Document vBDz46QVkb46ae990y6BLkOQ9

A R 226-3117 ^ 3 p |||. '.!''"/:*'n.';'!'c>-*"^^f,`',v^*p"1`;i!-T.rjiri';'-.' '- -V'^jiVi^*V1<.%fi-T^- - .v;"':!:'* ' .'..iv*!-Vl.,`;:.V i^ M ;} n|i|||j;:e "V^:i';'^7.WiVW`"H^'Srip'J-.^ S ^ E ^s i n i h -" :-;-t^;^|p|j|i| CONFIDENTIAL DPT441/984973 [ ALGAL GROWTH INHIBITION ASSAY I Sponsor DuPont Speciality Chemicals, Jackson Laboratory, Chambers Works, Deepwater, NJ 08023, USA. Research Laboratory Huntingdon Life Sciences Ltd., Eye, Suffolk IP23 7PX, ENGLAND. Draft Report Issued 20 January 1999 Final Report Issued 19 March 1999 Page 1 o f 29 Company Sanitized. Does not contain TSCA C B I D P T 4 4 1/984973 CONTENTS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS.................... 4 QUALITY ASSURANCE STATEMENT............................................................................ 5 CONTRIBUTING SCIENTISTS.......................................................................................... 6 SUMMARY........................................................................................................................... 7 INTRODUCTION. 8 TEST SUBSTANCE. 9 EXPERIMENTAL PROCEDURE. 10 MAINTENANCE OF RECORDS. 13 RESULTS. 14 CONCLUSIONS................ ................................................................................................... 15 REFERENCES............... ....................................;.................................................................. 15 FIGURE 1. Typical sample chromatogram................................................................................... 16 TABLES 1. Measured concentrations.............................................................................................. 2. Cell densities................................................................................................................ 3. Inhibition of growth...................................................................................................... 4. Environmental parameters........................................................................................... 17 18 19 20 :2 : Company Sanitized. Does not contain T SC A CBI DPT441/984973 CONTENTS - continued Page APPENDICES 1. Algal nutrient medium (OECD). . . .......................................................................... 21 2. The determination o U H H p aqueous m edia............................................ 22 :3 : Company Sanitized. Does 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 Laboratoiy 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. Study Director, Huntingdon Life Sciences Ltd. Date :4 : Company Sami QUALITY ASSURANCE STATEMENT The following have been inspected or audited in relation to this study D P T 4 4 1/984973 Study Phases Inspected Protocol Audit Process Based Inspections Counting of inoculum Formulation of test medium Sampling of test medium Experimental set-up Report Audit 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. m M O uuM Helen Comb, B.Sc.(Hons.), Principal Auditor, Department of Quality Assurance, Huntingdon Life Sciences Ltd. VACATOLA \C*Pic \ Date :5 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. Chief Chemist Andrew Robertson, B.Sc.(Hons.) Senior Chemist Richard Cubberley, B.Sc.(Hons.) Study Analyst Martin Nash, B.Sc.(Hons.) Scientific Officer DPT441/984973 SUMMARY DPT441/984973 The effect o f y ^ ^ m ^ o n growth of the unicellular green alga Selenastrum capricomutum was assessed imdernoiFaxemtr conditions. Throughourthe rep o rt the exposure concentrafrn a^H test results have been expressed in terms of the active ingredient (a.i.)jf The study was conducted in accordance with EEC Methods for Determintion of Ecotoxicity Annex to Directive 92/69/EEC (OJ. 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". Six replicate algal cultures, with an initial cell density o f 1 x 104/ml, were exposed t c ^ ^ H H H P ^ dispersed in algal nutrient medium at a nominal concentration of 100 mg a.i./l; to aid dispersion, ultrasound treatment was employed. The cultures were incubated for 72 hours in an orbital incubator under continuous illumination"at temperatures ranging from 23.2 to 23.7C. The measured concentration o f ^ ^ B B H N f jin unfiltered samples of medium ranged from 98.8 mg a.i./l at the start o f 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 (EiCS3 and ErC50) 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, not considered to require classification as the 72-hour EC50s are considered to oe greater than the overall mean measured concentration, 100 mg a.i./l. ( :7 : anltized.Doas not contain [SGA C31 Company San INTRODUCTION DPT441/984973 This study was designed to assess the effect alga Selenastrum capricomutum. the growth of the unicellular green 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 issued by 12 October 1998. Information provided by the Sponsor indicated that the solubility o f ^ m H | ^ | i n water w a s ^ i t i by weight at 35 - 40C and that its purity was m 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 gentie stirring to obtain an homogenous composition before use. The results o f the most recent laboratory reference test using potassium dichromate indicated that its 72-hour EbCso to Selenastrum capricomutum was 0.62 mg/1; this was within the range typically obtained in this laboratory (0.3 to 1 mg/1). :8 : _ Company Sanitized, oes not contain TSC.A CB I D P T 4 4 1/984973 Storage conditions: i Lot number: Expiry date: Purity: , Sample received: Room temperature L *3 2 years from date of receipt 23 June 1998 e> Company Sanitized. Does noit contain TSC C3l EXPERIMENTAL PROCEDURE DPT441/984973 TEST SPECIES Name Selenastrum capricomutum, Strain No. CCAP 278/4. Source Axemc, uni-algal, agar slope cultures were obtained from the Culture Collection o f Algae and Protozoa, Institute o f 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 contmuous illumination at nominal temperatures in the range 21 to 25C and typically had a cell uensity ui 1.2 x 106cells/ml. An aliquot of a primary culture was diluted using steriie algal nutrient medium to give a test inoculum with a cell density of 2.5 x 104cells/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 contain TSCA CRr Stability of test concentrations D P T 4 4 1/984973 The test concentration o f | |^ m 0 p jj^ Y a s 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 farthei^amples token for analysis. Additional samples were also taken from flasks containing 100 mg/l 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 o f 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 o f aqueous stock), the cell density in each flask was approximately 1 x 104 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./I. : 11 : Company Saniti!zed. Does not contain T SC A CSi Environmental conditions D P T 4 4 1/984973 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. 23 2C. The temperature was maintained at , 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. MEASUREMENT OF GROWTH 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 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 o f growth and is calculated using the equation: N +N -2N N +N -2N x t 1+ _ J ----- 2------ ~ ^ x [t 1 - t ]+....+ *. --- - 1- --------=-x [ t _ t ] Z n n -1 where A = area N,, = nominal cell densities at t,, n ,, n2 = measured cell densities at t,, tj N,, = measured cell densities at t^ t|> ^ = time o f first or second measurement (hours from start) . = time o f n* measurement (hours from start) = number of measurements taken after the beginning of the test Percentage inhibition of growth at the test concentration ( I J is calculated by comparing the area under the test curve (At) with that under the control curve (AJ as appropriate using the equation: A -A I = - * - ---- - x 100 aA C The EbCso ("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 E ^ o was not calculated because growth was not inhibited. : 12 : lompany Sanitized. Dosa r.o. c ;ain TSGA CBS  DPT441/984973 r 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,,-lnN0 . . t,,-t0 Where tDis the time at the beginning of the test. j The ErC50 ("x" - "y" h) is the median effect concentration for inhibition of growth based on a 1 comparison of growth rates from "x" to "y" hours. The ErC5owas not calculated because growth was not inhibited. The "no-observed effect concentration" could not be identified statistically using Dunnett's multicompanson test to compare the percentage inhibition in the test group with that for the control f cultures (Dunnett; C:W. 1955, 1964) because the mean' cell density f o r the exposure cdficeHShtioii j. was greater than that for the control group. 1 ' - . i. i 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 o f 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 cor.tain T SC A E RESULTS D P T 4 4 1/984973 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 lfig g g g g /flra n g e d from 98.8 mg a.i. /I at the start o f the test to 101 mg a.i./l after 72 hours, with an overall mean measured level of 100 mg a.iVl. After 72 hours, analysis of medium co n tain in g ^ H Q M H Q w h ic h had been incubated without algal cells gave similar results to test medium incubated in the presence of algal cells (99.7 mg a.i./l compared to lO ljng a.i./l); 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.i./l were significantly higher (Dunnett's test). The 72-hour median effect concentrations (E^C* and ErCS0) were not identified but must be greater than 100 mg a.i./l. 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.i./I. ' Under the ECGeneralClassification and Labelling Requirements for Dangerous Substances and P rep aratio n s,P M B B B IV jP not considered to require classification as the 72-hour ECS0s are considered to be greater than the overall mean measured concentration, 100 mg a.i./i. 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 22.TC . The test medium was a colourless dispersion with undissolved material visible at its surface and on the bases of the flasks. : 14 : Company Sanitized. Does not contain T S C A C3E CONCLUSIONS DPT441/984973 D 25 not found to be inhibitory to Selenastrum capricomutum when dissolved in algal nutrient medium at a mean measured level of 100 mg a.i./l. Uie 72-hour median effect concentrations (E^Csa and ErCS0) for inhibition of growth were not identified but must be greater than 100 mg a.i./l. The no-observed adverse effect concentration (NOEC) for inhibition o f 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 II "Classification on the Basis o fEnvironmentalE ffect p.62 - 64. : 15 : Company Sanitized. Does not contain TSCA CS FIGURE 1 D P T 4 4 1/984973 Typical sample chromatography - 400 mg/1 (100 mg a.i./l) taken on Day 0 o f the test : 16 : m __Can TABLE 1 Measured concentrations D P T 4 4 1/984973 Nominal conc$, (njg/1) control too 100 (no algae) Measured]! 0 hours %N nd nd - 98.9 98.6 99 -- Bconcentrations (me/1) 72 hours %N nd nd 103 99.5 101 99.8 99.5 100 %ti 103 - $ : in terms ofthe active ingrediem nd none detected (<2.5 mg active ingredient/1). *** %N mean measured concentration expressed as a percentage of the nominal concentration, %ti mean measured concentration after 72 hours expressed as a percentage of the mean starting concentration. Overall mean 100 99.7 : 17 : oes not contain T SC A C B l Com paq Sanitized. D P T 4 4 1/984973 TABLE 2 Cell densities - control and test cultures l' ^Mg^n^gurec^ Cell densities (cells x lOVml) concentration1(mg/1) 24 hours 48 hours 72 hours I Control R, 4.50 19.6 47.0 r2 r3 4.13 * 20.0 62.0 ** R, 3.75 . 18.3 54.6 R, 4.38 . 20.3 50.1 R 3.25 19.5 57.0 Mean 4.00 19.5 54.1 100 R, 4.13 23.6 75.1 R, 5.13 30.3 75.3 r3 3.50 28.0 67.1 j R< 2.88 34.0 70.4 R* 4.63 R 4.75 31.8 58.5 31.3 60.8 Mean 4.17 29.8 67.9 R , - R Replicates 1 -6 Flask not inoculated with algae in error. In terms of the active ingredienti Il 18 Company Sanitized. Dees not contain 7SC A CBl TABLE 3 Inhibition of growth D P T 4 4 1/984973 concentration^ (mg/1) Control R, r2 r3 R Rs R* 100 R, r2 r3 R, Rs R Area under Mean Growth rate Mean curve^@ 72 h (% Inhibition) (0-72 h) (% Inhibition) 1082 1263 * 1124 1134 1170 1507 1694. 1501 1670 1516 1535 1155 1571 (9) 1 5.347 5.732 * 5.556 5.436 5.615 5.998 6.002 5.842 5.909 5.651 5.705 5.537 5.851 m R,-R* Replicates 1 -6 * Flask not inoculated with algae in error. $ In terms of the active ingredient] # x 104 x 10'2 : 19 : Company Sanitized. Does not contain TisCA CEf TABLE 4 Environmental parameters temperature and pH D P T 4 4 1/984973 Mean measured Temperature C Control 100 Oh 72 h 24.3 23.5 - 24.0 24.4 23.6-24.1 $ In terms ofthe active ingredien pH Oh 72 h 7.9 7.6 - 7.7 7.9 7.5 - 7.7 1 : 20 Company Sanitized. Does not contain T S C CBi APPENDIX 1 Algal nutrient medium (OECD) D P T 4 4 1/984973 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 1: macro-nutrients n h 4ci MgCl2.6H20 CaCl2.2HjO MgS04.7H20 k h 2p o 4 Stock solution 2: Fe-EDTA FeCl3.6H20 Na2EDTA.2H20 Stock solution 3: trace elements h3b o 3 MnCl2.4H20 ZnCl2 CoC12.6H20 CuC12.2H20 Na2Mo04.2H20 Stock solution 4: NaHC03 NaHC03 Concentration in stock solution (g/I) 1.5 1.2 1.8 1.5 0.16 0.08 0.1 0.185 0.415 3 x IO'3 1.5 x 10 . IO5 7x 10'3 50 Volume of stock solution per litre of final medium (ml) Final concentration in test solution (mg/1) 10 15 12 18 15 1.6 1 0.08 0.1 ' 1 0.185 0.415 3x 10'3 1.5 x 10 10`5 7xW3 1 50 : 21 : Company Sanitized. Does not contain T3CA CB! D P T 4 4 1/984973 APPENDIX 2 The determination o: in aqueous media SAMPLE ANALYSIS Ttaaqueoussamples were diluted with sodium hydroxide and acetonitrile to bring the expected Q concentrations within the calibration range. Determination o f f l i m m f f t v a s by nigh performance liquid chromatography (HPLC) using a conductivity detectorf 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: Suppresser type: Regenerant composition: Flow rate: Injection volume: PLRP-S supplied by Polymer Laboratones 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 100 pi 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 condition! hromatographed as 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 Sanitized. Dosa not contain e 3C A CB! CALIBRATION SOLUTIONS Calibration solutions were prepared with the same batch o; the toxicity test solutions. Trout and Algae Studies D P T 4 4 1/984973 [used in the preparation of 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 8 ncentrations were determined using mean bracketing standards. The mean peak h e ig h ^ e s ^ n se s w e re calculated f o r ( j H | H Q i n bracketing standard chromatograms. g P m jm V H U c o n c e n t r a ti o n each sample was then calculated using the following equation: C o n cen tratio i^|m m ^m g /l) = ^ x ^ x p Where Y Z A F Detector response ti Mean detector response to bracketing standard. Concentration of bracketing standard (mg/1). Factor to take into account sample processing. The purity (active ingredient) of the test substance is_given in the Test Substance Data Sheet a J l B Nommal and fortified concentrations are reported a s B ^ H H A s supplied and in terms ofthe '' active ingredient. -- VALIDATION OF THE ANALYTICAL PROCEDURE The analytical procedure was validated by determining the linearity of response o f 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. : 23 . Dot r.Rf Validation recoveries ol TABLE 1 D P T 4 4 1/984973 rom fortified samples of dilution media Medium Dechlorinaled 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 2.483 124.1 124.1 - 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) Active ingredient content o:____ 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./l). 6 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 Sanitized. Does net contain T S C A CBI Stability TABLE 2 in dilution medium DPT441/984973 Storage conditions Procedural recovery1 Procedural recovery1 Light, sealed, room temperature 1 Light, sealed, room temperature 1 Dark, sealed, 4C 1 Dark, sealed, 4C 1 Procedural recovery2 Procedural recovery2 1Fortification level: 11.20 mg/1 as 2Fortification level: 11.65 mg/1 as]______ Results are given as percentage recoveries o: Time-point 0 hours 20 hours 110 - 112 - -' 103 - 106 - 109 - 93.1 - 09 - 98.9 liter storage for the indicated time. : 25 : Company Sanitized. Coes not contain TSC CtsS FIGURE 1 Standard Calibration fori ' (ca 40 - 500 m D P T 4 4 1/984973 Concentration (mj Run no.:DPT/439/0!3 Standard concentration (mg/I) 0.0 43.57 108.9 217.9 435.7 544.7 NOP: no observable peak. I) Peak height NOP 4075 10668 20734 38644 47864 1 : 26 : Company Sanitized. Does nc corstam T SC A CB1 FIGURE 2 Standard Calibration foim m m (ca 4 to SOm D P T 4 4 1/984973 Run no.:DPT/440/002 Standard concentration (mg/l) 0.0 4.720 11.80 23.60 47.20 59.00 NOP: no observable peak. Peak height NOP 3295 8657 18669 38224 48699 : 27 : Company Sanitized, Dees not contain T SC A CSI FIGURE 3 D P T 4 4 1/984973 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 # 84 DATA SAVED FIGURE 4 Typical chromatography - Unfortified algal medium CHANNEL A rWJECrr 09- 07- 78' T4 :5 5 :`r r STTTRE TO'~BIN 5 8 5 DATA SAVED TO BIN 8 85 : 28 : Company Sanitized. Does contain TSC CB1 FIGURES D P T 4 4 1/984973 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 DATA SAVED TO BIN # 75 : 29 : Company Sanitized. Does not contain T SC A CBi