysis of Variance (ANOVA) and Bonferroni t-test to the control and solvent control. The differences in the mean values among the treatment groups were greater than would be expected by chance. There was a statistically significant difference (P < 0.05) at the solvent control group when compared to the control. No statistically significant difference was seen at the geometric mean exposure concentration of 45.3 pg a.i./L when compared to the control and the solvent control. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 22 of 50 Project-No. 020719CK Study-No. DRE84111 4.2.2 Immobile Juveniles The number of immobile juveniles in the treatment groups is shown in table 4. No immobile juveniles were observed in the control group. Immobile juveniles in the solvent control and treatment group were counted only at the water renewals and not every 24 h as recommended by the guideline to prevent volatility of the test item. After 24 h the appearance of new born daphnids was checked and they were observed to be mobile. Therefore for the calculations the immobile juveniles were added to the juveniles alive. Table 4: Number of Immobile Juveniles Geometric mean exposure concentration [pg a.i./L] 45.3 Solvent Control Control Number of Immobile Juveniles 8 21 0 4.2.3 Number of Aborted Eggs Producing no Juveniles The number of aborted eggs in the treatment groups are shown in table 5. No aborted eggs were observed in the control group. Table 5: Number of Aborted Eggs Geometric mean exposure concentration [pg a.i./L] 45.3 Solvent Control Control Number of Aborted Eggs 12 6 0 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- Daphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 23 of 50 Project-No. 020719CK Study-No. DRE84111 4.2.4 First Appearance of Juveniles The first day of appearance of juveniles in individual groups is given in table 6. Table 6: First Appearance of Juveniles in the Individual Groups Geometric mean exposure concentration Day of First Appearance of Juveniles in Replicate No. [P9 a-DL] _1_ 2 3 4 5 6 7 8___9 10 45.3 11 9 9 9 9 9 9 9 9 10 Solvent Control 9999999999 Control 9999999999 First Appearance Mean Day 9.3 9.0 9.0 4.2.5 Immobilisation of the Parental No mortality of the parentals occurred at the control and solvent control. At the limit concentration mortality of the parentals came to 10 %. 4.2.6 Growth (Total Length and Dry Weight) of the Parent Animals Appearance of the parental animals was visually compared to the control and solvent control group. No significant deviations were noted in comparison to the control and solvent control group. At the end of the test the total length of each daphnia and the dry weight of all living daphnids at the control, solvent control and limit concentration were determined (Table 7). Table 7: Total Length and Dry Weight of the Parent Animals G.M.E.C. Total Length of the Parent Animals [mm] Replicate No. MV [pg a.i./L] 1 2 3 4 5 6 7 8 9 10 [mm] 45.3 Solvent Control Control 4.5 ~ 5.0 5.0 4.5 4.5 5.0 4.5 4.5 5.0 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.0 4.0 4.5 5.0 4.5 4.5 5.0 5.0 5.0 5.0 4.5 5.0 4.5 4.7 4.4 4.8 G.M.E.C. = Geometric mean exposure concentration MV = mean value Red. = Reduction, when compared to the control and solvent control N = number of daphnids alive = Not applicable N 9 10 10 Dry Wei ght [mg] MV 5.7 0.63 4.2 0.42 8.5 0.85 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 24 of 50 Project-No. 020719CK Study-No. DRE84111 4.2.7 Presence of Males No males were present neither in the control nor in the test groups during the test. 4.2.8 Occurrence of Ephippia (Winter Eggs) No ephippia occurred in control or test groups. 4.2.9 Water Parameters Table 11 shows the measured initial and final pH values of the control, solvent control and the limit concentration in one replicate per treatment group. Deviations of > 1.5 units in initial and final pH values are regarded as being significant. The Initial pH-values varied from 8.00 to 8.13 for the control, from 7.96 to 8.12 for the solvent control and from 7.93 to 8.04 for the limit concentration. The final pH-values varied from 7.33 to 8.62 for the control, from 6.97 to 7.66 for the solvent control and from 6.92 to 7.70 for the limit concentration. All deviations of initial and final pH-values were <1.5 units. The pH-values were all in a neutral range. The final oxygen concentrations of the measured replicates in the test media of the control, solvent control and limit concentration were found to be > 7.63 mg/L (Table 12). The total hardness was determined to be > 140 mg/L (Table 13). The temperature in the test vessels was within the range 20.1 to 22.0 C (Table 14). 4.2.10 Acute Immobilisation Test of the Reference Item The percentage immobility for the reference item was determined after 24 h. The EC100-value was determined directly from the test results. The EC-io-and EC50-valueswith 95 % confidence interval (Cl) were determined in a probability network by interpolation according to standard procedures. EC- values after 24 h of the reference item in mg/L: EC10 : 0.58 EC50 : 1.49 (Cl 1.18-1.87) EC-too : 5.80 The EC50-value of reference item potassium dichromate after 24 h is within the prescribed con centration range of 1.0 - 2.5 mg/L of quality criteria according to AQS: DIN Guideline 38412 L 30. 4.2.11 Conclusions The no observed effect concentration (NOEC) was determined to be the geometric mean exposure concentration 45.3 pg a.i./L. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 25 of 50 Project-No. 020719CK Study-No. DRE84111 5 Validity Criteria Mortality in the control and solvent control group did not exceed 20 % at the end of the test. The mean number of living offspring produced per parent animal surviving at the end of the test was > 60 in the control group. The coefficient of variation around the mean number of living offspring produced per parent in the control groups was < 25 %. 6 Literature / References AQS-Merkblatt: Bestimmung der nicht akut giftigen Wirkung von Abwasser gegenber Daphnien ber Verdnnungsstufen (DIN 38412 - L 30). Breitig G.& T mpling W. (1982): Methoden der Wasseruntersuchungen Bd.II, VEB Gustav Fischer Verlag, Jena. DIN 38402 Teil 51 (1986): Kalibrierung von Analysenverfahren, Auswertung von Analysenergebnissen und lineare Kalibrierfunktion fr die Bestimmung von Verfahrenskenngren DIN 38 412 Teil 11 (1982): Bestimmung der Wirkung von Wasserinhaltsstoffen auf Kleinkrebse Elendt B.P.(1990): Selenium Deficiency in Crustacea, Protoplasma 154, 2 5 -3 0 OECD Guideline 211 for Testing of Chemicals (1998): Daphnia magna STRAUS, Reproduction Test OECD Guideline 221 for Testing of Chemicals (revised draft document October 2000): Lemna sp. Growth inhibition test OECD Series on Testing and Assessment# 23 (2000): Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. W eber E. (1986): Grundri der biologischen Statistik, 9. Auflage 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- Daphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 26 of 50 Project-No. 020719CK Study-No. DRE84111 7 Observations at Test and Control Groups Table 8: Observations at the Control Group Date (2003) 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Replicate No. 1 10 Number of Juveniles 13 19 15 23 16 19 19 27 19 18 16 25 16 20 20 19 17 20 18 19 15 18 20 25 18 13 16 22 16 12 18 18 15 16 16 22 17 15 15 19 total 70 81 78 75 74 78 69 64 69 66 Mortality of Parental [N] Mortality of Parental [%] Immobile Juveniles 724 Unhatched Eggs -- no juveniles J juveniles observed, but not counted, because there was no water exchange 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- Daphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 27 of 50 Project-No. 020719CK Study-No. DRE84111 Table 9: Observations at the Solvent Control Group Date (2003) 01. 03. 04. 06. 07. 08. 09. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. 07. Day Replicate No. 1 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Number of Juveniles total 12 18 20 56 12 16 15 18 61 11 13 16 15 57 14 18 16 14 62 13 15 14 18 60 14 18 19 21 72 10 17 17 16 60 14 16 10 40 12 19 17 17 65 10 18 21 51 584 Mortality of Parental [N] Mortality of Parental [%] Immobile Juveniles Unhatched Eggs 13 21 -- no juveniles J juveniles observed, but not counted, because there was no water exchange 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- Daphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 28 of 50 Project-No. 020719CK Study-No. DRE84111 Table 10: Observations at the Limit Concentration -- no juveniles J juveniles observed, but not counted, because there was no water exchange M immobile female 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 29 of 50 Project-No. 020719CK Study-No. DRE84111 8 Water Parameters Comparison of initial (I) and final (F) values of the control, solvent control and limit concentration once per week. Table 11: pH-Values G.M.E.C. I F I F I F I [pg a.i./L] July 02. July 04. July 08. July 10. July 14. July 16. July 20. 45.3 8.02 7.21 7.93 6.92 8.04 7.61 7.96 Solvent control 8.07 7.03 7.96 7.11 8.05 6.97 8.12 Control 8.05 8.62 8.00 7.33 8.10 7.56 8.13 Water quality criteria of pH-value: Deviations < 1.5 units between the initial and final pH-values are regarded to be not significant. F July 21. 7.70 7.66 7.56 Table 12: Dissolved Oxygen Concentration [mg/L] G.M.E.C. I F I F [pg a.i./L] July 02. July 04. July 08. July 10. 45.3 8.42 7.63 9.31 8.54 Solvent control 8.48 8.38 9.19 9.44 Control 8.56 9.57 8.03 8.09 The dissolved oxygen concentration should be above 3 mg/L. I July 14. 8.38 8.37 7.97 F July 16. 7.76 7.90 8.91 I July 20. 8.80 8.94 8.46 F July 21. 7.70 7.88 7.94 Table 13: Total Hardness [mg/L] as CaCC^ G.M.E.C. I F I F [pg a.i./L] July 02. July 04. July 08. July 10. 45.3 169 167 179 171 Solvent control 166 176 179 174 Control 169 172 177 172 The total hardness should be more than 140 mg/L as CaCOs. I July 14. 165 166 165 F July 16. 163 163 167 1 July 20. 169 166 170 F July 21. 167 163 160 Table 14: Temperature [C] G.M.E.C. I F I F [pg a.i./L] July 02. July 04. July 08. July 10. 45.3 21.9 20.5 21.8 20.5 Solvent control 21.9 20.5 21.7 20.8 Control 22.0 20.1 21.5 20.9 The temperature should be within the range 18-22 C. G .M.E.C. = Geometric mean exposure concentration I July 14. 21.8 21.9 21.5 F July 16. 20.4 21.1 20.9 I July 20. 21.3 21.5 21.1 F July 21. 21.2 20.6 20.4 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 30 of 50 Project-No. 020719CK Study-No. DRE84111 9 GC - Analysis of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9, 10,10,10heptadecafluoro- 9.1 Method METHOD OF DETERMINATION Equipment Analyses of various concentrations of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro-were 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 Autosampler Detector Software CP-3800, Varian CTC Combi PAL, CTC A nalytics MS, Saturn 2000, Varian Saturn GC/MS Workstation 5.51, Varian Reagents fert.-butyl methyl ether 99.8 %, Batch 423370/1 31101, Fluka CONDITIONS OF ANALYSIS Column 1 Column 2 (for samplings started 2003-07-14) Chrompack CP-SIL 8 (Varian), Batch: 9016531,30 m, i.d.: 0.25mm, LOW BLEED / MS, Film thickness: 0.25 pm Factor Four VF-5ms (Var ian), Batch: 9024095, 30 m, i.d.: 0.25 mm, Film thickness: 0.25 pm (equivalent to Column 1) Injector Injector temperature Injection volume Temperature programme Splitless (30 psi pressure puls) for 1 min 250 C 1 pL Heating rate [C/min] -- 10 30 Temperature [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 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 31 of 50 Project-No. 020719CK Study-No. DRE84111 STANDARD PREPARATION OF STANDARDS PREPARATION OF TEST SAMPLES PREPARATION OF SAMPLES OF STOCK SOLUTION Test item was used as standard. A stock solution of the test item of 100 mg/L was prepared in fert.-butyl methyl ether, diluted (6 concentrations) and used for calibration. For calibration range see part 9.2.1. 5 mL fe/t-butyl methyl ether were given in a 50 mL conical shoulder bottle with glass stopper. 40 mL of the sample were filled into the bottle. After addition of approx. 6 g sodium chloride the bottle was closed and vigorously shaken for at least 2 min for extraction of the test item. After phase separation the ether phase was injected directly into the GC/MS. The extraction procedure included an enrichment factor of 8. Samples from the stock solutions with 1300 mg/L were diluted with tert.-butyl methyl ether prior to analysis. Dilution steps are listed in the following table. Table 15: Dilution of Samples of Stock Solution Sample Dilution Factor Sample Volume [mL] Stock Solution 1000 1) First dilution step 2) Second dilution step 0.11) 0.12) Dilution Medium [mL] 9.91) 0.92) SAMPLE STORAGE If necessary, all samples were stored at -20 2 C until start of analysis. METHOD VALIDATION 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 quantitation limits were adapted from DIN 32645. The method was previously validated as described in the report DAI84113. CALCULATION OF MEAN CONCENTRATIONS As an alternative two different mathematical procedures were chosen to calculate mean measured concentrations. 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 32 of 50 Project-No. 020719CK Study-No. DRE84111 Geometric mean exposure concentrations Geometric mean values have been calculated according to equation (1) as given in annex 2 of the OECD guidance document #23: Mean concentrt ion = ___ 1 T n-1 r y [(log(conc ;) + log(conc i+1)) (tk1 2(tn - =1 ( 1) ti = initial time < t2 < .....tn = final time conci = initial concentration, conc2 ....... . concn = final concentration From the three measuring intervals mean values have been calculated. Time weighted mean concentration For 3 renewal periods areas and time-weighted mean concentrations were calculated following equations (2) and (3) as described in OECD - guideline No. 221: Area = Cone 0 - Conci x days Ln(Conc 0) - Ln(Conc 1) (2) Cone 0 Cone 1 Days Initial concentration of each renewal period End concentration of each renewal period Duration of each renewal period TW Mean = Total Areas Total Days TW Mean = Time-weighted mean concentration (3) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 33 of 50 Project-No. 020719CK Study-No. DRE84111 9.2 Method Validation The method was validated and tested with satisfactory results with respect to linearity determination limit and detection limit. 9.2.1 Linearity The analytical system gave linear response in the range of 0.2 - 2 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 curves and chromatograms obtained for standard and test item are given in Figure 2 - Figure 9. 9.2.2 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). 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 34 of 50 Project-No. 020719CK Study-No. DRE84111 9.3 Results Table 16: 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- (sampling on days 0 and 2) S a m p lin q Date 2003-06-30 2003-07-02 Analysis Date Replicate Time Stock solution [h] 0 48 2003-06-30 2003-07-02 Solvent control 48 2003-06-30 2003-07-02 2003-07-171' Control 48 2003-06-30 2003-06-30 a.i. [mg/L] 1291 0.129 0.129 Meas. cone, [mg a.i./L] 1.487 1.530 1.795 1.876 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 1.180 1.271 1.183 1.222 1.289 1.273 0.722 0.679 0.731 0.729 0.621 0.691 Mean [mg a.i./L] 1.509 1.836 1.236 0.696 Calc, cone, [mg a.i./L] 1509 1836 0.15 0.087 RR [%] 117 142 116 67 Meas. cone. Calc. cone. LOQ 1) a.i. = measured concentration = calculated concentration, dilution or enrichment factor taken into account = Limit of quantitation = Reanalysed samples = active ingredient 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 35 of 50 Project-No. 020719CK Study-No. DRE84111 Table 16 (cont ): 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- (sampling days 0 - 2) S am p lin g Date Analysis Date Replicate 2003-06-30 2003-07-01 2003-07-17 2003-07-02 Time [h] 12 24 48 a.i. [mg/L] 0.129 0.129 0.129 Meas, cone, [mg a.i./L] 0.517 0.536 0.544 0.529 0.604 0.563 0.367 0.311 0.331 n.d. 0.387 0.360 n.d. n.d. n.d. 0.197 n.d. n.d. Mean [mg a.i./L] 0.549 0.351 0.197 Calc, cone, [mg a.i./L] 0.069 0.044 0.025 RR [%] 53 34 19 Ideas, cone. Calc. cone. LOQ 1) n.d. a.i. = measured concentration = calculated concentration, dilution or enrichment factor taken into account = Limit of quantitation = Reanalysed samples = not detected (peak spectrum match < 70 %) = active ingredient 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 36 of 50 Project-No. 020719CK Study-No. DRE84111 Table 17: 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- (sampling days 8 -10) Sampling Date Analysis I Replicate Date 2003-07-08 2003-07-10 2003-07-09 2003-07-10 Stock solution Time [h] 0 48 2003-07-08 2003-07-09 2003-07-10 2003-07-10 Solvent control 48 2003-07-08 2003-07-09 2003-07-10 2003-07-10 Control 48 2003-07-08 2003-07-09 2003-07-08 a.i. [mg/L] 1291 0.129 0.129 Meas, Mean Calc, cone, cone, [mg a.i./L] [mg a.i./L] [mg a.I./L] 1.184 1.169 1.177 1177 1.134 1.259 1.197 1197 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.984 0.903 0.951 0.968 0.949 0.12 0.927 0.962 0.566 0.611 0.618 0.540 0.578 0.072 0.560 0.570 RR [%] 91 93 93 56 Meas. cone. Calc. cone. LOQ a.i. = measured concentration = calculated concentration, dilution or enrichment factor taken into account = Limit of quantitation = active ingredient 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 37 of 50 Project-No. 020719CK Study-No. DRE84111 Table 17 (cont ): 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- (sampling days 8-10) Sampling Date Analysis Date Replicate 2003-07-08 2003-07-09 2003-07-09 2003-07-10 2003-07-10 Time [h] 12 24 48 a.i. [mg/L] 0.129 0.129 0.129 Meas, Mean Calc, cone, cone, [mg a.i./L] [mg a.i./L] [mg a.i./L] 0.445 0.498 0.459 0.435 0.444 0.056 0.411 0.415 0.296 0.299 0.275 0.251 0.277 0.035 0.284 0.254 0.170 n.d. 0.159 n.d. 0.169 0.021 n.d. 0.178 RR [%] 43 27 16 Meas. cone. Calc. cone. LOQ n.d. a.i. = measured concentration = calculated concentration, dilution or enrichment factor taken into account = Limit of quantitation = not detected (peak spectrum match < 70 %) = active Ingredient 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 38 of 50 Project-No. 020719CK Study-No. DRE84111 Table 18. 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- (sampling days 14 -16) Sampling Analysis Date ! Date 2003-07-14 2003-07-16 Replicate Stock solution Time [h] 0 48 2003-07-14 2003-07-16 Solvent control 48 2003-07-14 2003-07-16 2003-07-18 Control 48 2003-07-14 2003-07-14 a.i. [mg/L] 1291 0.129 0.129 Meas, Mean Calc, cone, cone, [mg a.i./L] [mg a.i./L] [mg a.i./L] 1.573 1.517 1.155 O 1.163 O 1.545 1.184 1545 1184 <LOQ <LOQ <LOQi2> <LOQ,2) <LOQ <LOQ <LOQ <LOQ 1.288 1.265 1.243 1.202 1.262 0.16 1.342 1.234 0.633 0.693 0.728 0.682 0.70 0.088 0.714 0.756 RR [%] 120 92 124 68 Meas. cone. Calc. cone. LOQ a.i. 1) 2) = measured concentration = calculated concentration, dilution or enrichment factor taken into account = Limit of quantitation = active ingredient = Reanalysed 2003-07-21 due to assumed dilution error; mean value of two Injections = Reanalysed 2003-07-21 due to assumed contamination of injector syringe; mean value of two injections 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 39 of 50 Project-No. 020719CK Study-No. DRE84111 Table. 18 (cont ): 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- (sampling days 14-16) Sampling Date Analysis Date Replicate 2003-07-14 2003-07-15 2003-07-18 2003-07-16 Time [h] 12 24 48 a.i. [mg/L] 0.129 0.129 0.129 Meas, Mean Calc, cone, cone, [mg a.i./L] [mg a.i./L] [mg a.i./L] 0.675 0.665 0.654 0.620 0.619 0.077 0.511 0.586 0.357 0.318 0.368 0.426 0.359 0.045 0.356 0.327 n.d. n.d. n.d. 0.152 0.152 0.019 n.d. n.d. RR [%] 60 35 15 Meas. cone. Calc. cone. LOQ n.d. a.i. = measured concentration = calculated concentration, dilution or enrichment factor taken into account = Limit of quantitation = not detected (peak spectrum match < 70 %) = active ingredient DR.U.NOACK-LABORATORIUM FUR ANGEWANDTE BIOLOGIE Report 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 40 of 50 Project-No. 020719CK Study-No. DRE84111 Table 19: Geometric Mean Exposure Concentration of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- 1sl Measuring period (days 0-2) 2nd Measuring period (days 8-10) 3rd Measuring period (days 14-16) Geometric mean exposure concentration Concentration [pg a.i./L] 48.9 39.8 47.2 45.3 Table 20: Time Weighted Mean Exposure Concentration of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- Time weighted mean exposure concentration Concentration [pg a.i./L] 64.3 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 41 of 50 Project-No. 020719CK Study-No. DRE84111 9.4 Calibration Curves Cone. [mg/L] Figure 3: Calibration Curve of the Test Item (date acquired 2003-07-09) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 42 of 50 Project-No. 020719CK Study-No. DRE84111 120000 - 100000 80000 3O o 60000 0) < 40000 20000 0 0 Cone. [mg/L] Figure 4: Calibration Curve of the Test Item (date acquired 2003-07-17) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 43 of 50 Project-No. 020719CK Study-No. DRE84111 9.5 Chromatograms GPrint Date: 18 Jul 2003 08:57:56 Target Compound Report ter#l from Sid 2,0-1.SMS Sample ID: Std 2,0 instrument ID: Measurement Type: Area Acquisition Date: 17.07.03 17:49 Calculation Date: 18.07.03 08:41 Sample Type: Calibration Inj Sample Notes: Divisor: 1.000000 Operator: Last Calibration: Calibration Type: Data File: Method: 18.07.03 08:41 External Standard c:V,, \std 2,0 -l.sms c:V.. dre8411_rep2.mt Multiplier: 1,000000 Parameter Compound Number Retention Time RT Offset Peak Name CAS Number Status Quan Ions Area Height Amount (RF) Fit Calibration Equation ^ Baseline Code Specification 6,570 +/- 0,250 69+395+483 >=10 >=700 Linear, Ignore, None Actual T 6,571 min. A0.00111 min. Fluowet 678-39-7 Status Pass 103971 Pass 35551 52299,051 880 Pass y= +5,4485e+4x-5617,4360 MB kCounjs Quart Ions: 69+395+463 merged Std 2,0 -1.SMS (Sample Spedami for Scan: 469 RT: 8.56min. BC |BP^73 (71539100%) Std 2.0 -1.SMS 75%-- I 50%-| 25% , l l 169 232 282 332 60r Reference SpectrumforFluowet 100% Scan: 440 RT: 6,269 min. std 2.0 -1 sms 73 4? 464 '40' 75%- 50%- 25%- 0%S f'S K T 1iso'""208" SB' a5a1 atT Dlff [Sample Spectrumfor Scan: 469 RT: e.56fTmin.B'Cminus 1 00% ;Ref Reference Spectrum for Fluowet ' W " 6^3 "641'"is" "l"' 6l7 ""e! TST 25T 30 Segment: 2 Time(min): 4.984-7,983 Scans: 300-617 Ionization: El Auto Preparation: None Channels:! 400 ' Figure 5: Chromatogram of Standard of Test Item 2.0 mg/L (acquired 2003-07-17) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 44 of 50 Project-No. 020719CK Study-No. DRE84111 Print Date: 21 Jul 2003 11:53:46 ( a Target Compound Report for #1 from DRE84111 A3 Oh R1 -1.SMS Sample ID: DR84111 A3 Oh R1 Instrument ID Measurement Type: Area Acquisition Date: 18.07.03 11:21 Calculation Date: 18.07.03 13:47 Sample Type: Analysis Inj. Sample Notes: Divisor: 1,000000 Operator: Last Calibration: Calibration Type: Data File: Method: 18.07.03 08:41 Externa) Standard c:\...dre84111 a3 Oh c:\... dreB411_rep2.mt Multiplier:_____ 1,000000 Parameter compound Number Retention Time RT Offset Peak Name CAS Number Status Ouan Ions Area Height Amount Fit Calibration Equation Baseline Code Sp-ec-i-fic-a-tio-n 6,571 +/- 0,250 69+395+463 >=10 > = 0,000 >=700 Linear, Ignore, None A1-ct-ua-l 6,565 min. A0.00609 min. Fluowet 678-39-7 S-ta-t-us- Pass 64627 Pass 25230 1,289 Pass 873 Pass y = +5,4485e+4x-5617,4360 MB kCnunts Quan Ions: 69+396+463 merged DR84111 A3 Oh R .SMS ISample Spectrum for Scan: 47;TRT:1}.568 rnrniiTS" BP73 (70481=100%) DRE84111 A3 Oh R1 -1.SMS 100%' 73 25 20 I 10 5 50% 25%J I 127 0%- ,|| LuJ ,'l 169 284 36 406 464 9 266 ....... 30 Reference opectrumTor Fluowet Scan: 440 RT: 6.269 min. etd 2.0 -1.sms 100% 73 400 m/z 75% 3 50% j 25%- 127 405 0% i!i J li o '" T ,,T'T2 ()g,r ' 'io " " z i b" 350 ' mlz Dlff Sample Spectrumfor Scar: 473 RT: 6.568 min. BC minus 100%- Ref Reference Spectrum for Fluowet J 50%- V " A " '50% 0j 6:2 6.3 6l4 6l6 eie 6.7 B'.S ' i o 1' 20" " 3 Segment: 2 Time(min): 4,964-7,986 Scans: 300-627 Ionization: El Auto Preparation: None Channels: 1 'i o Figure 6: Chromatogram of Sample at 0 h, day 14 0.13 mg/L (acquired 2003-07-18) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 45 of 50 Project-No. 020719CK Study-No. DRE84111 Print Date: 21 Jul 2003 11:53:56 6 , Target Compound Report for #1 from DRE84111 A3 46h R1 -1.SMS Sample ID: DRE84111 A3 48hR1 Instrument ID: Measurement Type: Area Acquisition Date: 18.07.03 22:05 Calculation Date: 21.07.03 10:56 Sample Type: Analysts Inj. Sample Notes: Divisor: 1,000000 Operator: Last Calibration: Calibration Type: Data File: Method: 18.07.03 08:41 External Standard c:\... dre84111 a3 48h c:\... dre8411_rep2.mt Multiplier: 1,000000 Parameter Compound Number Retention Time RT Offset Peak Name CAS Number Status Quan Ions Area Height Amount Fit Calibration Equation Baseline Code Specification 6,571 * ! - 0,250 69+395+463 >=10 >= 0,000 >=700 Linear, Ignore, None Actual Status 6,577 min. A0,00603 min. Fluowet 678-39-7 M Pass 0 Fail 0 0,000 Pass 69 Fail y= +5,4485e+4x -5617,4360 Status and Errors: M: Missing peatt. kCounts Quan Ions: 69*395+163 merged (No Results) RE8411.1 A3 48h R1 -1.SMS I |'l I ! " 6.2 ....S ' 6:7 j Segment: 2 Time(min): 4.985-7,983 Scans: 300-627 Ionization: El Auto Preparation: None Channels:! Figure 7: Chromatogram of Sample at 48 h, day 16 0.13 mg/L (acquired 2003-07-18) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 46 of 50 Project-No. 020719CK Study-No. DRE84111 Print Date: 22 Jut 2003 09:15:00 G. Sample ID: Instrument ID: DRE84111A3 48h Ktr Measurement Type: Area Acquisition Date: 21.07.03 19:00 Calculation Date: 22.07.03 09:02 Sample Type: Analysis Inj. Sample Notes: Divisor: 1,000000 Target Compound Report for #1 from DRE84111A3 48h Ktr -1.SMS Operator: Last Calibration: Calibration Type: Data File: Method: 22.07.03 09:02 External Standard c:\... dre84111a3 48h c:\... dre8411_rep2.mt Multiplier:________ 1,000000 Parameter Compound Number Retention Time RT Offset Peak Name CAS Number Status Quan Ions Area Height Amount Fit Calibration Equation Baseline Code Specification 6,572 +/- 0,250 69+395+463 >=10 >= 0,000 >=700 Linear, Ignore, None Actual Status 6,518 min. A0,05367 min. Fluowel 678-39-7 M Pass 0 Fail 0 0,000 Pass 316 Fail y= +6,8018e+4x -5702,8296 S tatus and Errors: M: Missing peak. kCounis Quan ions: 89+395+463 merged {No Results) DRE0411A3 40t>~Kr-1.SMS 1.0 0.0- 0.& - ""B S ...HUT"W A u Segment: 2 Time<mtn): 4,985-7,980 Scans. 300-617 Ionization: El Auto Preparation. None Channels: 1 Figure 8: Chromatogram of Control (48 h), day 16 (acquired 2003-07-21) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 47 of 50 Project-No. 020719CK Study-No. DRE84111 aPrint Date: 21 Jul 2003 11:53:55 Target Compound Report ter # 1 from ORE84111A3 48hLVKtr-1.SMS Sample ID: DRE84111A3 48hLVKtr Instrument ID: Measurement Type; Area Acquisition Date: 18.07.03 21:23 Calculation Date: 21.07.03 10:50 Sample Type: Analysis Inj. Sample Notes: Divisor: 1,000000 Operator: Last Calibration: Calibration Type: Data Pile: Method: 18.07.03 08:41 External Standard c:\... dre84111a3 48hl c A . dre8411_rep2.mt Multiplier: 1,000000 Parameter compound Number Retention Time RT Offset Peak Name CAS Number Status Quan )ons Area Height Amount Fit Calibration Equation Baseline Code Specification 6,571 +/- 0,250 69+395+463 >=10 >= 0,000 >=700 Linear, Ignore, None Actual Status 8,513 min. A0,05833 min. Fluowet 678-39-7 M Pass 0 Fai! 0 0,000 Pass 458 Fail y= +5,4485e+4x -5617,4360 ... biif' Ts"" 6:6' '"SiT" Anutas Segment: 2 Time(mln): 4,886-7,983 Scans: 300-627 Ionization: El Auto Preparation: None Channels: 1 Figure 9: Chromatogram of Solvent Control (48 h), day 16 (acquired 2003-07-18) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 48 of 50 Project-No. 020719CK Study-No. DRE84111 Print Date: 22 Jul 2003 09:15:00 Sample ID: DRE841HA3 48h SL2 Instrument ID: Measurement Type: Area Acquisition Date: 21.07.0319:43 Calculation Date: 22.07.03 09:02 Sample Type: Analysis Inj. Sample Notes: D iv is o r: 1 ,0 0 0 0 0 0 Parameter compound Number Retention Time RT Offset Peak Name CAS Number Status Quan Ions Area H e ig h t Amount Fit Specification 6,572 +/- 0,250 69+395+463 >=10 > = 0,000 >=700 Targe! Compound Report for #1 from DRE84111A3 48h SL2 -1.SMS Operator: Last Calibration: Calibration Type: Data File: Method: 22.07.03 09:02 External Standard c:\... dre84111a3 48h c:\... dre8411_rep2.mt Multiplier: Actual 1,000000 6,573 min. A0O0081 min. Fluowet 678-39-7 71609 26404 1,137 675 Status Pass Pass Pass Pass Figure 10: Chromatogram of Stock Solution (48 h), day 16 (acquired 2003-07-21) 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 49 of 50 Project-No. 020719CK Study-No. DRE84111 10 Certificate of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- Analysis G ih am GmbH, Week Gendorf CQG i Qualidtsmanagemenl D-84504 Burgturchen Fax: + + 4 9 8679 / 2676 C la ria n t Inspection certificate according to EN10204-3.1B Date: 13.11.2001 Page: 1 / 1 Our consignment Material Material-no. Baten No. 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10, 10-heptadecafluoro 104705 PQO/001 On the batch, of which the consignment -is a part, the following val-ues were determined. They conform to the agreed product specification. Inspection characteristic/-methodSpecification Result C8-Perfluoralkylethanol Capillary GC Sum PerfluoroaIky1ethanol Capillary GC Sum Perfluoralkylbutanol Capillary GC Sum Perfluoralkylethene Capillary GC Water content Karl Fischer DIN 51777 N-Methyipyrrolldone content Capillary GC A II O >-97,5 <TM0, 5 <=Q,2 AV i* io o 00 o 99,, 31 [a/a] 99,94[a/a] < 0,1%[a/a] < 0 , 1 % [a/a] < 0 , 1% < 0 , 1% The above particulars do not release the customer from the obligation to carry out an inspectxon of goods received. Ms. Baumgartner - Worksinspector 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-heptadecafluoroDaphnia magna Reproduction Test (21 d), Limit Test according to OECD Guideline 211 Page 50 of 50 Project-No. 020719CK Study-No. DRE84111 11 GLP-Certificate of Dr.U.Noack-Laboratorium n Niedersachsische* Umweltministerium Gute Laborpraxis/Good Laboratory Practice GLP-Bescheinigung/Statement of GLP Compliance (gem/according to 19 b Abs.1 Chemikaliengesatz) Eine GLP-lnspektion zur berwachung der Einhaltung der GLP-Grundsatze gem Chemikaliengesetz bzw. Richtlinie 88/320/EG wurde durchgefhrt in: Assessment of conformity with GLP according to Chemikaliengesetz and Directive 88/320/EEC at: X Prfeinrichtung/Test facility Prfstandort / Test site Dr. U. NOACK LABORATORIUM fr angewandte Biologie Kthe-Paulus-Str. 1 D-31157 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/accofding 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 Wasser und in der Luft, Prfungen zur Bioakkumuiation 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.mondi.year) 25. - 27. Juni 2001 / June 25"' - 27"', 2001 29. August und 21. September 2001 / August 29"' and September 21*', 2001 Die genannte Prfeinrichtung/Dor ge nannte PrOfstandort 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 P rfstndert die oben genannten Prfungen unter Einhaltung der GLP-Grundstze durchgefOhrt werden knnen. Niederschsisches Umweltministerium Referat 33 Archivstrae 2 30169 Hannover The above mentioned test facility / test site is included in the national G LP Compliance Programme and is inspected on a regular basis. Based on the inspection report it can be confirmed, that this test facility I te s t-s ite is able to conduct the aforementioned studies in compliance with the Principies of GLP. 11. Mrz 2002 Im Auftrge Or. Braedt POTTENGER LAW OFFICE Licensed in S e w Mexico and the Navajo Nation. 418 W. Broadway, Suite I Phone (505) 326-4233. FAX 326-1946 Farmington, NM 87401 NOTICE OF INTENT 2o a r-- TO: Mr. Benjamin F. Montoya, President Public Service Company of New Mexico do Patiick T. Ortiz. Registered Agent Alvarado Square, Mailstop 2708 Albuquerque, NM 87158 As an owner of the San Juan Generating Station near Waterflow, New Mexico, you are hereby put on notice per New Mexico law, NMSA 41-4-1 (1976), at seq., as to the possibility of litigation regarding torts and crimes committed by you and your agents against the people of the State of New Mexico. The alleged wrongful acts include the following, to-wit; 1. The discharge of sludge high in sulfates, heavy metals, human fecal material, and radioactive particles into the Shumway and Westwater Arroyos and ultimately the San Juan River and Colorado River systems on or around March 8th, 2001; 2. The failure to take any action to clean up same discharge; 3. The failure to investigate the source of same discharge or take acttons to prevent similar discharges in the future; 4. The discharge of some two hundred and fifty tons of salts and heavy metals into the Shumway and Westwater Arroyos and ultimately the San Juan River and Colorado River systems under the deceitful guise of "zero discharge-' each year; 5. The continuous and wilful poisoning of the drinking waters of the people of Waterflow, Hogback, and Shiprock, New Mexico, and other areas in between and in the circumference; 6 The failure to clean up the lands polluted by these discharges; 7 The wrongful death of Audrey Rogers, formerly of Waterflow, New Mexico; 8. Wrongful death of numerous and as yet un-ascertained residents by causing leukemia, cancer, and other illnesses through the discharge of chromium, barium, and other heaw NEW M EXICO DEPARTMENT OF AGRICULTURE VETERINARY DIAGNOSTIC SERVICES 700 Camino de Salud. NE Albuquerque. New Mexico 8 7 106 Telephone (505) 841-2576 co n A' s' May 25.2000 Mr. Glenn Saums NM Department of Environment Surface W ater Quality Bureau P.O. Box 26110 Santa F e , N e w Mexico 87502 Dear Mr. Saums: W e have concluded the necropsy and ancillary tests on four sheep received from Mr. Hunt's farm. A report on each sheep is enclosed. The sheep have varying conditions, ranging from severe to mild. The youngest sheep (that was received dead and partially scavenged) had a congenital heart defect that resulted in death. T he remaining sheep were received alive, ambulatory, and alert. These sheep were humanely euthanized. One ewe had multiple severe inflammatory lesions resulting from chronic bacterial infection. Another had moderate chronic pneumonia and bacterial lung abscesses. The last sheep had subdinical mild heart inflammation (myocarditis) of undetermined cause. These later two sheep (cases V0004550, VD004551) were in good nutritional status. No indication of acute or chronic toxicity was found. In summary, the examined sheep had various unrelated mild to severe conditions. These included congenital heart defect and infectious disease. All are relatively common diseases seen in New Mexico sheep. Please do not hesitate to call if you have any questions. Sincerely, Richard LaRock, DVM, Diplomate-ACVP Director - Veterinary Diagnostic Services RGL:ks ' L I C . ED 4 SPECIAL WATER QUALITY SAMPLING OF SHUMWAY ARROYO IN THE VICINITY OF THE R.G. HUNT RANCH NEAR WATERFLOW, SAN JUAN COUNTY, NEW MEXICO March 23, 2000 Introduction On March 23, 2000, at the request of the Deputy Secretary of the New Mexico Environment Department, the Surveillance and Standards Section of the Surface Water Quality Bureau conducted a special water quality sampling effort of Shumway Arroyo upstream o f its confluence with the San Juan River in the vicinity of the R.G. Hunt sheep ranch near Waterflow, New Mexico. New Mexico Water Quality Standards Shumway Arroyo is an unclassified surface water of the State. Existing and designated uses include livestock watering and wildlife habitat. Water quality standards applicable to Shumway Arroyo are set forth in sections 20.6.4.12 and 20.6.4.900 NMAC of the New Mexico water quality standards. Section 20.6.4.12 sets forth general narrative standards for all waters of the State. Section 20.6.4.900 sets forth specific numeric standards for the designated uses of livestock watering and wildlife habitat. Methods Water quality sampling methods used were in accordance with the EPA-approved Quality Assurance Project Plan for Water Quality Management Programs (QAPP). Sampling Stations Two sampling sites were selected for this sampling effort. Site 1. Shumway Arroyo just upstream from County Road 6800 (PNM road), located upstream of the H.G. Hunt sheep ranch. Site 2. Shumway Arroyo at State Highway 550 upstream of the San Juan River and downstream of the H.G. Hunt sheep ranch (note:, samples labeled as "Waterflow above Shumway" are the quality assurance replicates for those samples collected at site 2 in accordance with the QAPP) Water Quality Assessment Water quality data collected during the one-day sampling effort are appended to this document. A summary of the results is as follows: Site 1. Streamflow at this site was estimated to be approximately 1.0 cubic foot per second. The water was cool, very hard, highly oxygenated, slightly turbid and contained high to moderate concentrations of dissolved constituents. Most notable were sulfate at 4,060 mg/L and total Oklahoma Animal Disease Diagnostic Laboratory Accustom Report date: Owner: 01030601 3 29-01 /6J; HWaartte,rfRlo.Ow,, JNrM. (5 0 5 ) L 8-6050 HART. R.G, JR. Acct:51H) ATTN; HART, R.G., JR BOX 65 WATERFLOW. NM 87421 Oklahoma State University P.0. Box 7001 Stillwater. OK 74078-7001 (4051 744-6623 PAX: 744-8612 SUBMISSION SUMMARY Taken: co1, given Received: 3-8-01 Species Ovine Animals Tests Completed 11 1 * * AJDOENPED REPORT * * DIAGNOSIS AND COMMENTS 3-15 01/4:15o DIAGNOSES__________________________ DATE Mild to moderate nen-suppurgtive 3-15-01 m e n in g o e n c ep h a litis. Comments; The inflammatory lesions noted in the brain are more consistent w ith a viral or chlamydial agent. Specimens are being submirted for rabies exam at the CDC. There were no lesions noted consistent w ith a diagnosis of polioencsphslomalacia, Addendum: Rabies Results liom the Centers for Qisesse Control and Prevention: No rabies Virus antigen was detected by immunohistochemistry in the Brain tissue sections from 2 paraffin blocks submitted. CDC# A010227A A010227B Mike Niezgoda
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