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BACK TO MAIN NON-GLP STUDY: PFOS BACKGROUND SCREEN IN SELECTED WILDLIFE INTERNATIONAL, LTD. AVIAN AND AQUATIC FEED MATRICES WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C- 101-2 AUTHORS: Raymond L. Van Hoven, Ph.D. Willard B. Nixon, Ph.D. STUDY INITIATION DATE: January 18, 1999 STUDY COMPLETION DATE: September 11,2000 Submitted to: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, MN 55144 WildlifeInternational, Ltd. Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (4 10) 822-8600 [] Page 1 of 25 WILDLIFE INTERNATIONALLT,D. -2- BACK TO MAIN PROJECT NO.: 454C-101-2 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Non-GLP Study:PFOS Background Screen in SelectedWildlife International, Ltd. Avian and Aquatic Feed Matrices !' WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454C-101-2 .. .. STUDY DIRECTOR: .. Raym-ow nd I!Van Hoven, Ph.D. L. Scientist i' .A ,I MANAGEMENT: .. .L ., I . . 9 - !: Willard B. Nixon, Ph.D. I .. Manager, Analytical Chemistry ,. .. 3: 1.; DATE -1 I - DATE . WILDLIFE INTERNATIONALL.TD. -3- BACK TO MAIN . PROJECT NO. 454C-101-2 TABLE OF CONTENTS TitleICover Page .................................................................................................................................... 1 Report Approval .................................................................................................................................... 2 Table of Contents .................................................................................................................................. 3 Summary ............................................................................................................................................... 5 Introduction........................................................................................................................................... 6 Purpose.................................................................................................................................................. 6 Experimental Design.............................................................................................................................. 6 Materials and Met.hods........................................................................................................................... 7 Test Substance .......................................................................................................................... 7 Internal Standard....................................................................................................................... 7 Reagents and Solvents............................................................................................................... 7 Test Systems ............................................................................................................................. 7 Avian Feed ......................................................................................................................... 7 Aquatic Feed....................................................................................................................... 8 Test Procedures......................................................................................................................... 8 Calibration Curves .................................................................................................................... 9 Limits of Quantitation............................................................................................................... 9 Reagent Blanks ....................................................................................................................... 10 Results and Discussion ........................................................................................................................ 10 Avian Feed ............................................................................................................................... 10 Aquatic Feed............................................................................................................................. 11 Conclusions......................................................................................................................................... 11 TABLES Table 1 . Typical LCMS OperationalParameters............................................................................... 12 .. .. Table 2 - Determination of PFOS in Avian Feed ................................................................................ 13 .. Table 3 - Determinationof PFOS in Aquatic Feed ............................................................................. 14 -- BACK TO MAIN WILDLIFE INTERNATIONAL. LTD. -4- PROJECT NO.:454C-101-2 TABLE OF CONTENTS - Continued - FIGURES Figure 1 - A representative calibration curve for PFOS...................................................................... 15 Figure 2 - A representative ion chromatogram of a low-level (0.005 mg a.i./L) calibration standard... 16 Figure 3 - A representative ion chromatogram of a high-level (0.040mg a.i./L) calibration standard.. 17 Figure 4 - A representative ion chromatogram of a reagent blank sample from the avian feed screen. 18 Figure 5 - A representative ion chromatogram of an avian feed matrix blank sample ......................... 19 Figure 6 - A representative ion chromatogram of an avian feed matrix fortification sample................ 20 Figure 7 - A representative ion chromatogram of an artemia cyst aquatic feed sample. ...................... 2 1 .- Figure 8 - A representative ion chromatogram of a YCT aquatic feed sample. ................................... 22 !r ' Figure 9 - A representative ion chromatogram of a green-algae aquatic feed sample. ......................... 23 APPENDICES Appendix I - Diet Formulation - Wildlife International, Ltd. Game Bird Ration.................................24 , Appendix II - Personnel Involved in the Study ................................................................................... 25 BACK TO MAIN WILDLIFE INTERNATIONALLT,D. -5- PROJECT NO.: 454C-101-2 SPONSOR: SPONSOR' S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE F I N A L REPORT: SUMMARY 3M Corporation Rochelle Robideau Wildlife International, Ltd. Easton, Maryland WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: TEST DATES: 454C-10 1-2 PFOS Non-GLP Study: PFOS Background Screen in Selected Wildlife International, Ltd. Avian and Aquatic Feed Matrices Experimental Start - January 18, 1999 Experimental Termination - June 9, 1999 ___ TEST SYSTEMS: I. Avian Feed: Aquatic Feed: ~~ ~____ WLI Basal Ration (low-calcium diet) WLI Basal Ration + Limestone (high-calcium diet) Artemia Cysts Yeast, Cerophyll@,and Trout Chow (YCT) Green Algae (Selenastrum capricornutum) . , SUMMARY: PFOS concentrations were significantly below limits of quantitation in avian feed a. .* used by Wildlife International, Ltd. in avian toxicology studies. Low (ppb-level) concentrations of PFOS were measured in dry artemia cysts used as starting feed material in selected aquatic toxicology tests. The concentration levels anticipated in applied artemia (decapsulated and diluted in saltwater) are not expected to be of consequence in associated chronic effects testing. PFOS was not detected in YCT I: !! and green algae feed suspensions used by Wildlife International, Ltd. in chronic .. aquatic toxicology studies. L .: . -1.. 1.. .. I- BACK TO MAIN WILDLIFE INTERNATIONALLID., -6- PROJECT NO.: 454C-101-2 INTRODUCTION The integrity of environmental toxicology studies can be compromised if measurable quantities of the test substance under investigation are present in components of the test system. This is especially important in chronic or reproductive studies. In these studies, the potential for toxicological effects from a non-controlled source of the test substance introduced into the test system is greater than those espected in relatively shorter-term acute studies. Random, or non-controlled input sources, may bias the interpretation of toxicological effects from the introduction of controlled doses of the test substance into the test system. The types of feed used to sustain test species in chronic studies are a significant potential source of non-controlled input. Consequently, the screening of the feed to be used in anticipated toxicological effects testing is usefil to ensure the success of the studies. In this investigation, selected feed used by Wildlife International, Ltd. for avian and aquatic toxicology studies were screened for PFOS (perfluorooctane sulfonic acid, potassium salt) content. This study was conducted by Wildlife International, Ltd. and identified as Project Number 454C-101-2. The analyses of samples were performed at Wildlife International Ltd. analytical laboratory using high performance liquid chromatography with mass spectrometric detection (LCMS). Blank and fortified avian feed samples were prepared and analyzed between January 18 and February 3, 1999. Blank aquatic feed samples tests were prepared and analyzed between June 8 and 9, 1999. The raw data and a copy of the final report generated by Wildlife International, Ltd.are filed under Project Number 454C-101-2 in archives located on the Wildlife International, Ltd.site. PURPOSE The purpose of this study was to determine background concentrations of PFOS, if detectable, in selected avian and aquatic feed to be used in future environmental effect studies. EXPERIMENTAL DESIGN The testing consisted of either extraction (avian feed and artemia cysts) or dilution (YCT and green algae) of blank a i d o r fortified feed materials followed by PFOS analysis by high performance liquid chromatography with mass spectrometric detection (LCMS). BACK TO MAIN WILDLIFE INTERNATIONALLT,D. . -7- PROJECT NO.: 454C-101-2 Quantitation was performed with external standards of PFOS using concentrations that bracketed the expected instrumental concentrations of the samples. Internal standardization was used for avian feed screens only. For each sample set, one calibration curve was generated from the analyses of PFOS standard solutions. For avian feed screens, all calibration standards and samples contained the same nominal concentration (0,100 m a ) of the internal standard. MATERIALS AND METHODS Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675 upon receipt. The test substance, a white powder, was identified as: FC-95, Lot Number: 217, Expiration Date: 2008. The test substance had a reported purity of 98.9% and was stored under ambient conditions. Internal Standard The internal standard was received from 3M Corporation on July 2, 1998and was assigned Wildlife International, Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: IH, 1% 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619- 97-2. The standard was stored under ambient conditions. The internal standard is referred to hereafter as 8. 4HPFOS. 1: Rearrents and Solvents All solvents used were HPLC grade or equivalent. Test Svstems Avian Feed Two avian feed (game-bird ration) types were investigated. The feed types differ only in the r: :: I ..i omission or addition of supplemental calcium. Wildlife International, Ltd. (WLI) Basal Ration is a relatively low-calcium diet employed in acute toxicology studies. WLI Basal Ration + Limestone is a highcalcium diet used in pilot and reproductive studies. A calcium-enriched diet for female birds enhances egg production. The specifications for the ingredients common to these types of feed are presented in Appendix I. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. -8- PROJECT NO.: 454C-101-2 AQuatic Feed The feed used at Wildlife International, Ltd. for early life-stage studies with the fathead minnow (Pimephalespromelas) and life-cycle studies with the saltwater mysid (Mysidopsisbahin) consists of decapsulated artemia cysts (brine shrimp eggs) suspended in saltwater. Dry artemia cysts (Bonneville Artemia International, Salt Lake City, UT), prior to decapsulation and suspension in freshwater, were selected for the PFOS screen. Lifecycle studies with the cladoceran (Daphnia magna) employ freshwater suspensions of YCT (yeast, Cerophyll*, and trout chow) and green algae (Selenastrum capricornutum). These freshwater feed suspensions were taken for PFOS analysis. Test Procedures The analfical method for the analysis of PFOS in avian diet involved an extraction with methanol .. (HPLC grade, 99.9+%) and analysis by liquid chromatography mass spectrometry. Each type of avian diet was fortified at 1000mg a.i./kg PFOS (target nominal concentration)by dry mixing the feed with the test substance, and diluting the fortification to 1.00 mg a.i./kg PFOS (target nominal concentration) with .. the appropriate unfortified (blank) feed. For each avian feed type, duplicate 10-gram sub-samples of i- I - fortified (1.00 mg a.i./kg) and blank avian diets were transferred into 16-ounce French square bottles. One hundred milliliters of methanol was added to each bottle. The bottles were sealed with screw caps, placed on a tabletop shaker and shaken for a minimum of 30 minutes. The samples were vacuum filtered L .. using qualitative fiiter paper. The avian diet was rinsed three times with methanol into the filtrate. The filtrates were then transferred into 200-mL volumetric flasks and brought to volume with methanol. Final I. dilutions were performed by transferring five milliliters of each sample into 10-mL volumetric flasks .. partially-filled with NANOpure@water. The flasks were fortified with 100 pL of a 10 pg a.i./mL solution ._ of 4HPFOS (internal standard) in methanol. The solutions were brought to volume with NANOpure@ .. ._ water, mixed and filtered through a 13-mm, 0.2-pm Nylon@filter (Gelman Acrodisc@,lot 1572) into HPLC autosampler vials. The vials were capped and submitted for LCMS analysis. .. .I s: i.i The analytical method for the analysis of PFOS in artemia cysts consisted of extraction of replicate 10-gram samples of unfortified feed with 25 milliliters of methanol (HPLC grade, 99.9+%). Approximately three milliliters of each extract was removed from the methanol-saturated feed. A sub- sample of each 3 - I ~ Lextract was ampulated and submitted for LCMS analysis. The analpcal method for the analysis of PFOS in freshwater suspensions of YCT and green algae consisted of dilution prior to .. BACK TO MAIN WILDLIFE INTERNATIONALL.TD. -9- PROJECT NO.: 454C-101-2 LCMS analysis. Replicate one-milliliter sub-samples of each unfortified aqueous suspension were diluted to 10-mL final volumes with a solution of 50% methanol and 50% NANOpureO water containing 0.05% v/v formic acid and 0,100 mg/L of the internal standard. A sub-sample of each dilution was ampulated and submitted for LCMS analysis. Concentrations of PFOS were determined by reverse-phase high performance liquid chromatographyusing a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API lOOLC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using one of two Keystone Betasil C18columns (2-mm I.D., 3-pm particle size). A 100-mm column was used for avian feed screens and a 50-nun column was used for the aquatic feed screens. The instrument parameters are summarized in Table 1. Calibration Curves Calibration standards of PFOS, prepared in a 50% methanol : 50% NANOpure@water solution containing 0.100 mg 4HPFOS (internal standard)& and 0.05% formic acid (v/v), were analyzed with the samples. For avian feed samples, the calibration standards ranged in concentration from 0.005 to 0.040 mg a.i./L. For aquatic feed samples, the calibration standards ranged in concentration from 0.001 to 0.010 mg a.i./L. A minimum of three calibration standards was analyzed with each set of samples. The set of standards was injected at the beginning and end of each run, and one standard was injected, at a minimum, after every four samples. For avian feed samples, weighted (Ux) linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) of the calibration standards. For aquatic feed samples, weighted (l/x), linear regression equations were generated using peak area response versus PFOS concentration (internal standard not applied). A representative calibration curve is presented in Figure 1. The concentration of PFOS in the samples was determined by substitutingthe peak area response or peak area response ratio (as appropriate) into the applicable linear regression equation. Representative ion chromatogramsof low and high calibration standards are presented in Figures 2 and 3, respectively. Limits of OuantitzzLn The method limit of quantitation (LOQ) for analyses of avian feed samples was set at 0.20 mg a.i./kg. The LOQ was calculated as the product of the lowest calibration standard analyzed . .. . I. !' BACK TO MAIN WILDLIFE INTERNATIONALLT,D. - 10- PROJECT NO.: 454C-101-2 (0.005 mg a.i./L), the extraction volume-to-sample weight ratio (0.1 LjO.01 kg), and dilution factor of the matrix blank samples (4). The LOQ for analyses of artemia cyst samples was set at 0.0003 mg a.i./kg, calculated as the product of the lowest calibration standard analyzed (0.001 mg a.i./L), the extraction volume to sample weight ratio (0.025 L/O.O1 kg), and dilution factor of the samples (0.12). The LOQ for analyses of freshwater suspensions of YCT and green algae samples was set at . .. .( ._ 0.010 mg a.i./L, calculated as the product of the lowest calibration standard analyzed (0.001 mg a i & ) , and the dilution factor of the samples (10). Reagent Blanks Reagent blanks were analyzed to assess the presence of potential interferences. No chromatographic interferences were observed in the blanks at or above the limit of quantitation. For the avian feed screen, the reagent blank consisted of an aliquot of methanol diluted with NANOpure@water, fortified with internal standard, and filtered as for the study samples. For the aquatic feed screen, the reagent blank consisted of dilution solvent (a solution of 50% methanol and 50% NANOpure@water containing 0.05% v/v formic acid and 0.100 mgL of the internal standard). A representative ion chromatogram of a reagent blank sample from the avian feed screen is presented in Figure 4. I1 RESULTSAND DISCUSSION ._ Avian Feed The results of the avian feed screen are presented in Table 2. The PFOS concentration of all blank avian feed samples was significantly below the LOQ for the methodology (0.2 mg a.i./kg). The feed fortified at 1.00 mg a.i./kg had a mean PFOS recovery of 83.3 k 10%. No difference in analyhcal ......:>_ behavior was observed between the low- and highcalcium diet matrices. A representative ion i- : :I .:! chromatogram of an unfortified high-calcium diet sample is presented in Figure 5 . A corresponding representativeion chromatogram of a fortified sample is presented in Figure 6. BACK TO MAIN WILDLIFE INTERNATIONALLID,. - 11 - PROJECT NO.: 454C-10 1-2 Aquatic Feed The results of the aquatic feed screen are presented in Table 3. Concentrations of PFOS ranging - from 0.0009 to 0.001 mg a.i./kg were determined in dry artemia cysts. PFOS was not detected in either the YCT or green algae freshwater suspensions. Representative ion chromatograms of each aquatic feed matrix are presented in Figures 7-9. CONCLUSIONS PFOS concentrations were significantly below limits of quantitation in avian feed used by Wildlife International, Ltd. in avian toxicology studies. Low (ppb-level) concentrations of PFOS were measured in dry artemia cysts used as starting feed material in selected aquatic toxicology tests. The concentration levels anticipated in uppZied artemia (decapsulated and diluted in saltwater) are not expected to be of consequence in associated chronic effects testing. PFOS was not detected in YCT and green algae feed suspensions used by Wildlife International, Ltd. in aquatic toxicology studies. I . ,. .. BACK TO MAIN WILDLIFE INTERNATIONALLT,D. - 12- PROJECT NO.: 454C-101-2 Table 1 Typical LCMS Operational Parameters WSTRUMENT: Hewlett-Packard Model 1100 High PerformanceLiquid Chromatograph with a Perkin-Elmer API I OOLC Mass Spectrometer equipped with a Perkin-ElmerTurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil C18column (100 mm or 50 mm x 2 tmn I.D., 3-pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 10 minutes FLOW RATE: 0.220 d m i n u t e MOBILE PHASE: INJECTION VOLUME: 72% Methanol : 28% NANOpureB Water containing 0.1% Formic Acid 50.0 jd,:avian feed screen 25.0 pL: aquatic feed screen PFOS RETENTION TIME: Approximately 7.5 minutes (100-mm column) Approximately 3.6 minutes (50-mm column) INTERNAL STANDARD RETENTION T[ME: Approximately 4.9 minutes (100-mm column) PFOS MONITORED MASS: 498.6 amu INTERNAL STANDARD MONITORED MASS: 426.7 amu BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 15 - PROJECT NO.: 454C-101-2 Sample .. . Number (454C-10 1-2-) FS-MAB-1' FS-MAB-2' FS-MAS-1' FS-MAS-2I Table 2 Determination of PFOS in Avian Feed Concentration of P W i . K g ) Fortified Measured NA <LOQ~ NA <LOQ 1.oo 0.783 1.oo 0.724 Percent Recovery' NA NA 78.3 72.4 FS-MAB-3' NA <LOQ NA FS-MAB-4' NA <LOQ NA FS-MAS-3* 1.oo 0.873 87.3 FS-h4AS-4* 1.oo 0.95 1 95.1 Mean = 83.3% Standard Deviation = 10.0% N= 4 ~ 'Wildlife International, Ltd. Basal Ration (low-calcium diet). 'Wildlife International,Ltd. Basal Ration + Limestone(high-calcium diet). .. %e limit of quantitation (LOQ) was 0.20 mg a.i./kg calculated as the product of the lowest standard concentration (0.005 mg ., a.i&), the extraction volume to sample weight ratio (0.1 UO.01 kg), and the dilution factor of the matrix blanks (4). 'Percent recovery = [measured result (mg aikgynominal concentration(mg ai./kg)l x 100. l* .. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 14- PROJECT NO.: 454C-101-2 Table 3 Determination of PFOS in Aquatic Feed Sample Number (454C-10 1-2-1 ART- 1' ART-2' YCT-1' YCT-2* Concentration of PFOS Measured Measured (mg a.i./kg) (mg a.i./L) 0.0010 NA4 0.0009 NA NA ND' NA ND NA ND NA ND 'ART = Dry artemia cysts. Bonneville Artemia, Bonneville Artemia International, Salt Lake City, UT. 2YCT = Yeast, Cerophyll@,and Trout Chow. Freshwater suspension. Wildlife International,Ltd. Lot No. 99-9. 'GALG = Green algae (Selenusfrum cupricomufum).Freshwater suspension. Wildlife International, Ltd. Lot NO.99-22. %A = Not applicable. i. %D = Not detected Estimated instrument detection limit was 0.0001 mg a.i./L.. I . . ,. r- .. WILDLIFE INTERNATIONALLID,. - 15 - BACK TO MAIN PROJECT NO.:454C-101-1 Area .. .. . .. Figure 1. A representative calibration curve for PFOS. Intercept = 1964.6; Slope = 12284942; 3= 0.9920,weighted ( l / x ) . a. WILDLIFE INTERNATION~AmL,. - 16- BACK TO MAIN PROJECT NO.:454C-101-1 100 I 90 80 70 60 50 40 30 ,/ . r: 2 1 intensity: 797 cps 188 31 61 91 121 151 181 211 scan 1.25 2.45 3.66 4.86 6.07 7.27 8.48 Time I Figure 2. A representative ion chromatogramof a low-level (0.005 mg a.i./L)calibration standard. :.. :. WILDLIFE INTERNATIONLATDL. , - 17- BACK TO MAIN PROJECTNO.:454C-101-1 100 90 80 70 60 50 40 f t 10 intensity: 2977 cps 188 Figure 3. A representativeion chromatogramof a high-level (0.040mg a.i./L)calibration standard. .. WILDLIFE INTERNATIONALL,TD. -18- BACK TO MAIN PROJECT NO.:454C-101-1 1o e 52 90- I . .. . .. 1. i intensity: 27 cps 161 Figure4. A representative ion chromatogram of a reagent blank sample from avian feed screen (methanolextraction blank). WILDLIFE INTERNATIONLTADL. , - 19- BACK TO MAIN PROJECT NO.:454C-101-1 intensity: 508 cps 209 I I 3 2 100-p .I? A3 I I 8 31 125 61 2.45 IO0 I I 91 121 151 181 211 scan 3.66 4.06 6.07 7.27 8.48 Time Figure 5. A representativeion chromatogramof an avian feed matrixblank sample (454C-101-2-FS- MprB-4). WILDLIFE INTERNATIONLTADL. , - 20 - BACK TO MAIN PROJECTNO.:454C-101-1 .... .._._ , .: 100- intensity: 3440 cps ! 90- 80- 70- 60- 50- 40- 30- L. 157 1-1 A 1 6 ! .. 10 75 41 5 5 77 8-8 135 . . !I :.e 0-. -I I I- I I I I - .-I I I I scan 211 7 8.48 Time .. I. .- Figure6. A representative ion chromatogram of an avian feed matrix fortification sample (454C101-2-FS-MAS-4,1.00mg a.i.kgnominal concentration). WILDLIFE INTERNATIONLATDL., -21 - BACK TO MAIN PROJECT NO.:454C-101-1 intensity: 8643 cps 10 9 8 7 :. 6 5 4 3 2 1 . - 21 41 61 81 101 scan .. .. 0.88 1-72 2.55 3.39 4.23 Time --- r- Figure 7. A representative ion chromatogram of an artemia cyst aquatic feed sample (454C-101-1m-r-1). WILDLIFE INTERNATIONLATDL. , -22- BACK TO MAIN PROJECTNO.:454C-101-1 intensity: 274 cps 10 9 a 7 6 5 4 3 2 1 1 21 0.88 . I 41 1.72 I I 61 2.55 I t 81 3.39 . I 101 4.23 scan The Figure 8. A representative ion chromatogram of a YCT aquatic feed sample (454C-101-1-YCT-1). WILDLIFE INTERNATIONLIAD. L, - 23 - BACK TO MAIN PROJECTNO.:454C-101-1 / ! 9 I I intensity: 112 cps 3 2 1 .- 21 4-11 61 81 101 scan I.: .: 0.88 1 .M 2.55 3.39 4.23 Time ;. :. ti Figure9. A representative ion chromatogram of a green algae aquatic feed sample (454C-101-1. . , GALaG-1). :; ,. BACK TO MAIN WILDLIFE INTERNATIONALLT,D. - 24 - PROJECT NO.: 454C-101-2 APPENDIX I Diet Formulation Wildlife International, Ltd. Game Bird Ration' INGREDIENTS PERCENT (%) Fine Corn Meal Soy Bean Meal, 48% Protein Wheat Mid& Protein Base Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos Methionine Premix + Liquid Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)' Salt Iodized Total 44.83 30.65 6.50 6.00 4.00 3.00 2.50 0.90 0.60 0.35 0.32 0.25 0.10 100.00 VITAMIN AND MINER4L PREMJX AMOUNT ADDED PER TON Vitamin DS Vitamin A Riboflavin Niacin L . Pantothenic Acid Vitamin B12 !! t: Folic Acid Biotin Pyridoxine Thiamine Vitamin E L> Vitamin K (Menadione DimethylpyrimidinolBisuEte) Manganese Zinc Copper Ll:. '-. Iodine Iron Selenium 2,000,000I.C.U. 7,000,000 I.U. 6grams 40 &rams 10 grams 8mgs 600 mgs Umgs 1.2 grams 1.2grams 20,000 I.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 mgs !. 1 The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of 5% crude fiber. :. 2 Fermentation By-Products I. .. .. .. E BACK TO MAIN WILDLIFE INTERNATIONLATDL. , - 25 - PROJECT NO.: 454C-101-2 APPENDIX I1 Personnel Involved in the Study The follo\ving key Wildlife International Ltd. personnel were involved in the conduct or management of this study: 1. Raymond L. Van Hoven, Ph.D., Scientist 2. Willard B. Nixon, Ph.D., Manager, Analytical Chemistry 3. Jon A. MacGregor, B.S.,Scientist 4. Norman A. Glassbrook, M.S., Senior Chemist 5 . Ken B. Leach, B.S., Chemist