Document dYZx9B9EpRyQZ4MdbLvrENg70

R Z9.6-- /337 REPORT 96-HOUR FRESH WATER ALGAL GROWTH INHIBITION TEST WITH T-4127 C3 cz opo ~o rn ro cn -TO O --i r n -o O p i 0o 09 cn NOTOX Project 334351 NOTO* Substance 113607 - Page 1 of 37 - CCDD r T3 OD - .) / \ / \ tfVJA^ T-4127 STATEMENT OF GLP COMPLIANCE NOTOX Project 334351 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. Management: Ing. E.J. van de Waart M.Sc. Head of Genetic & Ecotoxicology o nnnn2 T-4127 QUALITY ASSURANCE STATEMENT NOTOX Project 334351 NOTOX B.V., 's-Hertogenbosch, The Netherlands 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) October 07-17, 2002 (Ecotoxicology) November 05-15, 2002 (Analytical Support) protocol inspection(s) (Study) December 06, 2001 report audit(s) (Study) April 29, 2003 REPORTING DATES October 21,2002 November 19, 2002 December 06, 2001 April 29, 2003 Head of Quality Assurance ' C.J. Mitchell B.Sc. Date: It, T-4127 NOTOX Project 334351 SUMMARY Selenastrum capricomutum, 96-Hour Fresh Water Algal Growth Inhibition Test with T-4127. The study procedures described in this report were based on the EEC Directive 92/69, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7, 1984; and ISO Standard 8692, First edition, 15 November 1989. 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 exponentially growing algae to a filtered solution prepared at a loading rate of 100 mg/l, which was magnetically stirred for three days before filtration. A blank-control was also included. Concentrations in the filtered solution decreased from 1.2 mg/l to below the limit of detection during the test. The biological results showed that approximately total inhibition of cell growth and reduction of growth rate occurred at the limit concentration. A final test was performed exposing exponentially growing algae 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 96 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. Since 100% effect was recorded in the lowest treatment throughout the test only samples taken from this level were analyzed. Results showed that the measured anion concentration varied between 77 and 87%, relative to the initial concentration. In the controls, cell density increased by an average factor of > 16 within 3 days. Further, temperature conditions remained within the ranges prescribed by the protocol. However, pH increased by more than 1.5 unit in the blank-control, but this was related to the air tightly capped vessels. Hence, this deviation did not affect the validity criterion for algal growth. T-4127 induced total inhibition of cell growth and total reduction of growth rate of Selenastrum capricomutum at concentrations < 0.05 mg/l. Hence, the 96h-EC50of this fresh water algae species exposed to T-4127 was < 1 mg/l (< 0.05 mg/l). 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. A T-4127 NOTOX Project 334351 PREFACE Sponsor Study Coordinator Study Monitor Testing Facility Aquatic Toxicology: Study Director Technical Coordinator Analytical Chemistry: Principal Scientist Study Plan 3M Corporate Toxicology 3M Center, Building 220-2E-02 P.O. Box 33220 ST. PAUL, MINNESOTA 55133-3220 U.S.A. Mrs. M. Mitchell Mrs. Dr. S. Beach 3M Environmental Technology and Safety Services 935 Bush Avenue, Building 2-3E-09 ST. PAUL, MINNESOTA 55144 U.S.A. NOTOX B.V. Hambakenwetering 7 5231 DD 's-Hertogenbosch The Netherlands Drs. M. Bogers Mrs. W. Koolen Ir. M.J.C. Brekelmans Start Project: December 05, 2001 Start of first exposure: July 01,2002 Completion last exposure: November 15, 2002 Completion Analysis: November 15, 2002 Completion project: May 13, 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. . Z. - nnAAC C T-4127 NOTOX Project 334351 PURPOSE The purpose of the study was to evaluate the test substance for its ability to inhibit the growth of green algae in a short-term experiment. GUIDELINES The study procedures described in this report were based on the ISO International Standard 8692: "Water quality - Fresh water algal growth inhibition test with Scenedesmus subspicatus and Selenastrum capricornutum", First edition, 15 November 1989. In addition, the procedures were designed to meet the test methods and validity criteria prescribed by the following guidelines: - European Economic Community (EEC), EEC Directive 92/69, Part C: Methods for the determination of ecotoxicity, Publication No. L383, C-3: "Algal Inhibition Test" adopted December, 1992. - Organization for Economic Co-operation and Development (OECD), OECD guideline for Testing of Chemicals, guideline No. 201: "Algae, Growth Inhibition Test", Adopted June 7, 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 Cell density is the number of cells per millilitre. Growth is the increase in cell density over the test period. Growth rate is the increase in cell density per unit time. It is derived from the slope under the growth curve in a logarithmic plot. Following from the mathematical nature of exponential growth, the measure of the specific growth rate is preferable over biomass. The ErC50 is the concentration of test substance that results in a 50% reduction in growth rate relative to the control. Total growth or biomass is defined as the increase in total cell density over the test period. It is derived from the area under the growth curve in a linear plot. The EBC5o is the concentration of test substance that results in a 50% inhibition of total cell growth relative to the control. No Observed Effect Concentration (NOEC) is the highest tested concentration at which the measured parameter(s) show(s) no statistically significant effect on algal growth relative to control values. - fi - a a a AC T-4127 NOTOX Project 334351 TEST SYSTEM Species Reason for selection Control of sensitivity Selenastrum caphcornutum, strain: NIVA CHL 1. This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species. The results of the most recent reference test with potassium dichromate (Merck, Art. 4864) are appended to this report. FRESH WATER ALGAE CULTURE Stock culture Stock culture medium Pre-culture Pre-culture medium Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 2C. M1; formulated using Milli-RO water and with the following composition: NaNOa 500 mg/l KjHPOi 40 mg/l M gS04.7H20 76 mg/l Na2CO3.10H2O 54 mg/l c 8h 8o 7 .h 2o 6 mg/l NH4NO3 330 mg/l CaCI2 .H20 36 mg/l C8H5F e 0 7 .xH20 6 mg/l H3 BO3 MnCI2.4H20 2.9 mg/l 1.81 mg/l ZnCI2 0.11 mg/l C uS 0 4.5H20 0.08 mg/l (NH4)8Mo70 24.4H20 0.018 mg/l 3 or 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use. M2; according to the ISO-Standard "Algal growth inhibition test" Nov. 1989; formulated using Milli-Q water preventing precipitation and with the following composition: NH4CI 15 mg/l MgCI2.6H20 12 mg/l CaCI2.2 H 2 0 18 mg/l M gS04.7H20 15 mg/l KH2P 0 4 1.6 mg/l FeCI3.6H20 Na2EDTA.2H20 80 //g/i 100 /'g/i -7 - T-4127 NOTOX Project 334351 h 3b o 3 MnCI2.4H20 ZnCI2 CoCI2.6H20 C uCI2.2H20 Na2M o04.2H20 NaHCOa Hardness (Ca+Mg) pH 185 415 ^g/i 3 Arg/I 1.5 pg/i 0.01 /rg/l 7 /rg/l 150 mg/l 0.24 mmol/l (24 mg CaCCyi) 8.3 0.2 PREPARATION OF TEST SOLUTIONS The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system were prevented as much as possible (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 three days. After the stirring period the mixture was clear but contained test substance particles, which settled at the bottom of the flask. The mixture 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. Note that the blank-control received the same treatment. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 104 cells/ml. 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 200 mg of T-4127 on a glass surface. After placing the carrier in a measuring flask 2 litre of test medium was added to obtain a loading rate of 100 mg/l. After the stirring period, the stock solution was transferred to a separation funnel and left to stabilise for 4 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. Note that the blank-control received the same treatment. After preparation, volumes of 80 ml were added to each replicate of the respective test concentration to reduce the headspace as much as possible. The algal medium contained extra NaHC03 (150 mg/l instead of 50 mg/l) to prevent depletion of C02. Subsequently, adequate volumes of an algal suspension were added to each replicate providing a cell density of 104 cells/ml. 000068 T-4127 NOTOX Project 334351 LIMIT TEST A limit test was performed exposing exponentially growing algae to a filtered solution prepared at a loading rate of 100 mg/l and a blank-control for a period of 72 hours. Sampling: Frequency Volume Storage at t-0 h, t=24 h and t=72 h. 10 ml from the approximate centre of the test vessel. Not applicable, all samples were analysed on the day of sampling. FINAL TEST: TEST CONCENTRATIONS T-4127 Controls Replicates 4.5, 10, 20, 45 and 100% of a WAF prepared at a loading rate of 100 mg/l. Test medium without test substance or other additives (Blank-control). 3 replicates of each test concentration. 6 replicates of the blank-control. 1 replicate of each test concentration and the blankcontrol for sampling purposes. 2 replicates of the highest test concentration without algae, filled to the rim and protected from light for the course of the test. TEST PROCEDURE AND CONDITIONS Test type Static Test vessels 100 ml, all-glass 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 M2-medium Cell density An initial cell density of 1 x 104 cells/ml. Test duration 96 hours Illumination Continuously using TLD-lamps of the type `Cool-white' of 30 Watt, with a light intensity within the range of 73 to 102 pE.m'2.s'1. Incubation During incubation the algal cells were kept in suspension by continuous shaking. - Q- a a a ACQ T-4127 NOTOX Project 334351 SAMPLING FOR ANALYSIS OF TEST CONCENTRATIONS Duplicate samples were taken from all dilutions and the blank-control directly after preparation, from separate vessels of the 4.5% dilution and the blank-control after 48 and 96 hours. The method of analysis is described in the appended Analytical Report. Sampling: Volume Treatment Storage 5 ml. 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 pH Temperature of medium At the beginning and at the end of the test. The pH of the solutions should preferably not deviate by more than 1.5 units during the test. Continuously in a temperature-control vessel. RECORDING OF CELL DENSITIES At the beginning of the test, cells were counted by microscope, using a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Varian Nederland BV., Houten, The Netherlands. Algal medium was used as blank. DATA HANDLING Calibration curve: Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The equation of this curve was then used to calculate the cell densities of the various test solutions at different points in time during the test period. Comparison of areas under the growth curves: The area below the growth curve was calculated using the formula: A = N r N0 Ni +N2 - 2 xN0 x(f2 - f 1) + Nn--i +Nn- 2 x.NQ fn-1) Where: A = area N0= nominal number of cells/ml at the start of the test N)= measured number of cells/ml at L Nn= measured number of cells/ml at tn f!= time of first measurements after beginning of the test fn= time of nth measurement after beginning of the test -10- T-4127 NOTOX Project 334351 The percentage inhibition of cell growth at each test concentration (lT) was calculated using the following formula: i Ac~ x100 ,T-- t r Where: Ac = area below the growth curve obtained in the control Ar = area below the growth curve at each test substance concentration Growth inhibition was calculated for the total period of 96h. Comparison of growth rates: The average specific growth rate (jj) for exponentially growing cultures was calculated as: . In A/n --In Af, tn~ t, The average growth rate at each test substance concentration was then compared to the control value and the percentage reduction in growth rate was calculated. RESULTS Limit test: The first test included samples prepared at a nominal loading rate of 100 mg/l filtered through a paper filter (ca. 5 //m). Samples were diluted with methanol in 1:1 (v:v) ratio and analyzed using the LCQduo LCMSMS (injection volume 100 //I). Concentrations in the filtered sample decreased from 1.2 mg/l to below the limit of detection during the test, see also Table 6 of the appended Analytical Report. The mean cell densities measured during the limit test are presented in Table 1. Table 2 presents the percentages growth inhibition and growth rate reduction per concentration. The results showed that approximately total inhibition of cell growth and reduction of growth rate occurred at the limit concentration. Table 1: Mean cell densities (x1(f cells/ml) during the limit test Loading rate T-4127 (m g/l) Blank-control 100; filtered 0 1.0 1.0 Exposure time (hours) 24 48 5.8 34.0 1.5 3.7 72 129.6 1.7 Table 2: Percentage reduction of growth rate and inhibition of total growth during the limit test Loading rate T-4127 (mg/1) Blank-control 100; filtered Cell growth (0-72 hrs) Mean growth rate Mean area (A) Inhibition (%) V (0-72 hrs) Reduction (%) 2448.16 85.36 96.5 0.06744 0.00721 89.3 -11 - T-4127 NOTOX Project 334351 Experimental conditions Table 3 shows the pH recorded at the beginning and the end of the test. The temperature of the test medium was 23.2C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 23.0 and 23.5C. Table 3: pH levels recorded during the limit test. Loading rate T-4127 (m g/l) Blank-control 100; filtered Exposure time (hours) 0 96 8.0 9.0 8.0 8.0 Final test: Measured test substance concentrations The results of analysis of the samples taken during the study are described in Table 7 of the appended Analytical Report. During the final test, sampling procedure was identical to that applied in the fish test. Because concentrations were expected to become below the limit of detection at the end of the test, it was decided to change sample pretreatment during the test and to test different injection volumes. Samples were taken in duplicate and each pretreated sample was injected in triplicate. The initial concentration in the water phase was 1.67 mg/l and thus higher than the water solubility limit of 0.6 - 0.9 mg/l. The concentrations measured at the start of the test are in Table 4. Table 4: Mean concentrations of duplicate samples taken at the start of the test T-4127 % of a WAF (100 mg/l) 4.5 10 20 45 100 Concentration T-4127 at the start of th e test in mg/l Sample 1 Sample 2 Mean 0.0671 0.0564 0.0617 0.152 0.160 0.1560 0.328 0.330 0.3292 0.754 0.742 0.7479 1.69 1.64 1.6670 Mean cell densities Algal density was determined every 24 hours for a total of 96 hours and no algal growth was recorded in any of the treatments, while algae cells grew exponentially during the first 72 hours in the control group. Between 72 and 96 hours growth rate decreased, resulting in a sigmoid curve (see Figure 1). Since 100% effect was recorded in the lowest treatment throughout the test only samples taken from this level were analyzed. The actual concentration remained rather stable (77-87% of initial), see Table 5. T-4127 NOTOX Project 334351 Table 5: Mean concentration in the lowest treatment during the test T-4127 % of a WAF (100 mg/l) 4.5 Concentration T-4127 0 0.0617 48 0.0474 96 0.0535 Table 6 shows mean cell densities measured at 24-hour intervals at the different concentrations of T-4127. The respective growth curves are shown in Figure 1 (see the Appendix I for the cell densities per replicate). Table 6: Mean cell densities (x 1(f cells/ml) during the final test T-4127 % of a WAF (100 mg/I) Blank-control 4.5 10 20 45 100 0 1.0 1.0 1.0 1.0 1.0 1.0 Exposure time (hours) 24 48 1.5 17.1 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 72 53.3 1.0 1.0 1.0 1.0 1.0 96 62.1 1.0 1.0 1.0 1.0 1.0 Experimental conditions Table 7 shows the pH recorded at the beginning and the end of the test. In spite of the addition of extra NaHC03to the M2-medium pH increased more than 1.5 units in the blank-control during the test period. This was related to the air tightly capped vessels as a result of the limited amount of NaHC03 in the solutions and the limited C 02exchange from the headspace. The temperature of the test medium was 23.1 C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22.6 and 23.8C. Table 7: pH levels recorded during the final test. T-4127 % of a WAF (100 mg/l) Blank-control 4.5 10 20 45 100 Exposure time (hours) 0 96 8.4 10.4 8.4 8.6 8.4 8.3 8.4 8.2 8.5 8.1 8.6 8.2 T-4127 NOTOX Project 334351 ACCEPTABILITY OF THE TEST 1. In the controls, cell density increased by an average factor of > 16 within 3 days. 2. Temperature conditions remained within the ranges prescribed by the protocol. However, pH increased by more than 1.5 unit in the blank-control, but this was related to the air tightly capped vessels. Hence, this deviation did not affect the validity criterion for algal growth. 3. Analysis of samples taken from the lowest treatment showed that the measured anion concentration varied between 77 and 87%, relative to the initial concentration. However, this concentration remained far more stable than similar concentrations in the fish and daphnia tests (NOTOX Projects 334338 and 334349). This was probably due to the difference in composition of test medium. CONCLUSION Under the conditions of the present study with Selenastrum capricornutum, T-4127 induced total inhibition of cell growth and total reduction of growth rate of this fresh water algae species at concentrations < 0.05 mg/l. Hence, the 96h-EC50 of Selenastrum capricornutum exposed to T-4127 was < 1 mg/l (< 0.05 mg/l). 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. -14- . M T-4127 Figure 1: Growth curves at different concentrations of T-4127. NOTOX Project 334351 T-4127 % of a WAF (100 mg/l) -- -- Blankcontrol --*--4 .5 - - -A- - - 10 --X --20 - - X - - 45 -- -- 100 n n n n rtc T-4127 NOTOX Project 334351 REFERENCE TEST Selenastrum capricornutum, fresh water algal growth inhibition test with potassium dichromate (NOTOX Project 356647). Start of first exposure: September 30, 2002 Completion last exposure: October 03, 2002 The study procedures described in this report were based on the EEC Directive 92/69, Publication No. L383 Part C-3 adopted December, 1992; OECD guideline No. 201, Adopted June 7, 1984; and ISO Standard 8692, First edition, 15 November 1989. This reference test was carried out to check the sensitivity of the test system used by NOTOX to POTASSIUM DICHROMATE (Merck, Art. 4864, Batch K28974764). Algae were exposed for a period of 72 hours to K2Cr20 7concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a blank-control. The initial cell density was 1.0 x 104cells/ml. Results: Calculation of % reduction in growth rate in the reference test: Concentration K 2C r 207 (m g/l) 0 0.18 0.32 0.56 1.0 1.8 3.2 Total cell growth: Mean Area Inhibition for 0-72h % 2000.16 1872.93 2068.42 1696.29 1222.95 355.99 147.24 6.4 - 3.4 15.2 38.9 82.2 92.6 Growth rate: Interval Reduction 0-72 h % 0.06480 0.06427 0.06564 0.06266 0.05723 0.03315 0.01067 0.8 - 1.3 3.3 11.7 48.8 83.5 Under the conditions of the reference study with Selenastrum capricornutum, potassium dichromate inhibited cell growth of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher and reduced growth rate at 1.0 mg/l and higher. The EC50for cell growth inhibition (EBC5o: 0-72h) was 1.1 mg/l with a 95 % confidence interval ranging from 0.78 to 1.6 mg/l. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/l. Hence, the EBC50: 0-72h for the present batch corresponds with this range. The EC50 for growth rate reduction (ErC5c>: 0-72h) was 1.9 mg/l with a 95 % confidence interval ranging from 1.6 to 2.2 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ErCS0: 0-72h for the present batch corresponds with this range. 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. T-4127 NOTOX Project 334351 APPENDIX I WORKSHEET DATA Table 11: Cell densities calculated from the individual extinction values Number of inoculated cells at t=0: 1x104 cells/ml T-4127 % of a WAF (100 mg/l) Blank-control 4.5 10 20 45 100 Vessel number 1 2 3 4 5 6 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Exposure time (hours) 0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 24 1.81 1.47 1.97 1.44 1.05 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 48 15.67 17.33 19.49 17.64 16.87 15.54 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 72 50.75 57.00 55.29 53.42 52.00 51.59 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 96 63.03 60.42 62.50 60.64 64.73 61.37 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 n n A A 'T ? ANALYTICAL REPORT 96 HOUR FRESH WATER ALGAL GROWTH INHIBITION TEST WITH T-4127; DETERMINATION OF THE CONCENTRATIONS NOTOX Project 334351 NOTOX Substance 113607 - Page 1 of 20- T-4127 REPORT APPROVAL PRINCIPAL SCIENTIST: NOTOX Project 334351 Ir. M.J.C. Brekelmans (Analytical Chemistry) T-4127 NOTOX Project 334351 PREFACE Analytical study Start: 01 July 2002 Completed: 15 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 M2-medium see main report Methanol HPLC-grade, Labscan, Dublin, Ireland Isopropanol (2-propanol) p.a., Merck, Darmstadt, Germany Ammonium acetate Factopur, Merck, Darmstadt, Germany 2.0 mM ammonium acetate 154 mg ammonium acetate in 1000 ml Milli-Q water Internal standard solution 50 pi of a solution of 1204 mg/l perfluorooctane sulfonate in methanol filled up to 5 ml with 50/50 (v/v) M2-medium/methanol. Isopropanol containing internal standard 140 pi 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. AAA/VOA T-4127 NOTOX Project 334351 SAMPLE PRETREATMENT PROCEDURE Analyses in July 2002 Each sample (10 ml) was quantitatively transferred to a 20 ml volumetric flask. A volume of 200 pi internal standard solution was added to the samples taken at 24, 48 and 72 hours, after which the flasks were filled up to the mark using methanol (internal standard concentration 120 pg/l). No internal standard was added to the flasks containing the t=0 samples. Analyses in November 2002 In order to prevent from adsorption, it was decided to change sample pre-treatment. Based on additional information supplied by the sponsor, each sample (5 ml) was quantitatively transferred to a 50 ml volumetric flask. A volume of 25 ml isopropanol containing internal standard was added to the 50 ml-flasks (internal standard concentration 164 pg/l). 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). The analytical method used in July 2002 was not sensitive enough to measure concentrations in pretreated samples from the final test. Therefore, a second method was used in November 2002. Analytical conditions for the analyses in July 2002 Column Mobile phase Flow Column temperature Autosampler temperature Injection volume Detection T-4127, anionic part Internal standard Betasil C18, 50 x 2.0 mm; dp= 5 pm (Thermo Hypersil Keystone) Gradient Eluens A: 2.0 mM ammonium acetate Eluens B: 100 % methanol Time (min) Eluens A (%) Eluens B (%) 0.00 90 10 1.00 90 10 5.50 5 95 10.00 5 95 11.00 90 10 13.00 90 10 300 pl/min ambient temperature ambient temperature 100 pi LCQ Duo mass spectrometer (Thermo Finnigan, San Jose, CA, USA) ESI negative mass-mass detection Position 3 Collision energy: 30% Isolation width: 1.5 M/z 512.0 - 388.2, 419.0 ESI negative mass detection Position 3 M/z 499.2 T-4127 NOTOX Project 334351 Analytical conditions for the analyses in November 2002 Column 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) Gradient Eluens A: 2.0 mM ammonium acetate Eluens B: 100 % methanol Time (min) Eluens A (%) Eluens B (%) 0.00 90 10 1.00 90 10 5.50 5 95 7.50 5 95 8.00 90 10 11.00 90 10 300 pl/min 25 C 4 C 10 pi 1 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 solutions Standard solutions of T-4127 were prepared in methanol. Calibration solutions for the analyses in July 2002 On each day of analysis, calibration solutions in 50/50 (v/v) methanol/M2-medium were made up from two standard solutions. Internal standard was added to a final concentration of 120 pg/l except for samples taken at t=0 hours. Inadvertently, no internal standard was added to these samples. Calibration solutions for the analyses in November 2002 On the day of analysis, calibration solutions in 1/5 (v/v) M2-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 methods used were only partly validated. Upon request of the sponsor, validation was not completed. The parameters validated are given below. Injection volumes of 20, 50 and 100 pi were tested but accuracy and precision was not improved and therefore it was decided to use 10 pi injection for analysis of samples. T-4127 NOTOX Project 334351 Validation of the analytical method used in July 2002 Specificity Blank M2-medium1was pretreated as specified in `sample pretreatment procedure' and subsequently injected in triplicate into the HPLC system. The resulting chromatograms were critically evaluated for interfering peaks by comparison with chromatograms of a test substance solution in the same medium. Interfering peaks are required to be s 30% of the LOQ. Linearity From two standard solutions (1020 and 1060 mg/l), eight dilutions were prepared in 50/50 (v/v) methanol/ M2-medium. This resulted in a concentration range of 0.0510 - 0.996 mg/l1internal standard. Each of these solutions was injected in triplicate. Responses were plotted against the concentrations. A linear regression program was used to calculate the regression line from the responses and concentrations. The correlation coefficient is required to be at least 0.99. Stability of solutions Solutions1of 0.0510 mg/l, 0.102 mg/l and 0.996 mg/l T-4127 were injected two times (in triplicate) over a 8.9, 8.4 and 5.4-hour time period, respectively. The maximum deviation of the responses was calculated for each concentration. Limit of detection (LOP) A 0.051 mg/l solution of the test substance in 50/50 (v/v) methanol/ M2-medium1was injected in triplicate. In each chromatogram, the test substance peak height was measured as well as the noise level of the system (both in mV). The LOD was calculated from the mean peak height and the mean noise level. Validation of the analytical method used in November 2002 Specificity Blank M2-medium12was pretreated as specified in `sample pretreatment procedure' and subsequently injected in triplicate into the HPLC system. The resulting chromatograms were critically evaluated for interfering peaks by comparison with chromatograms of a test substance solution in the same medium. Interfering peaks are required to be s 30% of the LOQ. Linearity From two standard solutions (782 and 874 mg/l), six dilutions were prepared in 1/5 (v/v) M2medium/isopropanol containing internal standard. This resulted in a concentration range of 0.00501- 0.0996 mg/l2. Each of these solutions was injected in duplicate. Responses were plotted against the concentrations. A linear regression program was used to calculate the regression line from the responses and concentrations. The correlation coefficient is required to be at least 0.99. 1 Internal standard was added to a final concentration 120 pg/l. 2 Internal standard was added to a final concentration of 164 pg/l. T-4127 NOTOX Project 334351 DATA HANDLING -VALIDATION OF THE ANALYTICAL METHOD Response: Peak area test substance R- - - Peak area internal standard Mean: 1 x = --y * xi where: xi : measured value n : number of measurements Standard deviation: Coefficient of variation: Maximum deviation: Linearity Validation of the analytical method (July 2002) Validation of the analytical method (November 2002) Recovery: Limit of detection (LOD): ( * '- x ) 2 Sn- X= 1 -- ------------- 1 ( -D (standard deviation / mean value) * 100% [(highest - lowest)/mean] * 100% where 'mean' is the mean value of the highest and the lowest value. A regression program was used to calculate the regression line or curve from the responses and concentrations. Regression analysis was performed using the least squares method. If necessary a (1/concentration) weighting factor was used. Regression curve: Y = f X2 + g X+ h Regression line: Y = a X +b where: Y : response X : concentration a : slope b : intercept f, g, h: regression constants (Concentration analysed1/ Concentration prepared) * 100% The limit of detection is defined as the concentration of T-4127 with a signal (peak height) of three times the noise level (S/N=3). 1 See `DATA HANDLING - SAMPLE ANALYSIS'. T-4127 NOTOX Project 334351 Limit of detection= ((3 * noise level)/ signal) * cone, where: noise level (N) : height of the noise [%] signal (S) : height of the test substance peak [%] cone. : concentration of test substance [mg/l] DATA HANDLING - SAMPLE ANALYSIS Calibration Response: Calibration curve: (July 2002) Peak area test substance R = --------------------------------------- Peak area internal standard A regression program was used to calculate the regression curve from the responses and concentrations. Regression analysis was performed using the least squares method. If necessary a (1/concentration) weighting factor was used. R = a* C+b R =f*C 2+ g*C + h R : response calibration solution C : concentration of test substance in calibration solution [mg/l] a, b, f, g, h: regression coefficients An each day of analysis, a calibration curve or line was constructed using eight concentrations injected in triplicate. The coefficient of correlation was > 0.99. Calibration curve: (November 2002) The response was correlated with the concentration of test substance, using linear regression analysis (least squares method) and a (1/concentration) weighting factor. R = a* C+b R= C= a= b= response calibration solution concentration in the calibration solution [mg/l] slope [l/mg] intercept On each day of analysis, two calibration solutions were used for quantification. Both calibration solution were injected (in duplicate) before and after a maximum of seven samples. Using the four responses, a calibration curve was constructed. T-4127 NOTOX Project 334351 Samples Recovery of recovery samples: Concentration relative to nominal: Concentration analysed -------------------------------- * 100 [%] Concentration prepared Concentration analysed ------------------------------- * 100 [%] Concentration nominal Concentration analysed: (Quadratic regression July 2002) Concentration analysed : (Linear regression July 2002) Concentration analysed: (November 2002) c - d . - g + Vg2 - 4 * f * ( h - R) [mg/l] 2*f (R-b) * d C = -------------[mg/l] a R : response sample [units] d : dilution factor a, b, f ,g ,h : regression constants (R-b) * d C = -------------[mg/l] a R : response sample [units] d : dilution factor a : slope [units*l/mg] b : intercept [units] T-4127 NOTOX Project 334351 RESULTS -VALIDATION OF THE ANALYTICAL METHOD The calculations for the validation tests were performed using not-rounded concentrations and responses. Therefore, some differences might be observed when calculating the statistical parameters using the values as mentioned in the tables. Validation of the analytical method used in July 2002 Specificity Figures 1 and 2 show chromatograms of a blank solution (50/50 (v/v) methanol/M2-medium) and of a 0.996 mg/l T-4127 solution, respectively. It was clear that blank chromatograms did not contain any interfering peaks at the position of the test substance. Linearity A quadratic regression was performed. The results are summarized in Table 1. The regression curve is shown in Figure 3. Table 1 Quadratic regression curve. Concentration [mg/l] Response 1 [units] 0.0510 0.0700 0.102 0.199 0.400 0.602 0.800 0.996 0.0123/ 0.0117/ 0.0120 0.0152/ 0.0144/ 0.0143 0.0213/ 0.0214/ 0.0224 0.0416/ 0.0418 / 0.0391 0.0804/ 0.0776/ 0.0774 0.1086/ 0.1088/ 0.1100 0.1325/ 0.1333/ 0.1384 0.1637/ 0.1553/ 0.1466 Triplicate measurements. From these results, it was concluded that there is a quadratic relationship (Y = 6.07669*1 O'5+0.220598*X-0.065148*X2; R2 = 0.9973) between response and concentration in the concentration range of 0.0510 - 0.996 mg/l though a deviation of more than 10% of the calibration points from the calculated line was observed at some concentrations. T-4127 Stability of the solutions The results are summarised in Table 2. Table 2 Stability of a 0.0510 mg/l T-4127 solution. Elapsed time [hours] Response 0.0 0.0123 0.2 0.0117 0.5 0.0120 8.4 0.0113 8.7 0.0111 8.9 0.0114 NOTOX Project 334351 Maximum deviation [%] 9.8 Table 3 Stability of a 0.102 mg/l T-4127 solution. Elapsed time [hours] Response 0.0 0.0213 0.2 0.0214 0.5 0.0224 8.0 0.0215 8.2 0.0217 8.4 0.0200 Maximum deviation [%] 11.1 Table 4 Stability of a 0.996 mg/l T-4127 solution. Elapsed time [hours] Response 0.0 0.1637 0.2 0.1553 0.5 0.1466 4.9 0.1617 5.2 0.1675 5.4 0.1542 Maximum deviation [%] 13.2 From these results, it was concluded that the solutions were stable (maximum deviation <20%) over at least a 8.9-hour time interval at a concentration of 0.0510 mg/l, at least a 8.4-hour time interval at a concentration of 0.102 mg/l and at least a 5.4-hour time interval at a concentration of 0.996 mg/l. T-4127 NOTOX Project 334351 Limit of detection (LOP) From three chromatograms of a 0.0510 mg/l solution of the test substance, the mean noise level (N) was determined to be 11.8%. From the same chromatograms, the test substance signal (S), i.e. the mean height of the test substance peak, was determined to be 100%. Using these values, the limit of detection (S/N=3) was calculated to be 0.0181 mg/l at an injection volume of 100 pi. Because samples from the ecotoxicological study were diluted by a factor of 2, the limit of detection for these samples is 0.0362 mg/l. Validation of the analytical method used in November 2002 Specificity Figures 4 and 5 show chromatograms of a blank solution (1/5 (v/v) M2-medium/isopropanol containing internal standard) and of a 0.100 mg/l T-4127 solution, respectively. It was clear that blank chromatograms did not contain any interfering peaks at the position of the test substance. Linearity The results are summarized in Table 5. The regression line is shown in Figure 6. Table 5 Linearity. Concentration [mg/l] Response 1 [units] 0.00501 0.00699 0.0100 0.0199 0.0559 0.0996 0.0867 / 0.0764 0.111 /0.0964 0.120/0.122 0.273 / 0.248 0.661 / 0.751 1.43/1.14 Slope Intercept with Y-axis Weighting factor R 12.6 0.0122 1/concentration 0.993 Duplicate measurements. Though duplicate injections showed a relatively large spread, it was concluded that there is a linear relationship between response and concentration in the concentration range of 0.00501 0.0996 mg/l if a (1/concentration) weighting factor is used. For quantitation of samples, calibration curves were constructed using two concentrations over only part of the linearity range tested. The calibration solutions are injected before and after the samples. T-4127 NOTOX Project 334351 RESULTS - SAMPLE ANALYSIS Samples July 2002 Table 6 shows the analytical results for the samples from the test performed in July 2002*. Inadvertently no internal standard was added to the samples pretreated on 01-Jul-02 and the calibration solutions. Table 6 Concentrations of T-4127 in test medium (Full test 1). Time of sampling [hours] Date of sampling [dd-mm-yy] Date of analysis [dd-mm-yy] Nominal [mg/l] Concentration Analysed1 [mg/l] Relative to nominal [%] 0 01-Jul-02 5 02-Jul-02 24 02-Jul-02 02-Jul-02 72 04-Jul-02 04-Jul-02 0 100 100 2 0 100 100 2 100 100 2 n.d. 1.209 1.537 n.d. 0.252 0.0624 3 <0.0362 4 <0.0362 4 n.a. 1.209 1.537 n.a. 0.252 0.0624 <0.0362 <0.0362 1 Mean of triplicate analysis. The maximum deviation between the responses calculated for each sample and was < 20%. 2 Without algae. 3 Calculated by extrapolation. 4 Concentrations below the limit of detection. 5 Inadvertently no internal standard was added to these samples and calibration solutions . n.d. Not detected. 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 334351 Samples November 2002 Table 7 shows the analytical results for the samples from the test performed in November 2002. Table 7 Concentrations of T-4127 in test medium (Full test 2). Time of sampling [hours] Date of sampling [dd-mm- yy] Date of analysis [dd-mm- yy] Nominal [mg/l] Concentration Analysed1[mg/l] Relative to nominal [%] 0 11-Nov-02 11-Nov-02 100 100 0 11-Nov-02 11-Nov-02 0 0 0.0721 0.0721 0.160 0.160 0.320 0.320 0.721 0.721 1.60 1.60 48 13-Nov-02 13-Nov-02 0 0 0.0721 0.0721 96 15-Nov-02 15-Nov-02 0 0 0.0721 0.0721 1.59 1.62 n.d. n.d. 0.0671 0.0564 0.152 0.160 0.328 0.330 0.754 0.742 1.69 1.64 n.d. n.d. 0.0471 0.0477 n.d. n.d. 0.0526 0.0543 n.a. n.a. 93 78 95 100 102 103 105 103 105 103 n.a. n.a. 65 66 n.a. n.a. 73 75 1 Mean of triplicate analysis. The maximum deviation between the responses calculated for each sample was < 20%. n.d. Not detected, n.a. Not applicable. T-4127 Samples July 2002 RT: 0.00 -12.99 NOTOX Project 334351 Time (min) Figure 1 HPLC chromatogram of a blank (50/50 (v/v) methanol/M2-medium containing internal standard). _ R elative A b un dan ce T-4127 100R T : 0 .0 0 - 1 2 . 9 9 90 Internai standard 80 70 60 50 40 NOTOX Project 334351 NL: 1 .2 0E 7 m /z= 4 9 8 .7 0 -4 9 9 .7 0 F: - c E S I Full m s [ 2 0 0 .0 0 -6 0 0 .0 0 ] M S 334351_020702b_25 NL: 1.65E 6 m /z= 3 8 7 .7 0 -3 8 8 .7 0 + 4 1 8 .5 0 -4 1 9 .5 0 F: - c E S I Full m s 2 5 1 2 .0 0 @ 3 0 .0 0 [ 1 4 0 .0 0 -5 0 0 .0 0 ] M S 334351_020702b 25 R elative A b un dan ce Figure 2 HPLC chromatogram of 0.996 mg/l T-4127 in 50/50 (v/v) methanol/M2-medium containing internal standard. rkfkA A Q O T-4127 NOTOX Project 334351 T-4127 Y = 6.07669e-005+0.220598*X-0.065148*XA2 RA2 = 0.9973 W: Equal Area Ratio Figure 3 Regression line for solutions in 50/50 (v/v) methanol/M2-medium containing internal standard: Responses against concentrations. T-4127 Samples November 2002 1 jagpar'gmwt.s" '- S -sr sTS" M 5 -" fgS - IstfL.; iliml 3 SSsrJ*u.Cs-. -as?S:-fTc" Test substance ISKffiS. ' MSbMSi_r*sc! 4* ti8!r1i s " NOTOX Project 334351 Internal standard I . ( .. I: I: Figure 4 HPLC-chromatogram of a blank (1/5 (v/v) M2-medium/isopropanoi containing internal standard). T-4127 SS" iis 'a ssrsSrSsar.s; I- <3 ir ! rttjaswMCsMaar1 5 2 b t .s# s sSK:t-a3Smlftatsess: t;1s = "ar= NOTOX Project 334351 Internal standard I_ ; Figure 5 LC-MSMS chromatogram of 0.100 mg/l T-4127 in 1/5 (v/v) M2-medium/isopropanol containing internal standard. a a a AQC T-4127 NOTOX Project 334351 H I I U H . H U i I . 1 A . 1 ! it I (T -t 1 ! M : ' I I I t t I * I 1 [ ' I I x ' w <t * < > * ) 1 - 1 2 x 0 0 1 2 ! ( r 0 0 9 2 S ) Figure 6 Regression line for solutions in 1/5 (v/v) M2-medium/isopropanol containing internal standard: Responses against concentrations.