Document m62DzYzzEGv1o05XqeOD4Zq0

- I3 K Ari? REPORT ACUTE TOXICITY STUDY IN DAPHNIA MAGNA WITH T-4127 NOTOX Project 334349 NOTOX Substance 113607 roc=o> CZ 0^0 -o m ro cn -S O --! m IH O f-ri oo ro cn Page 1 of 21 - r\ V-) .ir - r t CT* T-4127 STATEMENT OF GLP COMPLIANCE NOTOX Project 334349 NOTOX B.V., 's-Hertogenbosch, The Netherlands The study described in this report has been correctly reported and was conducted in compliance with the most recent edition of: The OECD Principles of Good Laboratory Practice which are essentially in conformity with: The United States Food and Drug Administration. Title 21 Code of Federal Regulations Part 58. The United States Environmental Protection Agency (FIFRA). Title 40 Code of Federal Regulations Part 160. The United States Environmental Protection Agency (TSCA). Title 40 Code of Federal Regulations Part 792. Study Director Drs. M. Bogers Management: Ing. E.J. van de Waart M.Sc. Head of Genetic & Ecotoxicology ,// ^ 2- T-4127 QUALITY ASSURANCE STATEMENT NOTOX B.V., 's-Hertogenbosch, The Netherlands NOTOX Project 334349 This report was audited by the NOTOX Quality Assurance Unit to ensure that the methods and results accurately reflect the raw data. The dates of Quality Assurance inspections and audits are given below. During the on-site inspections procedures applicable to this type of study were inspected. DATES OF QAU INSPECTIONS/AUDITS on-site inspection(s) (Process) May 13-31,2002 (Analytical Support) July 08-15, 2002 (Ecotoxicology) protocol inspection(s) (Study) January 31,2002 report audit(s) (Study) May 01,2003 REPORTING DATES June 04, 2002 July 17, 2002 January 31,2002 May 01,2003 Head of Quality Assurance C.J. Mitchell B.Sc. Date: i ^ _ S'- 0 3 -3 - T-4127 NOTOX Project 334349 SUMMARY Acute Toxicity Study in Daphnia magna with T-4127. The study procedures described in this report were based on the ISO International Standard 6341: "Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus Acute toxicity test, Third edition, 1996-04-01. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, C.2. "Acute Toxicity for Daphnia", and the OECD guideline No. 202 Part I: "Daphnia sp., Acute Immobilisation Test", Adopted April 4, 1984. T-4127 is a salt consisting of Perfluorooctanesulfonylmethylamide (anionic part) and Triphenylbenzylphosphonium (cationic part). Based on the response of the anionic part of the test substance water solubility of T-4127 was determined to be between 0.6 and 0.9 mg/l at 19.5 0.6C. The study started with a limit test exposing twenty daphnids per concentration to a filtered and an unfiltered solution, prepared at a loading rate of 100 mg/l and magnetically stirred for four days. A blank-control was also included. After 24 hours of exposure all organisms exposed to the filtered and the unfiltered solutions prepared at a loading rate of 100 mg/l became immobilised. A final test was performed exposing twenty daphnids per concentration to 4.5,10, 20, 45 and 100% of a Water Accommodated Fraction (WAF) prepared at a loading rate of 100 mg/l and a blank control for a maximum of 48 hours. All glassware used in the final test was pre-treated with dichlorodimethylsilane. Although vapour pressure was relatively low, based on the Henry constant evaporation was still a possible cause for decrease of test concentrations. Therefore the study was performed in air-tight vessels. Samples taken during the final test were added directly to isopropanol at a ratio of 1:5 to prevent any additional loss. The analytical program based on the anionic part showed that the actual concentrations could not be maintained at more than 80 % of the initial concentration, in spite of all precautions taken to prevent adsorption to glass and volatilization. In the control no mortality was observed. Further, all test conditions (pH, oxygen concentration and temperature) remained within the ranges prescribed by the protocol. Dilutions of a water phase of T-4127 originating from a loading rate of 100 mg/l induced 100% immobility of Daphnia magna. Chemical analysis of the anionic part of T-4127 showed that this corresponded with initial T-4127 concentrations down to 0.02 mg/l. Hence, the 48h-EC50 of Daphnia magna exposed to T-4127 was < 1 mg/l (< 0.023 mg/l) based on quantification of the anionic part and was already reached within 24 hours of exposure. Limiting the analytical support on only the anionic part of T-4127, validity of the test could not be ensured. There is a realistic possibility that the actual concentrations of the cationic part were higher and more stable than those of the anionic part. However, the sponsor did not agree with further research and decided to accept the highly toxic labeling (R50) based on the current results. -4 - T -4127 NOTOX Project 334349 PREFACE Sponsor Study Monitor 3M Corporate Toxicology 3M Center, Building 220-2E-02 P.O. Box 33220 ST. PAUL, MINNESOTA 55133-3220 U.S.A. S. A. Beach 3M Environmental Technology and Safety Services Building 2-3E-09 935 Bush Avenue St. Paul, MN 55144 (Address to which all communication is to be sent) Testing Facility Aquatic Toxicology: Study Director Technical co-ordinator Analytical Chemistry: Principal Scientist Study Plan NOTOX B.V. Hambakenwetering 7 5231 DD 's-Hertogenbosch The Netherlands Drs. M. Bogers Ing. B. van Wees Ir. M.J.C. Brekelmans Start Project: January 30, 2002 Start first exposure: July 01,2002 Completion last exposure: November 13, 2002 Completion Analysis: November 13, 2002 Completion project: May 09, 2003 TEST SUBSTANCE Identification Description Batch Composition Test substance storage Stability under storage conditions Expiry date Stability in water T-4127 Dark amber waxy solid D-2491 lot 2 '95 - 99% Fluoroelastomer curative <2% N-Methyl Perfluorooctanesulphonamide <1 % Methyl Alcohol <1% Ethyl Alcohol <1% Isopropyl Alcohol At room temperature in the dark Stable 31 March 2003 Not indicated The sponsor is responsible for all test substance data unless determined by NOTOX. PURPOSE The purpose of the toxicity test was to evaluate the influence of T-4127 on the mobility of Daphnia magna. T-4127 NOTOX Project 334349 GUIDELINES The study procedures described in this report were based on the ISO International Standard 6341: "Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus Acute toxicity test, Third edition, 1996-04-01. In addition, the procedures were designed to meet the test methods and validity of the following guidelines: European Economic Community (EEC), EEC directive 92/69, Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, C.2. "Acute Toxicity for Daphnia". Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No. 202 Part I: "Daphnia sp., Acute Immobilisation Test", Adopted April 4, 1984. ARCHIVING NOTOX B.V. will archive the following data for at least 10 years: protocol, report, test substance reference sample and raw data. No data will be withdrawn without the sponsor's written consent. DEFINITIONS Immobilisation: those animals not able to swim within 15 seconds after gentle agitation of the test vessel are considered to be immobile. EC50: the concentration estimated to immobilise 50% of the daphnia after a defined period of exposure. No Observed Effect Concentration (NOEC1: the highest tested concentration at which no effect (i.e. immobilisation) is recorded. TEST SYSTEM Species Reason for selection Validity of batch Characteristics Daphnia magna (Crustacea, Cladocera) (Straus, 1820) This system has been selected as an internationally accepted species. Daphnids originated from a healthy stock, 2ndto 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood. For the test selection of young daphnia with an age of < 24 hours. T-4127 NOTOX Project 334349 BREEDING Start of each batch With new-born animals, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel. Maximum age of the cultures 4 weeks Renewal of the cultures After 7 days of cultivation half of the medium twice a week. Temperature of medium 18-22C, constant within 1C Feeding Daily, a suspension of fresh water algae. Medium M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33). Composition of medium M 7: The following chemicals (analytical grade) are dissolved in tap water purified by reverse osmosis (milli-RO): Macro salts (mg/l): C a C I2.2 H 20 293.8 M g S 0 4.7H20 123.3 NaHCOg 64.8 Trace elements (mg/l): Macro nutrients (mg/l): ' KCI B Fe Mn Li, Rb and Sr Mo Br Cu Zn Co and 1 Se V Na EDTA.2H O 22 Na SiO . 9H20 23 NaNOg KH PO 24 K HPO 2t 5.8 0.125 0.05 0.025 0.0125 0.0063 0.0025 0.0016 0.0063 0.0025 0.0010 0.0003 2.5 10.0 0.27 0.14 0.18 Vitamins (pg/1): Thiamine B)2 Biotin 75.0 1.0 0.75 The hardness: 250 mg/l expressed as CaCOg and the pH: 8.0 0.2 after aeration. -7 - T-4127 NOTOX Project 334349 REFERENCE SUBSTANCE This report includes the results of a reference test with potassium dichromate. PREPARATION OF TEST SOLUTIONS The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface). T-4127 is a salt consisting of Perfluorooctanesulfonylmethylamide (anionic part) and Triphenylbenzylphosphonium (cationic part). Based on the response of the anionic part of the test substance water solubility of T-4127 was determined to be between 0.6 and 0.9 mg/l at 19.5 0.6C (NOTOX Project 340842, using the column elution method). Limit test A stock solution was prepared by weighing 100 mg of T-4127 on a glass surface. After placing the carrier in a measuring flask 1 litre of test medium was added to obtain a loading rate of 100 mg/l. The mixture was then magnetically stirred for four days. After the stirring period the mixture was clear but contained test substance particles, which settled at the bottom of the flask. Part of the mixture was used as such while another part was filtered through a paper filter (Schleicher and Schuell 604) to remove the larger undissolved test substance particles (ca. > 5//m). The final test solution was clear and colourless. Final test All glassware used in the final test was pre-treated with dichlorodimethylsilane to prevent loss of test substance due to adsorption to glass surfaces. A stock solution was prepared by weighing 500 mg of T-4127 on a glass surface. After placing the carrier in a measuring flask 5 litre of test medium was added to obtain a loading rate of 100 mg/l. Simultaneously, another measuring flask was prepared in the same way but without adding test substance (blank-control). After the stirring period, the solution prepared at a loading rate of 100 mg/l was transferred to a separation funnel and left to stabilise for 2% hours. Subsequently the Water Accommodated Fraction (WAF) was separated from the centre of the separation funnel. Lower test concentrations were prepared by dilution of the WAF. All final test solutions were clear and colourless. The test solutions originated from those used for the acute fish toxicity test (NOTOX Project 334338). LIMIT TEST A limit test was performed exposing twenty daphnids per concentration to a filtered and an unfiltered solution prepared at a loading rate of 100 mg/l and a blank-control. No samples were taken during the limit test. FINAL TEST: TEST CONCENTRATIONS T-4127 4.5, 10, 20, 45 and 100% of a WAF prepared at a loading rate of 100 mg/l. T-4127 NOTOX Project 334349 Control Test medium without test substance or other additives (Blank-control). TEST PROCEDURE AND CONDITIONS Test type Static Test duration Test vessels 48 hours 100 ml, all-glass. Pre-treated with dichlorodimethylsilane. Although vapour pressure was relatively low, based on the Henry constant evaporation was still a possible cause for decrease of test concentrations. Therefore the study was performed in air-tight vessels. Medium Number of daphnia Loading ISO, prepared in milli-RO water 20 per concentration 10 per vessel containing 80 ml medium Light 16 hours photoperiod daily Feeding No feeding Aeration No aeration of the test solutions. Introduction of daphnia Within 2 hours after preparation of the test solutions. SAMPLING FOR ANALYSIS OF TEST CONCENTRATIONS Duplicate samples were taken from the stock solution directly after preparation of the WAF as a specification of the origin of samples taken during the test. During the final test duplicate samples were taken from all concentrations and the blank-control at the start of the test, from the 4.5 and 10% dilutions after 24 hours of exposure and from the 4.5% dilution at the end of the test period. The method of analysis is described in the appended Analytical Report. Sampling: Volume T reatment Storage 5 ml from the approximate centre of the test vessels. Samples were added directly to isopropanol at a ratio of 1:5 to prevent any additional loss. Not applicable, samples were analysed on the day of sampling. MEASUREMENTS AND RECORDINGS Immobility (including mortality) At 24 hours and 48 hours. pH and dissolved oxygen At the beginning and at the end of the test, for all concentrations and the control(s). T-4127 NOTOX Project 334349 Temperature of medium Continuously in a temperature control vessel, beginning at the start of the test. RESULTS Limit test: Immobility Table 1 shows the responses recorded during the limit test. After 24 hours of exposure all organisms exposed to the filtered and the unfiltered solutions prepared at a loading rate of 100 mg/l became immobilised. Table 1: Incidence of immobility in the limit test: Loading rate T-4127 (m g/l) Blank-control 100; filtered 100; unfiltered Vessel num ber A B A B A B Number D a p h n ia exposed 10 10 10 10 10 10 Response at 24 h num ber 0 0 10 10 10 10 Total % 0 100 100 Response at 48 h num ber 0 0 10 10 10 10 Total % 0 100 100 Experimental conditions The results of measurement of pH and oxygen concentrations (mg/l) are presented in Table 2. The temperature of the test medium measured in the temperature control vessel varied from 19.2 to 19.9 C. Table 2: pH and oxygen concentrations during the limit test. Loading rate T-4127 (m g/l) Blank-control 100; filtered 100; unfiltered Start (t=0 h) pH 0 2 8.0 9.1 8.0 9.0 8.0 9.0 End (t=48 h) pH Oa 7.8 9.0 7.8 8.9 7.8 8.8 Final test: Measured concentrations The results of analysis of the samples taken during the study are described in Table 1 of the appended Analytical Report. During the final test the exposure consisted of a range of dilutions of a water fraction prepared at 100 mg/l containing 4.5 to 100% of this water fraction. Samples of 5 ml taken from these dilutions were added to 25 ml isopropanol and analyzed using the API300 LCMSMS (injection volume 10 yvl). Recovery samples were prepared in ISO-medium at 0.01 mg/l, 0.1 mg/l and 0.6 mg/l. The 0.01 mg/l concentration proved to be below LOD. Recoveries at 0.1 mg/l and 0.6 mg/l were relatively high (117% and 1 1 1 % respectively). T-4127 NOTOX Project 334349 Samples were taken in duplicate from the test solutions and each pretreated sample was injected in triplicate. The initial concentration in the water phase was 0.76 mg/l which corresponds with the water solubility of 0.6 - 0.9 mg/l (anionic part). Only the lowest treatment was sampled and analyzed during the whole exposure period. At the start of the test, the concentration of the lowest treatment was 0.023 mg/l, i.e. just above the limit of detection (LOD). After 48 hours it was below the LOD. Immobility Table 3 shows the responses recorded during the final test. After 24 hours 55% immobilization of daphnids was reached at the lowest treatment, while 100% effect was recorded in the 10% treatment and higher. After 48 hours also all daphnids at the lowest treatment were immobile. An acceptable 10% effect was recorded in the control replicates. Table 3: Acute immobilisation of daphnia after 24 and 48 hours in the final test. T-4127 Vessel Number Response at 24 h Response at 48 h % of a WAF (100 m g/l) num ber D a p h n ia exposed num ber Total % num ber Total % Blank-control A 10 B 10 0 (1 ) 0 0 1 (2) 1 10 4.5 A 10 5 (1 ) 55 10 100 B 10 6 (1 ) 10 10 A 10 10(1) 100 10 100 B 10 10 10 20 A 10 1 0 (5 ) 100 10 100 B 10 1 0 (1 ) 10 45 A 10 10 100 10 100 B 10 10 10 100 A 10 1 0 (1 ) 100 10 100 B 10 10 10 Between brackets: number of daphnids observed trapped at the surface. These organisms were reimmersed into the respective solutions before recording of mobility. Experimental conditions The results of measurement of pH and oxygen concentrations (mg/l) are presented in Table 4. The temperature of the test medium measured in the temperature control vessel varied from 20.2 to 20.9 C. Table 4: pH and oxygen concentrations during the final test. T-4127 % of a WAF (100 m g/l) Blank-control 4.5 10 20 45 100 Start (t=0 h) pH o2 8.0 8.7 8.0 8.4 7.9 8.4 7.8 8.4 7.9 8.2 7.8 7.6 End (t=48 h) pH o2 7.9 8.5 7.9 8.3 7.9 8.6 7.9 8.6 7.9 8.6 7.9 8.6 T-4127 NOTOX Project 334349 ACCEPTABILITY OF THE TEST 1. In the controls, not more than 10% of the daphnids became immobilised or trapped at the surface of the water. 2. All test conditions (pH, oxygen concentration and temperature) remained within the ranges prescribed by the protocol. 3. The analytical program based on the anionic part showed that the actual concentrations could not be maintained at more than 80 % of the initial concentration, in spite of all precautions taken to prevent adsorption to glass and volatilization. CONCLUSION Under the conditions of the present acute tests, dilutions of a water phase of T-4127 originating from a loading rate of 100 mg/l induced 100% immobility of Daphnia magna. Chemical analysis of the anionic part of T-4127 showed that this corresponded with initial T-4127 concentrations down to 0.02 mg/l. Hence, the 48h-EC50of Daphnia magna exposed to T-4127 was < 1 mg/l (< 0.023 mg/l) based on quantification of the anionic part and was already reached within 24 hours of exposure. Limiting the analytical support on only the anionic part of T-4127, validity of the test could not be ensured. There is a realistic possibility that the actual concentrations of the cationic part were higher and more stable than those of the anionic part. However, the sponsor did not agree with further research and decided to accept the highly toxic labelling (R50) based on the current results. T-4127 NOTOX Project 334349 REFERENCE TEST Start: November 04, 2002 End : November 06, 2002 48-hour Acute Toxicity Study in Daphnia magna with K2Cr207 (NOTOX Project 356669). The study procedures described in this report were based on the ISO International Standard 6341, the EEC directive 92/69, Part C.2. "Acute toxicity for Daphnia" and the OECD guideline No. 202: "Daphnia sp.t Acute Immobilisation Test", Adopted April 4, 1984. The reference test was carried out to check the sensitivity of the test system as used by NOTOX. Daphnia were exposed for a maximum of 48 hours to K2Cr20 7concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l and to a blank control. Ten daphnia were exposed per concentration. The reference substance, potassium dichromate (K2Cr20 7, art. 4864, batch no. K28974764) was obtained from Merck, Darmstadt, Germany. Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate: C o n c e n tra tio n K2Cr20 7 (m g /l) Number exposed % im m obile 24h 48h Expected response (%) after 48 hours 1 M inim al M axim al Blank-control 10 0 00 102 0.10 0.18 0.32 10 10 10 0 0 0 03 0 o3 0 o4 0 10 10 30 0.56 10 0 20 0 100 1.0 10 60 100 40 100 1.8 10 100 100 100 100 1 Based on historical data of the previous years (n>60). 2 A maximum response of 10% does not invalidate the results of the test. 3 Two daphnids were observed trapped at the surface of the test solutions. These daphnids were reimmersed in the respective solutions before scoring of mobility. 4 Slight precipitation and a floating layer were observed. The actual responses in this reference test with K2Cr20 7are within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of Daphnia magna was in agreement with the historical data collected at NOTOX. The 24h-EC50 was 0.93 mg/l with 95% fiducial limits of 0.82 - 1 .2 mg/l. The 48h-EC50 was estimated to be 0.63 mg/l with 20% immobility at 0.56 mg/l and 100% immobility at 1.0 mg/l. The protocol, raw data and report of this study are kept in the NOTOX archives. The test described above was performed under GLP conditions with a QA-check. ANALYTICAL REPORT ACUTE TOXICITY STUDY IN DAPHNiA MAGNA WITH T-4127; DETERMINATION OF THE CONCENTRATIONS NOTOX Project 334349 NOTOX Substance 113607 - Page 1 of 8 - T-4127 REPORT APPROVAL PRINCIPAL SCIENTIST: NOTOX Project 334349 lr. M.J.C. Brekelmans n /\A A r A T-4127 NOTOX Project 334349 PREFACE Analytical study Start: Completed: 11 November 2002 13 November 2002 PURPOSE The purpose of the analytical study was to determine the test concentrations and to validate the analytical method used. REAGENTS _________________________ ____________________ Milli-Q water Tap water purified by reversed osmosis and subsequently passed over activated carbon and ionexchange cartridges; Millipore, Bedford, MA, USA Methanol P.a., Merck, Darmstadt, Germany ISO-medium see main report Ammonium acetate Fractopur, Merck, Darmstadt, Germany 2.0 mM ammonium acetate 154 mg ammonium acetate in 1 litre of Milli-Q water Isopropanol (2-propanol) p.a., Merck, Darmstadt, Germany Isopropanol containing internal standard 140 pi of a solution of 2816 mg/l perfluorooctane sulfonate in methanol filled up to 2000 ml with isopropanol. INTERNAL STANDARD Identification number Name Description Batch number Purity Expiry Date Certified Storage conditions Supplier AS517 Perfluorooctane sulfonate (FC-95) White solid (determined at NOTOX) Lot 217 90.49% 21 February 2003 (determined at NOTOX) Yes At room temperature in the dark 3M The sponsor is responsible for all test substance data unless determined by NOTOX. SAMPLE PRETREATMENT PROCEDURE __________ Each sample (5 ml) was transferred quantitatively into a 50 ml volumetric flask and 25 ml of isopropanol containing internal standard was added (internal standard concentration 164 pg/l). AAAACC T-4127 NOTOX Project 334349 ANALYTICAL METHOD T-4127 is a salt consisting of Perfluorooctanesulfonylmethylamide (anionic part) and Triphenylbenzylphosphonium (cationic part). Upon request of the sponsor, quantitative analyses were only based on the anionic part of T-4127 using High Performance Liquid Chromatography with Mass Spectrometric detection (LCMSMS). Analytical conditions Column Column temperature Mobile phase Flow Column temperature Autosampler temperature Injection volume Detection Interface Monitored masses Betasil C18, 50 x 2.0 mm; dp= 5 pm (Thermo Hypersil, Keystone) 25C Time 2.0 mM Ammonium acetate Methanol (minutes) 0 1 (%) 90 90 (%) 10 10 5.5 5 95 7.5 5 8.0 90 11 90 95 10 10 300 pl/min 25 C 4 C 10 pi SCIEX MSMS system API-300 mass spectrometer (Applied Biosystems, Toronto, Canada) Turbo ionspray at 450C; N2flow rate of 7000 ml/min.; operated in negative ion mode MRM test substance m/z 512.1 --> 168.8 MRM internal standard m/z 499.0 --> 99.0 Standard and calibration solutions Standard solutions of T-4127 were prepared in methanol. On each day of analysis, calibration solutions in 1/5 (v/v) ISO-medium/isopropanol containing internal standard (internal standard concentration 164 pg/l) were made up from two standard solutions. VALIDATION OF THE ANALYTICAL METHOD The LCMSMS method used was only partly validated during NOTOX project 334338. Upon request of the sponsor, validation was not completed. T-4127 NOTOX Project 334349 DATA HANDLING - SAMPLE ANALYSIS Calibration Response (R): Peak area test substance R = --------------------------------------- Peak area internal standard Calibration curve: The response was correlated with the concentration of test substance, using linear regression analysis (least squares method) and a (1/concentration) weighting factor. Samples R = a*C+b R = response calibration solution C = concentration in thecalibration solution [mg/l] a = slope [l/mg] b = intercept On each day of analysis, two calibration solutions were used for quantification. Both calibration solutions were injected (in duplicate) before and after a maximum of six samples. Using the four responses, a calibration curve was constructed. Concentration analysed: Recovery of recovery samples: C = d* -- - [mg/l] a R = response calibration solution C = concentration in the calibration solution [mg/l] a = slope [l/mg] b ' = intercept Concentration analysed -------------------------------- * 100 [%] Concentration prepared Concentration analysed Concentration relative to n o m i n a l : ------------------------------- * 100 [%] Concentration nominal T-4127 NOTOX Project 334349 RESULTS -VALIDATION OF THE ANALYTICAL METHOD See NOTOX project 334338 "96-hour acute toxicity study In zebra-fish with T-4127 (static)". RESULTS - SAMPLE ANALYSIS Table 1 shows the analytical results for the samples from the ecotoxicity study*. Table 1 Concentrations of T-4127 in test medium (Full test 1). Time of sampling [hours] 02 02 24 48 Date of sampling [dd-mm-yy] 11-Nov-02 11-Nov-02 12-Nov-02 13-Nov-02 Date of analysis [dd-mm-yy] 11-Nov-02 11-Nov-02 12-Nov-02 13-Nov-02 Percentage of filtrate1 [%] Concentration Expected2 Analysed3 [mg/l] [mg/l] Relative to expected [%] 100 0.770 100 0.750 100 0.76 0.56 74 100 0.76 0.75 98 45 0.34 0.24 70 45 0.34 0.20 59 20 0.15 0.16 103 20 0.15 0.13 87 10 0.076 0.029 5 38 10 0.076 0.064 84 4.5 0.034 0.021 5 61 4.5 0.034 0.024 5'6 69 0 0 n.d. n.a. 0 0 n.d. n.a. 10 0.076 0.027 6 36 10 0.076 0.024 32 4.5 0.034 < LOD - 4.5 0.034 < LOD - 4.5 0.034 < LOD _ 4.5 0.034 < LOD - Percentage of a 5 pm filtered solution prepared at nominal concentration of 100 mg/l. 2 Based on the measured concentration (i.e. 0.760 mg/l) in the undiluted filtrate without algae. 3 Mean of triplicate analysis. The maximum deviation between the responses calculated for each sample was < 20% unless otherwise indicated. 4 Combined with N O TO X project 334338. 5 Calculated by extrapolation. Responses were >70% of the lowest calibration level. 6 Maximum deviation between responses was 23% and 27% , respectively, n.d. Not detected. The limit of detection (LOD) was 0.023 mg/l. n.a. Not applicable. All relative values were calculated using not-rounded concentrations. Therefore, some differences might be observed when calculating the relative values using the concentrations as mentioned in the table. T-4127 NOTOX Project 334349 I! am m iO i'r n * Internal standard S 70S 2 SO M B ,* ? 1 to o ty tf* 1 4 1 7 11 Figure 1 HPLC-chromatogram of a blank (1/5 (v/v) ISO-medium/isopropanol containing internal standard). Internal standard Figure 2 HPLC-chromatogram of 0.500 mg/l T-4127 in 1/5 (v/v) ISO-medium/isopropanol containing internal standard. T-4127 NOTOX Project 334349 Figure 3 Regression line for solutions in 1/5 (v/v) ISO-medium/isopropanoi containing internal standard: Responses against concentrations.