Document Ddvx5J9Gr2gzyRV56Dk199prn

AR226-3117 CONFIDENTIAL DPT441/984973 ALGAL GROWTH INHIBITION ASSAY Sponsor DuPont Speciality Chemicals, Jackson Laboratory, Chambers Works, Deepwater, NJ 08023, USA. Research Laboratory Huntingdon Life Sciences Ltd., Eve Suffolk D>23 7PX, ENGLAND. Draft Report Issued 20 January 1999 Final Report Issued 19 March 1999 Page 1 of 29 Company Sanitized. Does no( contain 7SCA CBI DPT441/984973 CONTENTS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS.................... 4 QUALITY ASSURANCE STATEMENT............................................................................ 5 CONTRIBUTING SCIENTISTS.......................................................................................... 6 7 SUMMARY.......................................................................................................................... 8 INTRODUCTION................................................................................................................. TEST SUBSTANCE............................................................................................................. 9 EXPERIMENTAL PROCEDURE........................................................................................ 10 MAINTENANCE OF RECORDS ........................................................................................ 13 14 RESULTS.............................................................................................................................. CONCLUSIONS................................................................................................................... 15 15 REFERENCES...................................................................................................................... FIGURE 1. Typical sample chromatogram ................................................................................... 16 TABLES 1. Measured concentrations ............................................................................................ 17 2. Cell densities............................................................................................................... 18 3. Inhibition of growth.............................--.................................................................... 19 4. Environmental parameters..................................--..........--.----..----...............--....... 20 Company Sanitized. Does not contain TSCA CB8 CONTENTS - continued APPENDICES 1. Algal nutrient medium (OECD). 2. The determination of |n aqueous media DPT441/984973 Page 21 22 Company Sanitized. Doss not contain TSCA CBl DPT441/984973 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS The study described in this report was conducted in compliance with the following Good Laboratory Practice Standards and I consider the data generated to be valid. The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No. 654). EC Council Directive, 87/18 EEC of 18 December 1986, (No. L 15/29). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. ,./(j2^-^ Eileen C. Daly, Nat. Diplonte^CEA Eire, Study Director, Huntingdon Life Sciences Ltd. j^^cLW Date Company Sanitized. Does not contain T3CA C3i QUALITY ASSURANCE STATEMENT The following have been inspected or audited in relation to this study DPT441/984973 Study Phases Inspected Protocol Audit Process Based Inspections Counting of inoculum Formulation of test medium Sampling of test medium Experimental set-up Report Andit Date of Inspection 07 August 1998 10 August 1998 02 October 1998 02 October 1998 30 November 1998 05 March 1999 Date of Reporting 07 August 1998 10 August 1998 02 October 1998 02 October 1998 01 December 1998 05 March 1999 Protocol Audit: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Process based inspections: At or about the time this study was in progress inspections of routine and repetitive procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated above. Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above. The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data. Helen Comb, B.Sc.(Hons.), Principal Auditor, Department of Quality Assurance, Huntingdon Life Sciences Ltd. J^,fMi>TC^A l^^^l Date :5 : Company sSa^nt.t^?.--Do^^c""135"^0^ CONTRIBUTING SCIENTISTS STUDY MANAGEMENT Eileen C. Daly, Nat. Diploma, NCEA Eire Study Director Karen Firth, Higher National Certificate (Applied Biology) Study Scientist Ben Smith, B.Sc.(Hons.), M.SC., C.Chem., M.R-S.C. i' Chief Chemist /l Andrew Robertson, 3.Sc.(Hons.) Senior Chemist Richard Cubberley, B.Sc.(Hons.) Study Analyst Martin Nash, B.Sc.(Hons.) Scientific Officer DPT441/984973 Ba^ed-Do^n-c^nTSCACBl Company sar.;r.^- SUMMARY DPT441/984973 o^jUURon The effect the growth of the unicellular green alga Selenastmm capricomvtum was assessed undemon-axenic' conditions. TnrDughouf'the''repon,'the exposure concgntratipn and test results have been expressed in terms of the active ingredient (a.i.)SHtf|BUBHl9 The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 3 "Algal Inhibition Test" and the OECD Guideline for Testing of Chemicals No. 201 "Alga, Growth Inhibition Test'. tqUmBpn Six replicate algal cultures, with an initial cell density of 1 x lOVml, were exposed dispersed in algal nutrient medium at a nominal concentration of 100 ing a.i./l; to aid dispersion, ultrasound treatment was employed. The cultures were incubated for 72 hours in an orbital incubator under continuous illummation'al temperatures ranging from 23.2 to 23.7C. The measured concentration of^f------lnm unfiltered samples of medium ranged from 98.8 mg a.i./l at the start of the test to 101 mg a.i./l after 72 hours, with an overall mean measured level of 100 mg a.i./l. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate. Compared to the control cultures, neither the area under the growth curve nor the growth rate were reduced at a mean measured level of 100 mg a.i./l. The 72-hour median effect concentrations (EbC;, and ErC,,,) were not identified but must be greater than 100 mg a.i./l. The "no-observed adverse effect concentration" for inhibition of growth was considered to be 100 mg a.i./l. Under the EC General Classification and Labelling Requirements for Dangerous Substances and Preparations,j||RB----qis not considered to require classification as the 72-hour EC,oS are considered tone greater than the overall mean measured concentration, 100 mg a.i./l. : 7 : Company cS--an--.ttiizzeead. Di- ^sno'^3'"130^ INTRODUCTION DPT441/984973 This study was designed to assess the effect ofJUmJJjon the growth of the unicellular green alga Selenastrum capricomutwn. The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 3 "Algal Inhibition Test" and the OECD Guideline for Testing of Chemicals No. 201 "Alga, Growth Inhibition Test". The protocol was approved by Huntingdon Life Sciences Management on 7 July 1998, by the Sponsor on 17 July 1998, and by the Study Director on 3 August 1998. The experimental phase of the study was conducted between 4 September and 2 October 1998 and the results of chemical analysis were issue'd By 12 October 1998. Information provided by the Sponsor indicated that the solubility ofgU|BH|jin water was^Hg by weight at 35 40C and that its purity was gHQ Throughout this report, the exposure concentration and the test results have been expressed in terms of the active ingredient. Also, the Sponsor indicated that at room temperature the test substance was a suspension in water and upon standing it would separate out into its component phases; accordingly, at the recommendation of the Sponsor, the suspension was wanned to 35 - 40C (in a water bath) with gentle stirring to obtain an homogenous composition before use. The results of the most recent laboratory reference test using potassium dichromate indicated that its 72-hour Et,C,o to Selenastrum capricomutum was 0.62 rag/I; this was within the range typically obtained in this laboratory (0.3 to 1 mg/1). 8 : Company Sanitized. Doss not conta'n TSCA C31 Identity: Chemical name: Appearance: Storage conditions: Lot number: Expiry date: Purity: Sample received: TEST SUBSTANCE DPT441/984973 Room temperature w 2 years from date of receipt M 23 June 1998 Company Ssaanniitii^zce"d-. Does not contain TSCA C3l EXPERIMENTAL PROCEDURE DPT441/984973 TEST SPECIES Name Selenastrum capricomutian. Strain No. CCAP 278/4. Source Axenic, uni-algal, agar slope cultures were obtained from the Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Cumbria, UK and arrived on 26 August 1998. Pre-culture The agar slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium (Appendix 1) was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (50 ml) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25C and typically had a cell density of 1.2 x 106 cells/ml. An aliquot of a primary culture was diluted using sterile algal nutrient medium to give a test inoculum with a cell density of 2.5 x 104 cells/ml before use. CULTURE MEDIUM Sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and OECD Procedure 201 (see Appendix 1). TEST SUBSTANCE PREPARATION Method of preparation Based on information provided by the Sponsor, the test substance was warmed (to c.38C) in a water bath and gently swirled to provide an homogeneous mixture before weighing. A concentrated stock was prepared by adding the test substance (800 mg, as received) directly to sterile algal nutrient medium (approximately 180 ml). This was treated with ultrasound for twenty minutes and its pH adjusted from 2.8 to 7.9 with 0.5 ml sodium hydroxide (1M). After adjustment to a volume of 200 ml, an aliquot (10 ml) of the stock was added to each test flask. - ^, : 10 : Company Sanitized; Does not confab TSCA CRr DPT441/984973 Stability of test concentrations The test concentration ofHBBHBIjvas measured using an HPLC method of chemical analysis (Appendix 2). Its stability in dilution medium under refrigerated storage conditions was determined before the start of the study. At the start of the definitive test, four samples (100 ml) were taken from the freshly-prepared control and test media; after 72 hours, the contents of the replicate flasks for each group were pooled and further samples token for analysis. Additional samples were also taken from flasks containing UH^------cat 100 mg/1 but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells. The samples were stored in a refrigerator before duplicates were transferred to the Huntingdon Research Centre, Cambridgeshire, for analysis; the other samples from each occasion of analysis remained in storage in case further . analysis was required. EXPOSURE CONDITIONS Experimental design A preliminary rangefinding test was followed by a definitive (limit) test with one test concentration plus an algal nutrient medium control group. Ten flasks were established for each control and test group. Six flasks from each group were incubated and the others were used for water quality measurements and chemical analysis at the start. Before the start of the test, the required number of test vessels (250 ml conical flasks), each containing algal medium (50 ml), were loosely stoppered with non-absorbent cotton wool, covered with aluminium foil which was secured by autoclave tape and sterilised by autoclaving (121C for 15 minutes). Following the addition of algal inoculum (40 ml) and the test substance (as a 10 ml aliquot of aqueous stock), the cell density in each flask was approximately 1 x 10" cells/ml. Each flask was then loosely plugged with non-absorbent cotton wool. The control cultures were prepared by adding aliquots (10 ml) of algal nutrient medium and test inoculum (40 ml) to the flasks containing sterile algal nutrient medium (50 ml). Test concentrations The rangefinding study used test concentrations of 1, 10 and 100 mg a.i./l. The definitive (limit) test employed a nominal concentration of 100 mg a.i./l. : 1l ' company Sanded. Does no? coital TSCA C5E DPT441/984973 Environmental conditions Conical flasks each containing control or test culture (100 ml) were placed in an illuminated orbital incubator according to a random number sequence. The cultures were incubated, without renewal of medium, for 72 hours under continuous illumination of approximately 7488 lux provided by 6 x 30 W "cool white" 1 metre fluorescent tubes. The temperature was maintained at 23 2C. The temperature and pH of control and test flasks at the start and end of the test were recorded. Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at a nominal 150 cycles per minute. The minimum, maximum and ambient temperature and light intensity in the test area were determined each day. i MEASUREMENT OF GROWTH I Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities \ measured using a haemacytometer (Improved Neubauer). The estimate of cell numbers in each I sample was based on the mean of four or eight consecutive counts. EVALUATION OF DATA The area under each growth curve (cell density v time) is taken to be an index of growth and is calculated using the equation: N -N N +N -2N A=--1----^xt -t 1. 2 1 +------2J-----x[- t1 -t N +N -2N ]+.....+--"-'----2-------x[Lt n ] n-1-1 where A N,, N,, N, Nn t,, t; tn n = area = nominal cell densities at to = measured cell densities at t,, t, = measured cell densities at t, = time of first or second measurement (hours from start) = time ofiia, measurement (hours from start) = number of measurements taken after the beginning of the test Percentage inhibition of growth at the test concentration (1^) is calculated by comparing the area under the test curve (AJ with that under the control curve (Ac) as appropriate using the equation: A -A I C =--c----'-xlOO 'A A The Ei,Cjo ("x" h) is the median effect concentration for inhibition of growth based on a comparison of areas under the growth curves after "x" hours. The Ei,C,o was not calculated because growth was not inhibited. Company S anitized^ . rDOrSr..f, t--^otc-o- r.ta'n TSCA CSS DPT441/984973 The average specific growth rate (p.) for each exponentially growing culture is also calculated from the appropriate section of the growth curve by the equation: // _lnN,-lnN^ . ^-to Where t^ is the time at the beginning of the test. The E^CSO("x" - "y" h) is the median effect concentration for inhibition of growth based on a comparison of growth rates from "x" to "y" hours. The E^Csowas not calculated because growth was not inhibited. The "no-observed effect concentration" could not be identified statistically using Dunnett's multicomparison test to compare the percentage inhibition in the test group with that for the control cultures" (Dunnett; C.W. 1955', 1964) because the mean" cell density fbrtne exposure concennatioh was greater than that for the control group. PROTOCOL DEVIATIONS One test flask was not inoculated with algal cells at the start of the test in error. However, this is not considered to have affected the integrity of the study. MAINTENANCE OF RECORDS All specimens, raw data and study related documents generated during the course of the study at Huntingdon Life Sciences, together with a copy of the final report will be lodged in the Huntingdon Life Sciences Archive. Such specimens and records will be retained for a minimum period of five years from the date of issue of the final report. At the end of the five year retention period the Sponsor will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the Sponsor's knowledge. : 13 : company Sanitized. Does net c^tam TSCA C3I RESULTS DPT441/984973 Chemical analysis The results of chemical analysis are given in Table 1 and an example chromatogram is illustrated in Figure 1. The mean measured concentration o^J--------|firangferodm 98.8 mg a.i. /I at the start of the test to 101 mg a.L/1 after 72 hours, with an overall mean measured level of 100 mg a.i71. After 72 hours, analysis of medium containmg^1--------||whicbahd been incubated without algal cells gave similar results to test medium incubated in the presence of algal cells (99.7 mg a.L/1 compared to 101 mg a.L/1); this indicates that the presence of algal cells had not affected the stability Algal growth Individual cell densities for each culture and the mean values are given in Table 2. The calculated area under the growth curve and average specific growth rate values are given in Table 3 and are expressed in terms of percentage inhibition by comparing the test group value with that of the control curve. Compared to the control cultures the area under the growth curve and the growth rate at a mean measured level of 100 mg a.L/1 were significantly higher (Dunnett's test). The 72-hour median effect concentrations (E^o and E,C,o) were not identified but must be greater than 100 mg a.L/1. The no-observed-effect concentration (NOEC) could not be identified statistically because the mean cell density for the exposure concentration was greater than that for the control group. Therefore, the "no-observed adverse effect concentration" was considered to be 100 mg a.L/1. Under the ECGeneral Classification and Labelling Requirements for Dangerous Substances and Preparations,|l|fjBBBqis not considered to require classification as the 72-hour ECsoS are considered to be greater than the overall mean measured concentration, 100 mg a.L/1. Observations No microscopic abnormalities of the cells were detected. Environmental parameters The measurements of water quality (temperature and pH) in control and test flasks are summarised in Table 4; they remained within acceptable limits throughout the study. The temperature of the incubator ranged between 23.2 and 23.7C. The test medium was a colourless dispersion with undissolved material visible at its surface and on the bases of the flasks. 14 Company Sanded. Does not contain TSCA C3E CONCLUSIONS DPT441/984973 ! not found to be inhibitory to Selenastrwn capricomutvm when dissolved in algal nutrient medium at a mean measured level of 100 mg a.i./l. The 72-hour median effect concentrations (Ei,Ca, and ,50) for inhibition of growth were not identified but must be greater man 100 mg a.i./l. The no-observed adverse effect concentration (NOEC) for inhibition of growth was considered to be 100 mg a.i./l. REFERENCES DUNNETT, C.W. (1955) "A multiple comparison procedure for comparing several treatments with a control". Journal of the American Statistical Association, 50, 1096-1121. DUNNETT, C.W. (1964) "New tables for multiple comparisons with a control". Biometrics, 20, 482-491. Official Journal of the European Communities Commission Directive (1 March 1991). Annex VI General classification and labelling requirements for dangerous substances and preparations. Part n "Classification on the Basis of Environmental Effect" p.62 - 64. :15: company Seized. Does ^ contain TSCA C3i FIGURE 1 DPT441/984973 Typical sample chromatography - 400 mg/1 (100 mg a.L/1) taken on Day 0 of the test , CHANNEL A INJECT 08-10-98 17:46:33 STORED TO BIN ft 75 i L___ 6.23 -' 16 : S3^ed.Does^e. ^.!..n...T..TSCSAfACWE,\ Company 5an;i.a<-"- TABLE 1 Measured concentrations DPT441/984973 Nominal conc$, (mg/1) control 100 100 (no algae) MeasuredMHBBHQconcentratiolis (mg/1) 0 hours %NT 72 hours %N nd nd - nd nd - 98.9 98.6 99 103 99.5 101 - - 99.8 99.5 100 Overall %ti mean - - 103 100 - 99.7 $ nd %N %ti : in terms of the active ingredient : none detected (<2.5 mg active ingredient/I). : mean measured concentration expressed as a percentage of the nominal concentration. : mean measured concentration after 72 hours expressed as a percentage of the mean starting concentration. : 17 : T2CAC31 not contain Compa^ Seized, i ,es DPT441/984973 TABLE 2 Cell densities - control and test cultures Mean measured Cell densities (cells x lOVml) concentration1 (mg/1) Control Pi R2 R, R, R5 R 24 hours 4.50 4.13 * 3.75 . 4.38 , 3.25 48 hours 19.6 20.0 * 18.3 20.3 19.5 72 hours 47.0 62.0 * 54.6 50.1 57.0 Mean 100 RI R, R, R< R5 R< 4.00 4.13 5.13 3.50 2.88 4.63 4.75 19.5 23.6 30.3 28.0 34.0 31.8 31.3 54.1 75.1 75.3 67.1 70.4 58.5 60.8 Mean 4.17 29.8 67.9 R, - Rs Replicates I - 6 * Flask not inoculated with algae m error, S In terms of the active mgredienQUj : 18 : Company- SanEtEzed. Dees no? coriEain 7SCA CB( TABLE 3 Inhibition of growth DPT441/984973 concentration'1' (mg/I) Control R, R2 R, R< R, Rs 100 Pi R, R, R, Rs R Area under curve^@72 h Mean (% Inhibition) Growth rate (0-72 h) 1082 1263 * 1124 1134 1170 1507 1694. 1501 1670 1516 1535 1155 1571 (0) 5.347 5.732 4: 5.556 5.436 5.615 5.998 6.002 5.842 5.909 5.651 5.705 R, - RS Replicates 1-6 * Flask not inoculated with algae in error. $ hi terms of the active ingredien # x 10" s x 10-2 Mean (% Inhibition) 5.537 5.851 W 19 Company Sanitized. Does not contain T-3CA CEf TABLE 4 Environmental parameters temperature and pH DPT441/984973 ^Mean measured -conc.,;>(^lg/l) Control 100 TemperatureC Oh 72 h 24.3 23.5-24.0 24.4 23.6-24.1 $ In terms of the active ingredient] P H Oh 72 h 7.9 7.6 - 7.7 7.9 7.5 - 7.7 : 20 : Company Ssaannsituizedu. Does not contain TSCA CBf APPENDIX 1 Algal nutrient medium (OECD) DPT441/984973 Four stock solutions were prepared according to the following table, using filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm). Stock solutions were sterilised by autoclaving (solutions 1-3) or by membrane filtration (solution 4) before being stored at 4C in the dark. Aliquots of stock solutions 1-4 were further diluted with the same diluent and autoclaved again to produce the working strength nutrient medium. The pH of the medium after equilibration with air is approximately 8. Nutrient Stock solution I: macro-nutrients NH4C1 MgCI^HsO CaCl,.2H,0 MgSO^.TH^O KH^ Stock solution 2: Fe-EDTA Concentration in stock solution (g/1) 1.5 1.2 1.8 1.5 0.16 Volume of stock solution per litre of final medium (ml) Final concentration in test solution (rag/I) 10 15 12 18 15 1.6 FeCl3.6H:20 Na^EDTA^H^O 0.08 0.1 1 0.08 0.1 Stock solution 3: trace elements HaBO, MnClz^H^O ZnCL; CoCI^Hp CuCl2.2H,0 Na^MoO.^HaO 0.185 1 0.415 3 x 10-3 1.5 xl0-3 . 10-5 7 xl0-3 0.185 0:415 3 xl0-3 1.5 x 10-3 10-5 7 xl0-3 Stock solution 4: NaHCO, NaHCO, 50 I 50 : 21 : Company Sanitized. Does not contain T3CA CBl APPENDIX 2 The determination ovWmSia aqueous media DPT441/984973 SAMPLE ANALYSIS The aqueous samples were diluted with sodium hydroxide and acetonitrile to bring the expected UHHHftI ofQJUHQwas concentrations within the calibration range. Determination by ligb performance liquid chromatography (HPLC) using a conductivity detectorl. CHROMATOGRAPHY INSTRUMENTATION AND CONDITIONS A high performance liquid chromatography system comprising autosampler, pump, conductivity detector, anion suppressor and data collection system was used. Column Type: Dimensions (1 x id): Temperature: Mobile phase Composition: Flow rate: Suppressor type: Regenerant composition: Flow rate: Injection volume: PLRP-S supplied by Polymer Laboratories 250 x 4.6 mm Ambient Acetonitrile : aqueous buffer solution (25 : 75% v/v) 1.0 ml/min 50 mN sulphuric acid 2.5 ml/min lOOu.1 Aqueous buffer solution: 2mM ammonium hydroxide/ImM sodium carbonate (made up in ultra high purity water) and filtered through 0.2 micron cellulose nitrate filter paper. Under the above conditions phromatographedas a single peak (Figure 3). ' A method contained in a fax dated 23 July 1998 from Kavsy D Dastur, Dupont Specialty Chemicals was modified to comply with Huntingdon Life Sciences standard operating procedures and instrumentation. : 22 : Company Sanitised. Doss not contain 73CA CB! DPT441/984973 CALIBRATION SOLUTIONS Calibration solutions were prepared with the same batch oj^HBBBH11^ in the preparation of the toxicity test solutions. Trout and Algae Studies Working calibration solutions in the nominal range 40 to 500 mg/1 (equal to 10 to 125 mg a.i./l) were prepared by volumetric dilution with acetonitrile: 100 mM sodium hydroxide (25:75% v/v) of a primary standard prepared in ultra high purity water. Daphnia Study Working calibration solutions in the nominal range 4.0 to 50.0 mg/1 (equal to 1.0 to 12.5 mg a.i./l) were prepared by volumetric dilution with acetonitrile : 100 mM sodium hydroxide (25: 75% v/v) of a primary standard prepared in ultra high purity water. CALCULATIONS oncentrations were determined using mean bracketing standards. The mean peak height responses were calculated forf^^^^^^^Buin bracketing standard chromatograms. ^fl^^^^^^^^^^l^^^m^^^i^c^^o^^n^ScHentration in ^^^^^^^^^^^^^^^^^w each sample was then *-' calculated using the following equation: Where Y Z A F Mean detector response to bracketing standard. Concentration of bracketing standard (mg/I). Factor to take into account sample processing. The purity (active ingredient) of the test substance is^iven in the Test Nominal and fortified concentrations are reported ^JUUHEf^ Substance Data supplied and in Sheet terms a^lj or the active ingredient. VALIDATION OF THE ANALYTICAL PROCEDURE The analytical procedure was validated by determining the linearity of response of the analytical system, specificity of chromatographic analysis, the limit of detection, and the method's accuracy and precision. During the course of the study the performance of the method was monitored by the analysis of quality control samples. "co^s-------0------^'"^"' Validation recoveries OB DPT441/984973 TABLE 1 from fortified samples of dilution media Medium Dechlorinated tap water Algal Elendt M4 Overall mean (RSD) Fortification level (mg/1) As supplied Control , 387.9 387.9 Control 535.5 530.0 214.2 212.0 Control 9.93 9.93 496.5 496.5 As active ingredient Control 96.98 96.98 Control 133.9 132.5 53.55 53.00 Control 2.483 2.483 124.1 1-24.1 Recovery as a % of fortification level ND 95.8 104 ND 92.0 101 92.6 95.5 ND 98.5 95.9 103 103 98.1 (4.2) RSD: relative standard deviation. ND: none detected; less than the limit of detection (trout and algal studies : 2.5 mg a.i./l; Daphnia study: 0.5 mg a.i./I). The limit of detection is defined as the analyte concentration in a processed sample which would give a peak equal to 3 x local base-line noise. : 24 : Company San'rt'zsd. Doss nci contain TSCA CBl TABLE 2 Stability ofl^UIHBf11 dilution medium DPT441/984973 Storage conditions Procedural recovery' Procedural recovery' Light, sealed, room temperature ' Light, sealed, room temperature ' Dark, sealed, 4C' Dark, sealed, 4C' Procedural recovery2 Procedural recovery2 ' Fortification level: 11.20 mg/1 as 2 Fortification level: 11.65 mg/1 as Results are given as percentage recoveries oi Time-point 0 hours 20 hours 110 - 112 - - 103 106 - 109 - 93.1 - 109 - 98.9 storage for the indicated time. : 25 : Company Sanded. Does not contain TSCA CBS N) 01 0 0 3 o 0 ^ w 0) N ro a. ^ ^ w 0 ^ 0 CO z 0 r? o I Ico I: w ;0 sr <3 a Ct I..J ^ (-> 4^ ts) ~ A. I*) ^ 0 ^ <J' ~-> w '0 ~~1 -~1 \0 4^ l*> 0 in o -~) B^ '2, ^ r '^ y ^ w ^J -- ~-1 00 0 0 1,, Z_ s- oo o\ y> .>. '~i w E o\ ~ CT< '-" Q ^ 4^. 4S>. 00 Response I a o y0 0 0 0 l/i .fe, M _ >0 ^1 ^ " ^ :<) 0 0\ 00 ts> 0 3 ^ '3 ^ ^ S ^ ^ 55 S % ^[- z o ^ & 5 K S ~> l^ "o 2. Response FIGURE 3 DPT441/984973 Typical calibration chromatography - 217.9 mg/1 (54.47 mg/1 as a.i.) CHANNEL A INJECT 09-09-98 14:45:47 STORED TO BIN ft 84 DATA SAVED TO BIN 84 FIGURE 4 Typical chromatography - Unfortified algal medium CHANNEL ft L fNJECT" 09-07-78- 14:75 :TT STnREnTO'-BTRr S5 DATA SAVED TO BIN 8 B5 Company Sanitized. Does ncS contain TSCA CB1 DPT441/984973 FIGURES Typical sample chromatography - algal medium fortified at 535.5 mg/1 (133.9 mg/1 as a.i.) CHANNEL A INJECT 09-09-98 13:19:59 STORED TO BIN 75 r_____. f=^~6.36 DATA SAVED TO BIN 75 : 29 : Company Sanitized. Doss not eor.Sa'n TSCA CBE