Document Gzjdr73gVg885woaeNaoe5Bam

AR226-3116 unting pgi 4i=>/ytad!-:! %t M^i 0 V J' not conta* TSCACBl ' c o m p a s sanM M d- DOeS -riiv CONFIDENTIAL DPT440/992118 ACUTE TOXICITY TO DAPHNIA MAGNA 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 25 January 1999 Final Report Issued 4 March 1999 Page I of 28 Com pany Sanitized. Doss not contain T S C A CB I I iiI CONTENTS COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS QUALITY ASSURANCE STATEMENT........................................................ CONTRIBUTING SCIENTISTS...................................................................... SUMMARY........................................................................................................ INTRODUCTION....................................... ...................................................... TEST SUBSTANCE........................................................................................... EXPERIMENTAL PROCEDURE..................................................................... MAINTENANCE OF RECORDS...................................................................... RESULTS............................................................................................................. CONCLUSIONS................................................................................................. REFERENCE...................................................................................................... FIGURE 1. Typical sample chromatogram................................................................. TABLES 1. Measured concentrations........................................................................... 2. Cumulative immobilisation........................................................................ 3. Environmental parameters......................................................................... DPT440/9921I8 Page 4 5 6 7 8 9 10 13 14 15 15 16 17 18 19 V :2 : Com pany Sanitized. Does not contain T S C A C3fl APPENDICES CONTENTS-continued DPT440/992118 Page 1. Elendt M4 medium ................... ^ .......................................................................... 2. The determination o n H ^ ^ H H I R i n aqueous media............................................. 20 21 :3: Com pany Sanitized. D oes not contain i S C A CEL DPT440/992118 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 (Official Journal No. L 15/29). OECD Principles o f Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98) 17. Study Director, Huntingdon Life Sciences Ltd. Date :4 : Company QUALITY ASSURANCE STATEMENT The following have been inspected or audited in relation to this study DPT440/992118 Study Phases Inspected Protocol Audit Process Based Inspections Formulation of test media Sampling of test media Experimental set-up Daphnia observations Report Audit Date of Inspection 06 August 1998 02 October 1998 02 October 1998 15 December 1998 11 November 1998 19 February 1999 Date of Reporting 06 August 1998 02 October 1998 02 October 1998 15 December 1998 11 November 1998 19 February 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. 3 A A & r o U IPPfX Date :5 : Company Sanitized. Doss not conta:n T 3 C A CB CONTRIBUTING SCIENTISTS STUDY MANAGEMENT Eileen C. Daly, Nat. Diploma, NCEA Eire. Study Director Deborah A. Blacoe, B.Sc.(Hons.), Ph.D. Study Scientist Karen A. 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 DPT440/992118 :6 : ;sa itize d .O o * s n ,t contain TSCA C3i Company SUMMARY DPT440/992118 The acute toxicity o f ^ R 9 H H Q to Daphnia magna was assessed under static exposure conditions. Throughout this report, the exposure concentrations and the test results have been expressed in terms of the active i n g r e d i e n t j i ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ W i 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 2 "Acute Toxicity for Daphnia" and the OECD Guideline for Testing of Chemicals No. 202, Part I "Daphnia Acute Immobilisation Test". Groups o f twenty Daphnia, less than 24 hours old, were exposed for 48 hours nominal concentrations of 4.27, 9.39, 20.7, 45.5 and 100 mg/1 (as active ingredient). The test media were prepared in Elendt M4 medium and to aid dissolution ultrasound treatment was employed. The measured concentrations o f ^ H B H H ^ H l i n samples unfiltered of media indicated that the intended exposure concentrations were adequately achieved (between 85 and 97% of their nominal values) and maintained (between 93 and 100% of their starting values). The overall mean measured levels were 4.01, 8.92, 18.6, 40.2 and 85.9 mg a.iA Observations o f the Daphnia in each control and test vessel were made after 24 and 48 hours. After 48 hours, 20% immobility had occurred at the highest concentration (85.9 mg a.i./l); the highest concentration at which no immobility had occurred was 40.2 mg a.i./l. The 48-hour EC*, could not be calculated but must be >85.9 mg a.i./l and the "no-observed-effect concentration" (NOEC) was 40.2 mg a.iA Under the EC. General Classification and Labelling Requirements for Dangerous Substances and P r e p a r a t i o n s , not considered to require classification as the 48-hour EC*, is considered to be greater than the highest nominal concentration, 100 mg a.i./l (mean measured level of 85.9 mg a.i./l). :7 INTRODUCTION DPT440/992118 Theobje ' - EC,) o ' was to determine the acute toxicity (48 hour median effect concentration [o Daphnia magna at 20C. The study was conducted in accordance with EEC Methods for Determination o f Ecotoxicity Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 2 "Acute Toxicity for Daphnia" and the OECD Guideline for Testing of Chemicals No. 202, Part I "Daphnia Acute Immobilisation 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 o f the study was conducted between l September and 20 November 1998 and the results of chemical analysis were issued by 8 December 1998. Information provided by the Sponsor indicated that the solubility o f j H | m Q i n water was 25% by weight at 35 - 40C and that Thr0UP ")Ut this report, the exposure concentrations and 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 warmed to 35 - 4GC (in a water bath) with gentle stirring to obtain an homogenous composition before use. The sensitivity of juvenile Daphnia cultured in this laboratory is periodically assessed using the reference substance potassium dichromate. The results for the most recent test performed prior to this study indicated that its 48-hour EC#, to Daphnia magna was 0.57 mg/1; this was within the range typically obtained in this laboratory (0.3 to 0.8 mg/1). :8: Compa TSCACBl  Identity: Chemical name: iI Appearance: Storage conditions: Lot number: Expiry date: Purity: Sample received: TEST SUBSTANCE DPT440/992118 Pale-yellow slurry Room temperature in the dark 3 2 years from date of receipt CM 23 June 1998 D :9 : I contain TSCACS* Company sanaasd-D ossno. EXPERIMENTAL PROCEDURE DPT440/992118 TEST ORGANISM Daphnia magna (Straus) used in this study were cultured in-house and were obtained from a strain originating from the Institute National de Recherche Chimique Appliqu (IRChA), France. Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 1.5 litres o f Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 2C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end o f each light phase. The culture medium was renewed at least twice each week. Cultures were fed daily with a suspension of the unicellular green alga, Chlorella vulgaris, to provide 0.1 to 0.2 mg carbon per daphnid, per day. Culture conditions ensure that the stock animals reproduce by parthenogenesis. The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by the gravid (egg-bearing) adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24 hours old, were used in the test. DILUTION MEDIUM The test organisms were maintained and the tests conducted in Elendt M4 medium (Appendix 1). The medium was prepared in reverse osmosis water. TEST SUBSTANCE PREPARATION Method of preparation Based on information provided by the Sponsor, the test substance was wanned (to c.38C) in a water bath and gently swirled to provide a homogenous mixture before weighing. At all concentrations, the test media were individually prepared by adding the appropriate weights of substance (17.1, 37.6, 82.8, 182 and 400 mg) to dilution medium in a volumetric flask (1 1); to aid dissolution, the contents o f the flasks were treated with ultrasound for thirty minutes before being made up to volume with dilution medium. : 10 : Com pany Sanitized. Does ns' contain 7 S C A C3s DPT440/992118 Stability of test concentrations The test concentrations measured using an HPLC method of chemical analysis (see Appendix 2). The stability of the test substance in dilution medium under refrigerated storage conditions was determined before the start of the study (Table 2, Appendix 2). At the start of the definitive test, four samples (100 ml) were taken from the freshly-prepared control and test media; after 48 hours, the contents of the test vessels from each group were pooled and further samples were taken for analysis. They were stored in a refrigerator until two samples from each set were transferred to Huntingdon Research Centre, Cambridgeshire; one sample per concentration was analysed and all of the other samples remained in storage at Eye Research Centre in case further analysis was required. EXPOSURE CONDITIONS Experimental design A rangefinding test was conducted, followed by a definitive (limit) test with one test concentration (100 mg a.i./l) plus a control (dilution medium). Because 60% immobility occurred in the limit test it was repeated and 50% immobility was observed. Another test was then undertaken employing five test concentrations (4.27, 9.39, 20.7, 45.5 and 100 mg a.i./l) but this gave variable results. It was suspected that repeated wanning (to 38C) of the small volume of test substance in the sub-sample container used for the aquatic studies may have affected the chemical characteristics of the test substance. Therefore, a new sub-sample of the test substance was obtained and a definitive test was conducted with five test concentrations and the results are reported here. Twenty Daphnia, four replicates o f five animals per vessel, were exposed in each control and test group. The first instar Daphnia were placed in groups of five, at random into glass dishes containing 100 ml of medium to give a loading o f 20 ml medium per organism. The dishes were loosely covered. Test concentrations The preliminary tests employed test concentrations w'ithin the range 1 to 100 mg/1 (as active ingredient). The definitive test concentrations, which were selected based on the results of the preliminary tests, were: Nominal concentrations: 4.27, 9.39, 20.7,45.5 and 100 mg active ingredient/1. (Expressed in terms of the test substance as received: 17.1, 37.6, 82.8, 182 and 400 mg/1) Medium renewal Daphnia were exposed to the test or control conditions for a period of 48 hours without renewal of test media. : 11 : Com pany Sanitized. DOe=S not contain T S C A C B l DPT440/992118 Environmental conditions The temperature o f the test area was maintained at 20 2C and constant to within 1C during the study. Temperature was continuously monitored in an additional vessel containing the same volume of dilution medium. A photoperiod o f 16 hours light: 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. No supplementary aeration was employed and no feed was given during the exposure period. The temperature, pH and dissolved oxygen levels o f control and test media were recorded at the start and at the end of the study. CRITERION OF EFFECT Daphnia were considered to be immobile if they were unable to swim within approximately 15 seconds following gentle agitation of the test vessel. The numbers of mobile, immobile and floating Daphnia were counted 24 and 48 hours after the start of the study. EVALUATION OF DATA The "no-observed-effect concentration" (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% is considered to be significant. PROTOCOL DEVIATIONS None. : 12 : Com pany Sanitized. Does rsGt contain T S C A CB! DPT440/992118 MAINTENANCE OF RECORDS All specimens, raw data and study related documents generated during the course o f 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 : RESULTS DPT440/992118 Chemical analysis The results of chemical analysis are given in Table 1 and an example chromatogram is illustrated in Figure 1. Concentrations are expressed in terms of the measured levels of the active ingredient. Results for unfiltered samples o f j|j iH I H ^ in d i c a t e d that the intended exposure concentrations were adequately achieved (between 85 and 97% of nominal values) and were maintained (between 93 and 100% of starting values). The overall mean measured levels were 4.01,8.92, 18.6,40.2 and 85.9 mg a.i./l. Immobility Observations of the Daphnia in each control and test vessel, made after 24 and 48 hours, are listed in Table 2. . After 48 hours, 20% immobility occurred at the highest exposure concentration (85.9 mg a.i/1) and the highest concentration at which no immobilisation had occurred was 40.2 mg/1. The 48-hour EC could not be calculated but must be >85.9 mg a.i./l. Under the General Classification and Labelling Requirements for Dangerous Substances and Preparations,M pPH H H M U is not considered to require-classification as the 48-hour ECM is considered to be greater than the highest nominal concentration, 100 mg a.i./l (mean measured level of 85.9 mg a.i./l). Environmental parameters The measurements o f water quality (temperature, pH, dissolved oxygen and total hardness) are summarised in Table 3; they remained within acceptable limits during the study. The test media were clear and colourless. : 14 : Company Sanitized. Does not contain T S C A CBt CONCLUSIONS DPT440/992118 The 48-hour ECWo f ^ B B B M ^ |f o r the immobilisation of Daphnia magna could not be calculated but must be >85.9 mg active ingredient/1. The `no-observed-effect concentration' ol ingredient/1. I^jwith Daphnia magna was 40.2 mg active REFERENCE Official Journal o f 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 fEnvironmental Effect" p.62 - 64. : 15 : Company Sanitized. Doss not contain TSCA CBi DPT440/992118 FIGURE 1 Typical sample chromatogram 37.6 mg/1 (9.39 mg a.i71) taken from Day 2 of the study CHANNEL A INJECT 0 1 -1 2 -9 8 19:52:24 STORED TO BIN 0 57 DATA SAUED TO BIN # 57 : 16 : Com pany Sanitized. Does not conta:n TSC A GBl TABLE 1 Measured concentrations DPT440/992118 Nominal conc.,5 (mg/l) 0 4.27 9.39 20.7 45.5 100 Measure 0 hours 48 hours nd nd 4.14 3.87 [97] [91] 8.93 8.90 [95] [95] 1-8.7 18.5 [90] [89] 41.5 38.9 [91] [85] 86.2 85.5 [86] [86] C oncentrations (mg/l) % ti Overall mean -93 4.01 (94) 100 8.92 (95) 99 18.6(90) 94 40.2 (88) 99 85.9 (86) $ in terms of the active ingredienti__ nd none detected (< 0.5 mg active ingredient/l). % ti measured concentration after 48 hours expressed as a percentage of the starting concentration. [ ] measured concentration expressed as a percentage of the nominal concentration, ( ) overall mean measured concentration expressed as a percentage of the nominal concentration. 17 Company Sanitized. Decs ne{contain TSCA CBi DPT440/992118 TABLE 2 Cumulative immobilisation Nominal conc. mg/1 (measured) Control (nd) 4.27(4.01) 9.39 (8.92) 20.7(18.6) 45.5 (40.2) 100 (85.9) Cumulative numbers of immobile Daphnia 24 hours 48 hours R. r2 Rj R< total % R, R2 Rj R. total % 000000000 000 0 00000000 000 0 0 0 000 0 0 0 0 0 0 0000000000 00 000000000 000 0 0 0 0 0 0 2 2 0 0 4 20 $ as active ingredienti nd none detected (< 0.5 mg active ingredient/1) R replicate number : 18 : Company Sanitised. D s n.-t co"ta!n TS! :A C**l TABLE 3 Environmental parameters temperature, pH and dissolved oxygen DPT440/992118 ! Nominal conc.,$ mg/l Temperature C Dissolved oxygen pH %ASV j (measured) Oh 48 h Oh 48 h Oh 48 h ii Control (nd) 19.7 19.8 7.8 7.3 95 92 4.27(4.01) 20.0 19.9 7.8 7.8 93 91 ! 9.39 (8.92) 20.0 20.0 7.7 7.6 92 92 II 20.7(18.2) 19.9 19.9 7.6 7.8 92 92 45.5 (40.2) 20.2 19.9 7.5 7.8 91 91 100 (85.9) 19.8 20.0 7.2 7.8 89 89 *' nd ASV : as active ingredient : none detected (<0.5 mg active ingredient/l). : air saturation value. The total hardness and alkalinity o f the batch of Elendt M4 medium used were 240 mg/l and 65 mg/l as CaCO, respectively. Continuous monitoring o f an additional vessel containing diluent = 19.9 to 20.4C. II I i ! i i : 19 : Com pany Sanitized. Does not contain TSCA CBI APPENDIX 1 ELENDT M4 MEDIUM Trace elements H,BOj MnCl,.4H,0 LiCl RbCl SrCl2.6H20 NaBr N a,M o 0 4.2HjO CuC1.2H20 ZnClj CoCl,.6H20 KI Na2.S e 0 3 NH4VOj Fe-EDTA solution Macro nutrients CaCl2.2H20 M gS04.7H,0 KCI NaHCOj Buffer nutrients N a2.S i0 j.9 H 20 NaNOj k h 2p o 4 k 2h p o 4 Vitamins Thiamine hydrochloride Cyanocobalamine (B12) Biotin mg/1 2.86 0.36 0.31 0.071 0.152 0.016 0.063 0.0165 0.013 0.010 0.0033 0.0022 0.00058 3.50 mg/1 294 123 5.80 64.8 mg/1 10 0.274 0.143 0.184 mg/1 0.075 0.0010 0.00075 The above analytical grade reagents are dissolved in reverse osmosis water. DPT440/992118 : 20 : Com pany Sanitized. Does not contain T S C A CBF DPT440/992118 APPENDIX 2 THE DETERMINATION O F | 1 H M I M m N AQUEOUS MEDIA SAMPLE ANALYSIS The aqueous samples were diluted with sodium hydroxide and acetonitrile to bring the expected ^ liH H ta ^ c o n c e n tra tio n s within the calibration range. Determination o f g P ^ H I H H J w a s by high performance liquid chromatography (HPLC) using a conductivity detector1. 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 Laboratories 250 x 4.6 mm Ambient Acetonitrile : aqueous buffer solution (25 : 75% v/v) l.Oml/min 50 mN sulphuric acid 2.5 ml/min 100 pi Aqueous buffer solution: 2mM ammonium hydroxide/lmM sodium carbonate (made up in ultra high purity water) and filtered through 0.2 micron cellulose nitrate filter paper. Under the above conditions :hromatographed as a single peak (see Figure 3). 1A 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. : 21 : Company Sanitized. Do as not contain 7 S C A CBf DPT440/992118 CALIBRATION SOLUTIONS Calibration solutions were prepared with the same batch of the toxicity test solutions. jused in the preparation of 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 ^ ^ M IB B B Ij^ o n c e n tra tio n s were determined using mean bracketing standards. The mean peak height responses were calculated f o r ^ p ^ M M ^ i n bracketing standard chromatograms. T h e n |^ m ^ D c o n c e n t r a t i o n in each sample was then calculated using the following equation: Concentration1 mg/1) = X i l i i x F Where Y Z A F Detector response tol Mean detector response to bracketing standard. Concentration of bracketing standard (mg/1). Factor to take into account sample processing. The p u r i t y f p | H | H H f l ^ o f the test substance i^given in the' ;st Substance Data Sheei Nominal and fortified concentrations are reported as| s supplied and in terms of the active ingredient. VALIDATION OF THE ANALYTICAL PROCEDURE The analytical procedure was validated by determining the linearity o f 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. : 22 : Company Sanitized. D oss net contain TSG A C B f Validation recoveries ol DPT440/992118 TABLE 1 rom fortified samples of dilution media Medium Fortification level (mg/1) Recovery as a % of fortification level As supplied As active ingredient I Dechlorinated tap water Control Control ND 387.9 96.98 95.8 387.9 96.98 104 Algal Control Control ND 535.5 133.9 92.0 530.0 132.5 101 214.2 53.55 92.6 212.0 53.00 I Elendt M4 Control Control 95.5 ND 9.93 2.483 98.5 9.93 2.483 95.9 496.5 124.1 103 496.5 124.1 103 Overall mean (RSD) 98.1 (4.2) Active ingredient content of RSD: relative standard deviation. ND: none detected; less than the limit of detection (trout and algal studies : 2.5 mg a.i./l; Dctphnia study: 0.5 mg a.i./l). The limit o f detection is defined as the analyte concentration in a processed sample which would give a peak equal to 3 x local base-line noise. : 23 : Sanilissd. D oss not contain TSC Company Stability oi TABLE 2 in dilution medium DPT440/992118 Storage conditions Procedural recovery 1 Procedural recovery 1 Light, sealed, room temperature 1 Light, sealed, room temperature 1 Dark, sealed, 4C ' Dark, sealed, 4C 1 Procedural recovery2 Procedural recovery 2 ' Fortification level: 11.20 mg/l as 2Fortification level: 11.65 mg/l Results are given as percentage recoveries ol Time-point 0 hours 20 hours 110 - 112 - - 103 - 106 - 109 - 93.1 - 109 - 98.9 ifter storage for the indicated time. : 24 : Com pany Sanitised. Doss not contain T S C A CBS DPT440/992118 FIGURE 1 Standard Calibration fo; (ca 40 - 500 mg/I) Concentration (mg Run no.:DPT/439/013 Standard concentration (mg/1) 0.0 43.57 108.9 217.9 435.7 544.7 NOP: no observable peak Peak height NOP 4075 10668 20734 38644 47864 . : 25 : Company San1 ;ed. Does nei contain ; 3CA CB: FIGURE 2 Standard Calibration for (ca 4 to 50 mg7) DPT440/992118 Run no.:DPT/440/002 Standard concentration (mg/1) 0.0 4.720 11.80 23.60 47.20 59.00 NOP: no observable peak Peak height NOP 3295 8657 18669 38224 48699 ' : 26 : Contains TSCA CBlj FIGURE 3 DPT440/992118 Typical calibration chromatography - 217.9 mg/I (= 54.47 mg/I as a.i.) CHANNEL A INJECT 09-09-98 14:45:47 STORED TO BIN # 84 DATA SAVED TO BIN # 84 FIGURE 4 Typical chromatography - Unfortified algal medium __ ____________CHANNEL A' W J E C T 09-09-70 14:55: XT STORED TO'BIN B 85 Y DATA SAVED TO BIN # 85 : 27 : Com pany Sanitized. Does net contain t S C A C3 DPT440/992118 FIGURE 5 Typical chromatography Sample of algal medium fortified at 535.5 mg/l (= 133.9 mg/1 as a.i.) CHANNEL A INJECT 09-09-98 13:19:59 STORED TO BIN 11 75 DATA SAVED : 28 : Com pany Sanitized. Ceas net contain T S C A C B l