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AR226-1429 DR.U.NOACK-LABORATORIUM FR ANGEWANDTE BIOLOGIE 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- Earthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil _______________________________ acc. to OECD Guideline 207 for Testing of Chemicals (adopted April 04, 1984) RECEIVED OPPT NCIC 2003 July 18 1:00PM Sponsor Telomer Research Project ASAHI GLASS CLARIANT DAIKIN INDUSTRIES LTD DUPONT Author Martina Noack Test Facility DR.U.NOACK-LABORATORIUM FR ANGEWANDTE BIOLOGIE Kthe-Paulus-Str. 1 D-31157 Sarstedt Laboratory Project ID Project-No. Study-No. Study Plan-No. 020719CK RRA84111 RRA8411- Page 1 of 33 Date 2003-04-14 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 2 of 33 Project-No. 020719CK Study-No. RRA84111 Statement of GLP Compliance Title 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil Guideline Test Item Test Facility OECD No. 207 (April 4, 1984) 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: P00/001) DR.U.NOACK-LABORATORIUM FR ANGEWANDTE BIOLOGIE Kthe-Paulus-Str.1, D-31157 Sarstedt Phone: (+49) 050 66 / 706 70, Fax: (+49) 050 66 / 706 789 E-mail: info@noack-lab.de Deviations from GLP Principles None 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. 2003-04-14 (Date) .............................................................. (M. Noack, Study Director) 2003-04-14 (Date) ................................... ................................... (Dr. Udo Noack, Head 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 3 of 33 Project-No. 020719CK Study-No. RRA84111 Statement of the Quality Assurance Unit Title 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- Earthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil Guideline OECD No. 207 (April 4, 1984) for Testing of Chemicals Test Item 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro(Batch: P00/001) Study Director Martina Noack The study was verified as follows: inspection study plan process based report dates 2002-11-11 2002-11-13 2002-11-07 2003-01-14 2003-01-30 2003-03-06 2003-04-10 date of report 2002-11-11 2002-11-13 2002-11-07 2003-01-14 2003-01-30 2003-03-07 2003-04-10 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. 2003-04-14 ...pp....................................................... Gudrun Mhrmann-Kalabokidis 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 4 of 33 Project-No. 020719CK Study-No. RRA84111 Personnel Involved Study Director: Martina Noack (Biologist) Technical Staff: Kirsten McVean Gwendolin Kraetzig Petra Makus Barbara Wienker Quality Assurance Unit: Deputy: Gudrun Mhrmann-Kalabokidis (Biologist) Susanne Becker (Biologist) Head of Test 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 5 of 33 Project-No. 020719CK Study-No. RRA84111 Table of Contents Title Page Statement of GLP Compliance Statement of Quality Assurance Unit Personnel Involved Table of Contents List of Tables and Figures 1 Summary 2 Characterisation Data of the Test Item 3 Method 4 Results 4.1 Mortality 4.2 Behaviour and Pathological Symptoms 4.3 Live Weight of the Earthworms 4.4 Physical Data 5 Validity Criteria 6 Discussion and Conclusions 7 Literature / References 8 Live Weight Data of the Earthworms 9 Physical Data 10 Data of the Reference Item 11 Certificate of Test Item Analysis 12 GLP Certificate of DR.U.NOACK-LABORATORIUM Table 1: Table 2: Table 3: List of Tables and Figures Summary of All Significant Effects in the Test Concentrations Earthworm Mortality in [%] after 7 and 14 Days of Exposure Earthworm Behaviour and Pathological Symptoms after 7 and 14 Days of Exposure Page 1 2 3 4 5 5 7 10 11 17 17 19 20 21 22 22 22 23 28 29 32 33 8 17 19 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 6 of 33 Project-No. 020719CK Study-No. RRA84111 Table 4: Mean Loss of Live Weight: Comparing Test Start to Test End 20 Table 5: Mean Live Weight on Day 14 Compared to Control Group 21 Table 6: Live Weight on Day 0 (Control) 23 Table 7: Live Weight on Day 0 (Solvent control) 23 Table 8: Live Weight on Day 0 (10 mg/kg) 23 Table 9: Live Weight on Day 0 (32 mg/kg) 24 Table 10: Live Weight on Day 0 (100 mg/kg) 24 Table 11: Live Weight on Day 0 (320 mg/kg) 24 Table 12: Live Weight on Day 0 (1000 mg/kg) 25 Table 13: Live Weight on Day 14 (Control) 25 Table 14: Live Weight on Day 14 (Solvent control) 25 Table 15: Live Weight on Day 14 (10 mg/kg) 26 Table 16: Live Weight on Day 14 (32 mg/kg) 26 Table 17: Live Weight on Day 14 (100 mg/kg) 26 Table 18: Live Weight on Day 14 (320 mg/kg) 27 Table 19: Live Weight on Day 14 (1000 mg/kg) 27 Table 20: pH-Values and Moisture of the Test Media 28 Table 21: Light Intensity in the Incubation Room 28 Table 22: Reference Item: Mortality of the Earthworms after 7 and 14 Days 29 Table 23: LC-Values after 14 d for 2-Chloroacetamide 30 Table 24: Mean Loss of Live Weight 30 Table 25: Reference item: Mean Live Weight on Day 14 Compared to Control Group 31 Figure 1: Course of Live Weights 7 Figure 2: Concentration-Effect Relationship after 14 Days of Exposure 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 7 of 33 Project-No. 020719CK Study-No. RRA84111 1 Summary The acute effects of 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- (batch no. P00/001) on earthworm Eisenia fetida Savigny were determined according to OECD Guideline No. 207 at DR.U.NOACK-LABORATORIUM FR ANGEWANDTE BIOLOGIE in 31157 Sarstedt, Germany from 2002-12-13 to 2002-12-27. The study was conducted under static conditions over a duration of 14 days. The test item was applied once at the test start. Forty test organisms with an individual weight of 0.30 to 0.60 g, divided into four replicates, were tested per concentration, solvent control and control in artificial soil as described by the guideline. The study was conducted with the nominal concentrations: 10 - 32 - 100 - 320 - 1000 mg/kg *). *) All data given in mg/kg refer to dry weight of artificial soil No significant mortality was seen in the concentrations 10 - 320 mg/kg after 14 days of exposure. The concentration 1000 mg/kg resulted in a mean mortality of 45 % after 14 days. The LC50-value was greater than the nominal test item concentration of 1000 mg/kg dry weight of artificial soil. 0,6 Mean Weight [g] 0,5 * * 0,4 * 0,3 0,2 0,1 0,0 ontrol ontrol C ent C Solv Figure 1: Course of Live Weights 10 32 100 Treatment [mg/kg] 320 1000 * statistically significant difference (ANOVA, DUNNETT'S Method, p < 0.001) Day 0 Day 14 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 8 of 33 Project-No. 020719CK Study-No. RRA84111 The course of live weights is given in Figure 1. The percent loss in live weight of the surviving adults came to a maximum of 4 % in the solvent control group and to 4 - 34 % in the treatment groups. In the control group there was no loss in live weight (Table 4, Figure 1) The mean live weights of the surviving earthworms of the treatment groups were compared by a suitable statistical method (ANOVA, One Way Analysis of Variance, DUNNETT'S test) for significance of differences (a = 0.05) from controls. A statistically significant difference in live weight was found for the test concentrations 100 mg/kg when compared to the control (P = < 0.001), (Table 5). Other effects (pathological symptoms, changes in behaviour of the worms) of the test item were reported after 7 and 14 days of exposure. Pathological symptoms were found for concentrations 100 mg/kg (Table 3). A summary of all significant effects in the test concentrations is given in Table 1. Table 1: Summary of All Significant Effects in the Test Concentrations Effects Mortality Mean loss of live weight Mean live weight compared to control Other symptoms (behaviour and pathological symptoms) Summary of all test effects --- = no significant difference +++ = significant difference * = NOEC # = LOEC Concentrations [mg/kg] 10 32 100 320 1000 --- --- --- --- +++ --- --- --- +++ +++ --- --- +++ +++ +++ --- --- +++ +++ +++ --- ---* +++# +++ +++ 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 9 of 33 Project-No. 020719CK Study-No. RRA84111 The no observed effect concentration (NOEC) for 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro- with respect to mortality, live weight and other symptoms of Eisenia fetida was found to be 32 mg/kg after 14 days of exposure. The lowest observed effect concentration (LOEC) was 100 mg/kg (Table 1). Results of the reference test with 2-Chloroacetamide performed from 2001-11-30 to 2001-12-14 showed that the method is sensitive and valid according to the guidelines DIN ISO 11268-1 (1997) and OECD No. 207 (1984) (Part 10). The LC50 of the reference item 2Chloroacetamide was in the range 15 - 60 mg/kg. Validity criteria required by the guideline were fulfilled. Percent mortality in the control group in the reference test and the final definitive test was within the required value of 10 %. The average loss of biomass of the worms in the control group did not exceed 20 %. 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 10 of 33 Project-No. 020719CK Study-No. RRA84111 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: 99.8 % (a/a), sum perfluoroalkylethanol 99.9 % (a/a) Appearance Yellowish solid wax Water solubility 134 g/L at 12 degree C, Std 29 g/L, n=8 (DuPont, 2002) 137 g/L at 25 degree C, Std 53 g/L, n=17 (DuPont, 2002) Stability in water Density Stable at test conditions 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 11 of 33 Project-No. 020719CK Study-No. RRA84111 3 Method GUIDELINE OECD Guideline No. 207, adopted April 4, 1984. TYPE AND PURPOSE OF THE STUDY Determination of the effects of the test item on mortality and live weight development of the earthworm species Eisenia fetida in artificial soil. TEST SYSTEM Reason for the selection of the test system Source Culture Feeding Eisenia fetida Savigny 1826 (Annelida, Lumbricidae) Subspecies: Eisenia fetida andrei According to the guideline Eisenia fetida is suitable for this kind of study. Biologische Bundesanstalt fr Land- und Forstwirtschaft, Messeweg 11/12, D-38104 Braunschweig Organisms of the species Eisenia fetida are cultured at the test facility in covered plastic vessels (volume 60 L) containing potting compost at a temperature of 20 2C in the dark. The earthworms were fed with dried litter of stinging nettle and porridge oats portioned according to the feeding rate and to the density of worms in the vessels. TEST ITEM Pretreatment 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, weighed out, dissolved in tert.-methyl-butyl-ether and mixed with 10 g quartz sand. After the solvent had volatized out of the quartz sand, the sand was mixed into the artificial soil to produce the desired test item 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 12 of 33 Project-No. 020719CK Study-No. RRA84111 Test concentrations Vehicle Solvent 1000 - 320 - 100 - 32 - 10 mg/kg dry weight (factor 10) Test item content [g] nominal actual Test Concentration [mg/kg] 10 32 100 320 1000 23 23.1 73.6 73.5 230 230.1 736 735.7 2300 2300.3 10 g quartz sand tert.-methyl-butyl-ether Solvent [mL] actual Solvent control 15 Test Concentration [mg/kg] 10 32 100 320 3 4 6 10 1000 15 CONTROL SOLVENT CONTROL REFERENCE ITEM Item purity Batch No. Stability Test concentrations Test duration Artificial soil and 10 g quartz sand moistened with demineralized water (621 mL) without test or reference item was used as control medium. Artificial soil and 10 g quartz sand treated with tert.-methyl-butylether in a manner similar to the test item treatments and moistened with demineralized water without test item was used as solvent control medium. 2-Chloroacetamide (FLUKA) 99.7 % (HPLC) 374979/1 14199 2002-06-07 15 - 30 - 60 mg/kg dry mass 2001-11-30 to 2001-12-14. 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 13 of 33 Project-No. 020719CK Study-No. RRA84111 TEST METHOD Replicates Number and age of the earthworms Test medium Test container Application technique Feeding Four replicates were used per concentration of the test item and per control treatment. 10 adult earthworms (with clitellum) were used per replicate. They had been acclimatised to artificial soil two days prior to test start. At start of the test, they had an individual live weight of 0.37 to 0.60 g. Artificial soil was used as described by the test guideline. It consisted of the following components: - 10.5 % peat (air dried and finely ground) - 20 % kaolin (kaolinite content 84 - 87 %) - 69 % quartz sand (sand with particle size of 50-200 m: > 50 %) - 0.5 % calcium carbonate (CaCO3) Demineralized water was added (1080 mL) to give an overall moisture content of 27 % of the dry weight (about 25 % of the max. water capacity) of the soil. The definitive moisture content, determined the day before application of the test item by drying a sample at 105 C and re-weighing, was 26 %. The measured pHvalue of 5.94 was in the recommended range of 6.0 0.5. Glass dishes (volume 1.5 L) were used as test vessels. During the test they were covered with perforated plastic film to prevent the test substrate from drying. Test item was incorporated into the artificial soil 24 h prior to test start. The concentrations of the test item are related to the dry weight of the soil. The test item was weighed out and dissolved in tert.-methyl-butylether. The test item stock solution was mixed with 10 g quartz sand. After the solvent had volatized out of the quartz sand, the treated quartz sand was carefully mixed into the artificial soil with a mixer to insure a homogeneous distribution of the test item in the soil. The amount of quartz sand added was the same for each test concentration, solvent control and control treatment. After 24 h the test substrate was added to the test vessels. No food was added to the test vessels during the toxicity test. 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 14 of 33 Project-No. 020719CK Study-No. RRA84111 Temperature Photoperiod Light intensity 18 - 22 C 16 h 400 - 800 lx TYPE AND FREQUENCY OF MEASUREMENTS AND OBSERVATIONS The maximum water holding capacity of the soil was determined before adaptation of the earthworms (actual 106 %, according to ISO DIS 1128-2 / Annex C). At the start and at the end of the test, pH and moisture content of the test substrate were determined in every treatment and control using mixed samples of all replicates. The live weight of the earthworms was measured individually on the day of application and on day 14. Mortality, behaviour and morphological changes of the earthworms were recorded 7 and 14 days after application. TEST DESCRIPTION Preparations: Day -2: Day -1: Start: A preliminary NON-GLP test was performed with 3 concentrations (10 - 100 - 1000 mg/kg). Effects on mortality and the mean live weight of the earthworms were evaluated. Control replicates were tested under the same conditions as the test groups. Based on the results of the preliminary test, the main study was performed with five concentrations from 10 to 1000 mg/kg with a geometric factor of 10. Artificial soil was produced and moistened with demineralized water to about 25 % of the maximum water holding capacity of the artificial soil. It was adjusted to a pH of 5.94. Earthworms were placed on the artificial soil separately moistened to about 35 % of the soil dry weight (adaptation). The test item was heated to 50 C, weighed out, dissolved in tert.-methyl-butyl-ether and mixed with 10 g quartz sand. After the solvent was volatized out of the quartz sand, the treated quartz sand was mixed with the artificial soil. The treated test substrate was added to the test vessels. 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 15 of 33 Project-No. 020719CK Study-No. RRA84111 Day 7: Day 14: Earthworms were washed in dechlorinated tap water, weighed individually and placed on the surface of the test medium. The pH and moisture content were determined using pooled samples of each treatment and control group. The test vessels were covered with perforated plastic film. Earthworms were taken out of the test vessels and observed with respect to mortality and morphological and behavioural changes. Thereafter, live earthworms were replaced on the surface of the test substrate and test vessels were covered with perforated film. Earthworms were taken out of the test vessels and observed with respect to mortality and morphological and behavioural changes. The individual live weight of the survivors was measured. Soil pH and moisture content of every test item treatment and of the controls were measured using pooled samples. VALIDITY CRITERIA The mortality in the control group should not exceed 10 % at the end of the test. The average loss of biomass of the worms in the control groups should not exceed 20 %. STATISTICS AND CALCULATIONS NOEC, live weights Significant differences in worm live weight after 14 days were determined in comparison with the control group using One Way Analysis of Variance and the Multiple Comparison Method of DUNNETT'S. When running a One Way Analysis of Variances a Normality and Equal Variance Test were done first. P-Values for both Normality and Equal Variance Test were < 0.05. The a-value for ANOVA and DUNNETT'S test was a=0.05. The calculations were carried out using SigmaStat rel. 2.03 (1992-1997), SPSS INC. LC50 -values and confidence intervals for mortality Calculations for the reference item were carried out via probit analysis using software SigmaPlot rel. 2000 (2000), SPSS CORPORATION. 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 16 of 33 Project-No. 020719CK Study-No. RRA84111 TIME OF THE STUDY DEVIATIONS FROM THE GUIDELINE DEVIATIONS FROM THE STUDY PLAN REPORTING ARCHIVING Probit values according to WEBER (1986). Calculation of the confidence intervals for LC50 were carried out using standard procedures according to BREITIG & TMPLING (1982). The LC50-value for the test item control could not be determined at the tested concentration range. The study was carried out from 2002-12-13 to 2002-12-27. None None The final report is issued as paper copy (5 originals) and an electronic record (1 original). The following will be 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 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 17 of 33 Project-No. 020719CK Study-No. RRA84111 4 Results 4.1 Mortality The percentage mortality after 7 and 14 days of all replicates is given in Table 4. Table 2: Earthworm Mortality in [%] after 7 and 14 Days of Exposure 4 replicates (n = 40) Nominal conc. [mg/kg] 1 Control 0 Solvent control 0 10 0 32 0 100 0 320 0 1000 0 M O R T A L I T Y [%] 7 day 2 3 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MV 1 0 0 0 0 0 0 0 0 0 0 0 10 0 40 14 day 2 3 4 MV 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2.5 40 50 50 45 MV = mean value No significant mortality was seen in the tested concentrations 10 - 320 mg/kg after 14 days of exposure. The highest tested concentration of 1000 mg/kg resulted in a mortality of 45 %. The LC50- value was not determinable. 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 18 of 33 Project-No. 020719CK Study-No. RRA84111 Probit 8 7 6 5 4 3 2 0,5 1,0 1,5 2,0 2,5 3,0 3,5 log conc. Figure 2: Concentration-Effect Relationship after 14 Days of Exposure 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 19 of 33 Project-No. 020719CK Study-No. RRA84111 4.2 Behaviour and Pathological Symptoms The earthworms in the control, the solvent control and the tested concentrations 10 to 32 mg/kg showed normal behaviour with normal reactions to mechanical stimulus and negative phototactic reactions after placing them on the substrate surface after 7 and 14 days of exposure. In the concentrations 100 to 1000 mg/kg pathological symptoms and abnormal reactions were observed at 7 and 14 days. The data are given in Table 3. Table 3: Earthworm Behaviour and Pathological Symptoms after 7 and 14 Days of Exposure 4 replicates (n = 40) Conc. [mg/kg] Control Effect a) 1 10/10 DAY 7 Replicate 2 3 10/10 10/10 4 10/10 1 10/10 DAY 14 Replicate 2 3 10/10 10/10 4 10/10 Solvent control a) 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10 a) 10/10 10/10 10/10 10/10 10/10 9/10 10/10 10/10 e) - - - - - 1/10 - - 32 a) 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 100 a) 7/10 5/10 4/10 5/10 - - - - c) - - 1/10 - - - -* - g) 2/10 2/10 1/10 1/10 2/10 2/10 1/10 1/10 h) 2/10 4/10 6/10 4/10 10/10 10/10 10/10 10/10 320 e) - - - - 1/10 - - - g) 1/10 - 1/10 2/10 2/10 1/10 1/10 2/10 h) 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 1000 c) - 1/10 - e) - - - - - - -* - - 5/10 4/10 6/10 - g) 2/10 1/10 - 1/10 1/10 3/10 3/10 2/10 h) 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 a) no obvious pathological symptoms c) no negative phototactic reaction e) spontaneous segmentation and separation g) ulcer and skin bleeding h) worms hard, not elastic * after 14 days no worm was on the surface of the test medium 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 20 of 33 Project-No. 020719CK Study-No. RRA84111 4.3 Live Weight of the Earthworms The worms had an individual weight of 0.37 to 0.60 g at test start in all concentrations and control groups. Therefore, all weights were in the recommended range. The data of the treatment, solvent control and control groups are given in Tables 6 to 12. The mean weights across replicates for each concentration and control and the pooled residual standard deviation were calculated for data at 14 days. The tables 15 to 21 present data for individual weight of the earthworms on day 14 as well as the mean values and the standard deviations for all replicates of test and control groups. The percent loss of live weight was 4 % in the solvent control group and 4 to 34 % in the treatment groups. Only in the tested concentrations 320 mg/kg the loss of live weight was 20 %. The live weight of the control group remained at the starting level (Table 4). Table 4: Mean Loss of Live Weight: Comparing Test Start to Test End Concentration [mg/kg] Control Solvent Control 10 32 100 320 1000 Test start [g/worm] 0.52 0.52 0.52 0.52 0.52 0.52 0.50 Mean live weight Test end [g/worm] 0.52 0.50 0.50 0.53 0.45 0.39 0.33 Loss* [%] 0 4 4 -2 13 25 34 * loss 20 % biologically not significant (according to DIN ISO 11268-1 (1997)) 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 21 of 33 Project-No. 020719CK Study-No. RRA84111 The mean live weights of the surviving earthworms after 14 days of exposure of the treatment groups were compared to the control by One Way Analysis of Variance for significance of differences (a = 0.05). In the One Way Analysis of Variance the differences in the mean values among the treatment groups were greater than would be expected by chance. To isolate the groups that differ from the others the multiple comparison method of DUNNETT'S was used. There was a statistically significant difference at the concentrations 100 mg/kg (P < 0.001, Table 5). Table 5: Mean Live Weight on Day 14 Compared to Control Group 4 replicates, n=40 Conc. [mg/kg] Mean live weight [g] Replicate Comparison versus control 1 2 3 4 Overall MV CV Statistically SD significant* Control 0.51 0.52 0.54 0.51 0.52 0.05 9.29 - Solvent Control 0.50 0.50 0.51 0.48 0.50 0.05 9.95 No 10 0.49 0.49 0.51 0.50 0.50 0.06 11.25 No 32 0.52 0.52 0.53 0.54 0.53 0.06 11.45 No 100 0.45 0.43 0.46 0.46 0.45 0.06 13.17 Yes 320 0.39 0.39 0.40 0.39 0.39 0.05 12.99 Yes 1000 0.36 0.35 0.32 0.29 0.33 0.04 13.12 Yes * statistically significant difference (ANOVA, DUNNETT'S Method, p < 0.001) 4.4 Physical Data Physical data are given in part 9. Moisture content of test medium was 51.6 - 53.4 % of dry weight at test start and 50.4 - 51.5 % at test end (Table 20). Values for pH were 5.08 - 5.58 at test start and 5.45 - 5.75 at test end (Table 20). During the test the temperature in the incubation room remained in the range 20 2 C. Testing was done a with a mean light intensity of 491 lx for a photoperiod of 16 h (Table 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 22 of 33 Project-No. 020719CK Study-No. RRA84111 5 Validity Criteria Conditions for the validity of the test were as follows: The mortality in the control group did not exceed 10 % at the end of the test. The average loss of biomass in the control group did not exceed 20 % at the end of the test. 6 Discussion and Conclusions Mortality was seen at the test concentrations of 320 and 1000 mg/kg after 14 days of exposure (Table 2). The live weight was statistically significantly decreased at concentrations 100 mg/kg when compared to control group during the 14 days of exposure (ANOVA, DUNNETT'S Method, p < 0.001). Pathological symptoms were seen at concentrations 100 mg/kg (Table 5). No test item related mortality, pathological symptoms and reduction in live weight were seen at concentrations 32 mg/kg. Finally the highest tested concentration with no effect (NOEC) of the test item with respect to mortality, body weight and other symptoms was found to be 32 mg/kg. The lowest observed effect concentration (LOEC) was found to be 100 mg/kg. Effects of the reference item 2-Chloroacetamide on earthworms show the method is sensitive and valid (Part 10). 7 Literature / References (1) BBA (1984)-Verfahrensvorschlag: ,,Toxizittstest am Regenwurm Eisenia fetida (Savigny) 1826 in knstlichem Boden" (LC 50, 14 Tage), Braunschweig. (2) BREITIG, G., TMPLING, W. (editors, 1982): Methoden der Wasseruntersuchungen Bd.II. VEB GUSTAV FISCHER VERLAG, Jena. (3) DIN ISO 11268-1 (APRIL 1997), WIRKUNGEN VON SCHADSTOFFEN AUF REGENWRMER. (4) OECD Guideline 207 for Testing of Chemicals: Earthworm, Acute Toxicity Test adopted April 4, 1984. (5) WEBER, E., Grundri der biologischen Statistik, 9. Auflage (1986). (6) DuPont, Determination of physico-chemical parameters of Perfluorooctylethanol, CAS No. 678-39-7, Unpublished results, Update Feb. 15, 2002 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 23 of 33 Project-No. 020719CK Study-No. RRA84111 8 Live Weight Data of the Earthworms Table 6: Live Weight on Day 0 (Control) Rep. 1 1 0.42 W E I G H T g O F E A R T H W O R M NO. 2 3 4 5 6 7 8 9 0.49 0.51 0.51 0.52 0.60 0.55 0.57 0.52 10 0.52 2 0.60 0.47 0.44 0.47 0.54 0.51 0.51 0.46 0.52 0.55 3 0.44 0.51 0.50 0.48 0.58 0.60 0.49 0.57 0.46 0.53 4 0.48 0.49 0.51 0.41 0.43 0.55 0.51 0.47 0.60 0.60 sum 5.21 5.07 5.16 5.05 20.5 n mean std. dev. 10 0.52 0.05 10 0.51 0.05 10 0.52 0.05 10 0.51 0.06 40 0.52 Table 7: Live Weight on Day 0 (Solvent control) Rep. 1 2 3 4 1 0.57 0.40 0.44 0.48 W E I G H T g O F E A R T H W O R M NO. 2 0.48 3 0.46 4 0.49 5 0.51 6 0.60 7 0.56 8 0.53 9 0.47 0.60 0.51 0.50 0.42 0.53 0.50 0.59 0.45 0.50 0.44 0.56 0.56 0.48 0.56 0.59 0.58 0.49 0.46 0.47 0.58 0.54 0.53 0.53 0.59 10 0.52 0.55 0.54 0.51 sum 5.19 5.05 5.25 5.18 20.8 n mean std. dev. 10 0.52 0.05 10 0.51 0.07 10 0.53 0.06 10 0.52 0.04 40 0.52 Table 8: Live Weight on Day 0 (10 mg/kg) Rep. 1 2 3 4 1 0.49 0.50 0.49 0.48 W E I G H T g O F E A R T H W O R M NO. 2 0.44 3 0.46 4 0.55 5 0.60 6 0.57 7 0.48 8 0.55 9 0.60 0.45 0.45 0.60 0.46 0.52 0.47 0.50 0.56 0.45 0.46 0.53 0.60 0.47 0.48 0.52 0.55 0.51 0.44 0.60 0.49 0.42 0.46 0.50 0.60 10 0.51 0.60 0.60 0.59 sum 5.25 5.11 5.15 5.09 20.6 n mean std. dev. 10 0.53 0.06 10 0.51 0.06 10 0.52 0.05 10 0.51 0.07 40 0.52 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 24 of 33 Project-No. 020719CK Study-No. RRA84111 Table 9: Live Weight on Day 0 (32 mg/kg) Rep. 1 2 3 4 1 0.40 0.57 0.49 0.50 W E I G H T g O F E A R T H W O R M NO. 2 0.46 3 0.58 4 0.55 5 0.59 6 0.54 7 0.43 8 0.44 9 0.51 0.43 0.48 0.60 0.60 0.55 0.50 0.56 0.55 0.60 0.58 0.43 0.55 0.45 0.48 0.55 0.43 0.58 0.55 0.50 0.57 0.52 0.55 0.46 0.59 10 0.45 0.52 0.60 0.45 sum 4.95 5.36 5.16 5.27 20.7 n mean std. dev. 10 0.50 0.06 10 0.54 0.05 10 0.52 0.07 10 0.53 0.05 40 0.52 Table 10: Live Weight on Day 0 (100 mg/kg) Rep. 1 2 3 4 1 0.52 0.48 0.52 0.46 W E I G H T g O F E A R T H W O R M NO. 2 0.57 3 0.42 4 0.40 5 0.53 6 0.46 7 0.60 8 0.55 9 0.60 0.47 0.60 0.57 0.60 0.43 0.47 0.53 0.55 0.52 0.53 0.51 0.49 0.51 0.50 0.49 0.56 0.45 0.55 0.52 0.41 0.52 0.58 0.59 0.48 10 0.60 0.52 0.53 0.51 sum 5.25 5.22 5.16 5.07 20.7 n mean std. dev. 10 0.53 0.05 10 0.52 0.06 10 0.52 0.02 10 0.51 0.06 40 0.52 Table 11: Live Weight on Day 0 (320 mg/kg) Rep. 1 2 3 4 1 0.45 0.41 0.50 0.56 W E I G H T g O F E A R T H W O R M NO. 2 0.57 3 0.49 4 0.45 5 0.60 6 0.58 7 0.53 8 0.52 9 0.50 0.50 0.60 0.59 0.57 0.59 0.44 0.44 0.54 0.46 0.51 0.51 0.51 0.58 0.46 0.56 0.47 0.45 0.47 0.53 0.55 0.52 0.47 0.45 0.48 10 0.50 0.51 0.60 0.57 sum 5.19 5.19 5.16 5.05 20.6 n mean std. dev. 10 0.52 0.05 10 0.52 0.07 10 0.52 0.05 10 0.51 0.05 40 0.52 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 25 of 33 Project-No. 020719CK Study-No. RRA84111 Table 12: Live Weight on Day 0 (1000 mg/kg) Rep. 1 2 3 4 1 0.53 0.40 0.45 0.42 W E I G H T g O F E A R T H W O R M NO. 2 0.49 3 0.51 4 0.48 5 0.60 6 0.52 7 0.60 8 0.46 9 0.49 0.43 0.51 0.50 0.51 0.43 0.52 0.53 0.53 0.55 0.51 0.46 0.52 0.51 0.60 0.49 0.46 0.44 0.43 0.60 0.48 0.60 0.37 0.40 0.40 10 0.57 0.60 0.45 0.44 sum 5.25 4.96 5.00 4.58 19.8 n mean std. dev. 10 0.53 0.05 10 0.50 0.06 10 0.50 0.05 10 0.46 0.08 40 0.50 Table 13: Live Weight on Day 14 (Control) Rep. 1 1 0.58 W E I G H T g O F E A R T H W O R M NO. 2 3 4 5 6 7 8 9 0.54 0.47 0.62 0.48 0.49 0.54 0.47 0.49 10 0.43 2 0.53 0.48 0.55 0.45 0.49 0.52 0.54 0.61 0.54 0.47 3 0.51 0.57 0.47 0.61 0.52 0.56 0.57 0.58 0.51 0.46 4 0.58 0.43 0.52 0.47 0.52 0.54 0.52 0.50 0.50 0.53 sum 5.11 5.18 5.36 5.11 20.8 n mean std. dev. 10 0.51 0.06 10 0.52 0.05 10 0.54 0.05 10 0.51 0.04 40 0.52 Table 14: Live Weight on Day 14 (Solvent control) Rep. 1 2 3 4 1 0.45 0.48 0.61 0.48 W E I G H T g O F E A R T H W O R M NO. 2 0.47 3 0.52 4 0.52 5 0.46 6 0.54 7 0.52 8 0.55 9 0.45 0.54 0.57 0.47 0.49 0.46 0.59 0.37 0.52 0.54 0.43 0.55 0.52 0.54 0.44 0.45 0.51 0.53 0.44 0.46 0.49 0.49 0.52 0.46 0.43 10 0.54 0.47 0.51 0.45 sum 5.02 4.96 5.10 4.75 19.8 n mean std. dev. 10 0.50 0.04 10 0.50 0.06 10 0.51 0.06 10 0.48 0.03 40 0.50 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 26 of 33 Project-No. 020719CK Study-No. RRA84111 Table 15: Live Weight on Day 14 (10 mg/kg) Rep. 1 2 3 4 1 0.49 0.39 0.42 0.44 W E I G H T g O F E A R T H W O R M NO. 2 0.47 3 0.52 4 0.44 5 0.37 6 0.59 7 0.43 8 0.52 9 0.52 0.46 0.47 0.49 0.49 0.55 0.60 0.49 0.49 0.48 0.53 0.51 0.48 0.48 0.58 0.61 0.43 0.49 0.45 0.55 0.57 0.53 0.44 0.44 0.56 10 0.51 0.51 0.54 0.50 Sum 4.86 4.94 5.06 4.97 19.8 n mean std. dev. 10 0.49 0.06 10 0.49 0.06 10 0.51 0.06 10 0.50 0.05 40 0.50 Table 16: Live Weight on Day 14 (32 mg/kg) Rep. 1 2 3 4 1 0.48 0.50 0.57 0.60 W E I G H T g O F E A R T H W O R M NO. 2 0.47 3 0.64 4 0.58 5 0.44 6 0.59 7 0.40 8 0.55 9 0.55 0.54 0.55 0.45 0.44 0.57 0.53 0.54 0.56 0.49 0.50 0.67 0.57 0.61 0.48 0.44 0.46 0.55 0.57 0.57 0.58 0.42 0.51 0.49 0.58 10 0.49 0.55 0.56 0.49 sum 5.19 5.23 5.35 5.36 21.1 n mean std. dev. 10 0.52 0.07 10 0.52 0.05 10 0.54 0.07 10 0.54 0.06 40 0.53 Table 17: Live Weight on Day 14 (100 mg/kg) Rep. 1 2 3 4 1 0.45 0.35 0.43 0.51 W E I G H T g O F E A R T H W O R M NO. 2 0.47 3 0.38 4 0.31 5 0.51 6 0.33 7 0.51 8 0.50 9 0.52 0.41 0.45 0.45 0.47 0.51 0.39 0.44 0.34 0.43 0.48 0.42 0.50 0.46 0.41 0.49 0.51 0.42 0.42 0.44 0.36 0.50 0.41 0.57 0.47 10 0.50 0.48 0.50 0.48 sum 4.48 4.29 4.63 4.58 18.0 n mean std. dev. 10 0.45 0.08 10 0.43 0.06 10 0.46 0.04 10 0.46 0.06 40 0.45 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 27 of 33 Project-No. 020719CK Study-No. RRA84111 Table 18: Live Weight on Day 14 (320 mg/kg) Rep. 1 1 0.45 W E I G H T g O F E A R T H W O R M NO. 2 3 4 5 6 7 8 9 10 0.46 0.42 0.34 0.41 0.40 0.35 0.47 0.37 (0.22) sum 3.89 2 0.34 0.45 0.39 0.40 0.38 0.34 0.36 0.33 0.44 0.47 3.90 3 0.34 0.43 0.40 0.41 0.40 0.38 0.43 0.37 0.49 0.37 4.02 4 0.33 0.35 0.42 0.37 0.37 0.42 0.41 0.37 0.45 0.41 3.90 15.7 () = earthworm dead, but could be found, not decomposed, included in calculations n mean std. dev. 10 0.39 0.07 10 0.39 0.05 10 0.40 0.04 10 0.39 0.04 40 0.39 Table 19: Live Weight on Day 14 (1000 mg/kg) Rep. W E I G H T g O F E A R T H W O R M NO. 1 2 3 4 5 6 7 8 9 10 1 0.38 0.40 0.41 0.33 0.32 0.31 - - - - 2 0.30 0.34 0.40 0.39 0.36 0.29 - - - - 3 0.32 0.36 0.29 0.31 0.31 - - - - - 4 0.33 0.29 0.28 0.30 0.26 - - - - - - not determinable, earthworms decomposed sum n mean std. dev. 2.15 2.08 1.59 1.46 7.28 6 0.36 0.04 6 0.35 0.05 5 0.32 0.03 5 0.29 0.03 22 0.33 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 28 of 33 Project-No. 020719CK Study-No. RRA84111 9 Physical Data Measurement of pH values and moisture content of test medium was based on pooled samples of all replicates per treatment. Moisture values are given in % of the dry weight of the test medium. Table 20: Conc. [mg/kg] Control Solvent control 10 32 100 320 1000 pH-Values and Moisture of the Test Media pH-value day 0 5.20 5.08 5.31 5.58 5.18 5.26 5.29 day 14 5.75 5.45 5.45 5.74 5.57 5.47 5.46 Moisture [%] of dry weight day 0 53.3 52.4 53.4 52.8 52.8 51.6 53.1 day 14 50.7 50.5 50.4 51.4 51.5 51.1 51.1 Table 21: Light Intensity in the Incubation Room (Measurement in [lx]) Measurement on day Measurements [lx] 1 2 3 4 2002-12-16 512 432 421 598 Mean [lx] 491 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 29 of 33 Project-No. 020719CK Study-No. RRA84111 10 Data of the Reference Item 2-Chloroacetamide is used as reference item in the Acute Toxicity Test in Artificial Soil with earthworms. The reference test was performed at the test facility from 2001-11-30 to 2001-12-14. The study no. was RRA12011. The reference item was applied once at test start. The study was conducted with the nominal concentrations: 15 - 30 - 60 mg/kg The mortality for the reference item was determined after 7 and 14 days in percent (Table 22). Table 22: Reference Item: Mortality of the Earthworms after 7 and 14 Days Conc. [mg/kg] 1 M O R T A L I T Y [%] 7 d 14 d 2 3 4 MV 1 2 3 4 MV Control 0 0 0 0 0 0 0 0 0 0 15 0 0 0 10 2.5 0 0 0 10 2.5 30 100 80 90 60 82.5 100 90 90 60 85 60 100 100 100 100 100 100 100 100 100 100 The lowest tested concentration resulting in 100 % mortality (14d LC100) after 14 days of exposure was determined directly from the test results to be 60 mg/kg. The mean lethal concentration (14 d LC50 ) was determined to be 23.5 mg/kg with a confidence interval ranging from 21.8 to 25.2 mg/kg estimated by probit analysis, so that the mean lethal concentration was in the recommended range of the guidelines (DIN ISO 11268-1; OECD 207) (Table 23). Finally, the concentration with no effect (NOEC) of the reference item with respect to mortality and other effects was found to be 15 mg/kg after 14 days of exposure. 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 30 of 33 Project-No. 020719CK Study-No. RRA84111 Table 23: LC-Values after 14 d for 2-Chloroacetamide LC - Values LC10 LC50 LC100 Concentration [mg/kg] 17.3 23.5 60.0 --- = not determined Confidence interval [mg/kg] --- 21.8 - 25.2 --- The earthworms in the control and test group 15 mg/kg showed normal behaviour with normal reactions to mechanical stimulus and negative phototactic reactions after placing them on the substrate surface. Obvious pathological symptoms were observed at 30 mg/kg. The worms had an individual weight of 0.30 to 0.60 g at test start in all concentration and control groups. Therefore all weights were in the recommended range. The mean live weights across replicates for each concentration and control were calculated (Table 24). During the test the weight of the earthworms decreased in the treatment and control groups due to the fact, that there was no feeding at all in the test period. The percent loss in live weight came to 8 % in the control, to 6 % in the concentration 15 mg/kg and up to 37 % in the concentration 30 mg/kg. The loss of live weight was significant in the tested concentration 30 mg/kg (> 20%), (Table 24). Table 24: Mean Loss of Live Weight Concentration [mg/kg] Test start [g/worm] Control 15 30 60 0.49 0.49 0.49 0.49 --- = * = n.s. = sig. = dead live weight loss > 20 % not significant (loss 20 %) significant (loss > 20 %) Mean Live weight Test end [g/worm] Loss [%] 0.45 8 0.46 6 0.31 37 --- --- Significance* n.s. n.s. sig. --- 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 31 of 33 Project-No. 020719CK Study-No. RRA84111 The mean live weight after 14 days of the concentrations 15 and 30 mg/kg was then compared with the control mean using One Way Analysis of Variance. The differences in the mean values among the treatment group 30 mg/kg were determined to be statistically significant, P < 0.001 (One Way Analysis of Variances, Dunnett's Method), Table 25. Table 25: Reference item: Mean Live Weight on Day 14 Compared to Control Group Conc. [mg/kg] 1 Mean live weight [g] Replicate 2 3 4 Comparison versus Control Significance* Control 0.45 0.45 0.44 0.45 0.45 - 15 0.46 0.47 0.45 0.45 0.46 no 30 0.42 0.35 0.27 --- 0.31 yes 60 --- --- --- --- --- --- --- = * = no = dead One Way Analysis of Variance (DUNNETT'S Method, p< 0.001) no statistically significant difference The environmental conditions were as follows: Moisture content of test medium was 32.7 - 33.6 % of dry weight at the test start and 31.5 32.5 % at the test end. Values of pH were 5.52 - 5.62 at the test start and 5.20 - 5.29 at the test end. During the test the temperature was in the range of 18 - 20 C. Testing was done with a light intensity of 428 to 535 lx (mean of 753 lx) for a photoperiod of 16 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 32 of 33 Project-No. 020719CK Study-No. RRA84111 11 Certificate of Test Item Analysis 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-heptadecafluoroEarthworm (Eisenia fetida), Acute Toxicity Test in Artificial Soil, according to OECD Guideline 207 Page 33 of 33 Project-No. 020719CK Study-No. RRA84111 12 GLP Certificate of DR.U.NOACK-LABORATORIUM